cocaine exposure throughout adolescence

Cecília Juliana Moreira Alves

Cocaine exposure throughout adolescence: a hormonal neurochemical and behavioural study in the rat

Dissertação de Candidatura ao grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Orientador- Professor Doutor Pedro Manuel Ribeiro da Rocha Monteiro Professor Adjunto Escola Superior de Tecnologias da Saúde do Porto, Instituto Politécnico do Porto Co-orientadora- Professora Doutora Liliana Maria de Carvalho e Sousa Professora Associada Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto

Preceitos Legais

Nesta dissertação foram utilizados os resultados dos trabalhos publicados ou em

preparação abaixo indicados. A autora desta dissertação declara que interveio na

concepção e execução do trabalho experimental, na interpretação dos resultados e na

redacção dos resultados publicados ou em preparação, sob o nome de Alves CJ:

In this dissertation were used experimental results published or under preparation stated

below. The author of this dissertation declares that participated in the planification and

execution of the experimental work, in the data interpretation and in the preparation in the

work stated below, under the name Alves CJ:

Publicações em resumo:

Alves CJ, Magalhães A, Summavielle T, de Sousa T, Tavares MA, Monteiro PRR. 2007.

Dopaminergic system response to na anxiogenic situation in rats chronically exposed to

cocaine during adolescence. Neurotoxicology and Teratology 29:402-403.

Alves CJ,

Magalhães A, Summavielle T, Tavares MA, Monteiro PRR. 2006. Effects of

chronic cocaine administration on behavior of adolescent rats in the elevated plus-maze

test. Neurotoxicology and Teratology 28:412- 427.

Publicações por extenso:

Alves CJ, Magalhães A, Summavielle T, Melo P, de Sousa L, Tavares MA, Monteiro

PRR. 2008. Hormonal, neurochemical, and behavioral response to a Forced Swim Test in

adolescent rats throughout cocaine withdrawal. Ann.N.Y.Acad.Sci 1139:366-373.

Alves CJ, Magalhães A, Melo P, de Sousa L, Tavares MA, Monteiro PRR, Summavielle

T. 2009. Cocaine impaired risk-assessment in adolescent rat. (in preparation).

Alves CJ

, Magalhães A, Melo P, de Sousa L, Tavares MA, Summavielle T, Monteiro

PRR. 2009. Altered social behaviour in young adult rats exposed to cocaine throughout

adolescence. (in preparation).

III

Aos meus pais

Agradecimentos

Gostaria de agradecer a todos aqueles que de alguma forma contribuíram para

que a realização deste trabalho fosse possível.

Ao Professor Doutor Pedro Monteiro, meu orientador, que proporcionou a minha

entrada no mundo da ciência, por todo o seu apoio, pela confiança que depositou em

mim e pelo seu optimismo.

À Professora Doutora Liliana de Sousa, minha co-orientadora, por me ter recebido

no seu grupo, pelo apoio e disponibilidade que sempre manifestou para me ajudar, em

especial agradeço a sua ajuda nos estudos comportamentais que integram esta

dissertação, e a sua ajuda com a análise estatística dos resultados.

À Professora Doutora Maria Amélia Tavares, por me ter recebido no seu grupo,

pelo apoio e interesse demonstrados.

À Teresa, por toda a sua ajuda, por encontrar sempre solução para os problemas

que foram surgindo durante a realização deste meu trabalho, por tudo o que me ensinou

e pela sua preocupação com o meu futuro, ficarei para sempre grata.

Ao Pedro e à Ana gostaria de agradecer, mais do que pela sua imensurável ajuda

com trabalho, por tornarem fácil, mesmo em momentos de desânimo, a ida para o

laboratório. À Ana por acreditar em mim, pela sua incansável paciência para me ouvir, e

por tantas vezes me chamar à razão. Ao Pedro pela sua boa disposição, pelos elogios

que fizeram aumentar o meu ego e por me empurrar sempre para a frente. Foi em vós

que tantas vezes fui buscar alento para seguir adiante. Aos dois pelo o que me ajudaram

a crescer durante este período. Para sempre nutrirei uma profunda amizade por vós.

À Joana, por toda a ajuda, em especial por me ter ensinado as técnicas de

dissecção do cérebro indispensáveis à realização deste trabalho.

À Lorena pela sua preocupação com os meus problemas e pelo seu empenho em

tentar ajudar-me sempre, pelos bons momentos e pela amizade.

À Raquel e à Ema que tão importantes foram nos primeiros tempos deste trajecto,

por toda a ajuda.

À Manuela, embora curto o período que passou no laboratório a amizade nasceu

forte e ficará para sempre.

À Aline, à Sílvia, à Danira ao Venâncio e a todos os outros que ao longo destes

anos foram passando pelo laboratório pela vossa ajuda, pela companhia e pela amizade.

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Ao Luís e à Nita por todos os fins-de-semana que passaram comigo no IBMC, ao

Luís pela ajuda nas recolhas, à Nita pela ajuda na injecção dos animais, e aos dois pela

vossa amizade.

À minha família, por tudo, por acreditarem em mim, por estarem sempre lá. Vós

sabeis, sois tudo para mim.

À Fundação para a Ciência e a Tecnologia e ao Programa de Financiamento

Plurianual do IBMC pelo apoio financeiro para a execução deste trabalho.

VIII

Abbreviations List

5-HIAA 5-hydroxyindoleacetic acid

5-HT Serotonin ; 5-hydroxytryptamine

5-HT1A Serotonin-1A receptors

5-HT1B Serotonin-1B receptors

5-HT2 Serotonin-2 receptors

5-HT3 Serotonin-3 receptors

AChE Acetylcholinesterase

ACTH Adrenocorticotropic hormone

ANOVA Analysis of variance

AR Androgen receptor

AVP Arginine vasopressin

cAMP cyclic-AMP

CNS Central nervous system

CPP Conditioned place preference

CRH Corticotrophin-releasing hormone

CRH-R1 Corticotrophin-releasing hormone receptor 1

Ct Threshold cycle

CTA Conditioned aversive properties

D1 Dopamine receptor 1



DA Dopamine

DAT Dopamine transporter

DHT Dihydrotestosterone

DNA Deoxyribonucleic acid

DOPAC 3,4-Dihydroxyphenylacetic acid

e.g. As an example

EPM Elevated plus maze test

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Fab Fragment, antigen binding

Fc Fragment, cristallizable

FSH Follicle-stimulating hormone

FST Forced swim test

GABA Gamma-aminobutyric acid

GAPDH Glyceraldahyde-3-phosphate-dehydrogenase

GluR1 Glutamate AMPA receptor subunit GluR1

GnRH Gonadotropin-releasing hormone

GnRH-R Gonadotropin-releasing hormone receptor

GR Glucocorticoid receptor

HPA Hypothalamus-pituitary-adrenals

HPG Hypothalamus-pituitary-gonads

HPLC-EC High-performance liquid chromatography with electrochemical detection

HPT Hypothalamus-pituitary-testes axis

HVA 4-Hydroxy-3-methoxyphenylacetic acid ; homovanillic acid

i.p. Intraperitoneal injection

i.v. Intravenous injection

LH Luteinizing hormone

LHβ Luteinizing hormone β-subunit

LTP Long-term potentiation

MAO Monoamine oxidase

MR Mineralocorticoid receptor

mRNA Messenger ribonucleic acid

mPFC Medial prefrontal cortex

Nac Nucleus accumbens

NR1 Glutamate NMDA receptor subunit NR1

PCR Polymerase chain reaction

PND Postnatal day

POMC Proopiomelanocortin

X

PTSD Post-traumatic stress disorders

PVN Paraventricular nuclei

qRT-PCR Quantitative real-time PCR

SA-HRP Streptavidin-horseradish peroxidase

s.c. Subcutaneous injection

S.E.M. Standard error of the mean

SERT Serotonin transporter

SN Substantia nigra

SN-VTA Ventral mesencephalon (substantia nigra and ventral tegmental area)

TH Tyrosine hydroxilase

UV Ultraviolet

VTA Ventral tegmental area

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Resumo

A adolescência é um período em que os sistemas neurobiológicos sofrem

importante reorganização através de processos de produção e eliminação de sinapses,

do desenvolvimento dos sistemas de neurotransmissores e de processos de

mielinização. Durante este período do desenvolvimento, os níveis hormonais alteram-se

de forma drástica em resultado do início da puberdade. Estas alterações reflectem-se

também nas competências cognitivas, psicológicas e sociais. Diversos factores

ambientais podem interferir no desenvolvimento neurobiológico e psico-fisiológico dos

adolescentes. Estudos epidemiológicos mostram que é frequentemente nesta fase que se

iniciam as experiências com drogas de abuso. Paradoxalmente, o estudo dos efeitos da

exposição a drogas de abuso, tem sido realizado principalmente em animais adultos, e

apenas pontualmente no adolescente. No entanto, as características ontogenéticas

desde período e o facto da adolescência representar um período propício para o início do

uso de drogas de abuso, afiguram-se importantes argumentos para que se encare a

adolescência como uma idade chave para o estudo dos efeitos destes compostos. Assim,

com este trabalho pretendeu-se contribuir para a caracterização dos efeitos da cocaína

no adolescente. Para atingir este objectivo foram desenvolvidos vários protocolos

experimentais a fim de caracterizar os efeitos neuroquímicos, hormonais e

comportamentais, a curto e a longo prazo, da exposição crónica a cocaína durante a

adolescência.

Neste trabalho foi utilizado um modelo animal de exposição crónica repetida a

cocaína. Ratos Wistar machos e fêmeas foram tratados diariamente, entre o dia pós-natal

(PND) 35 e o 50, com 3 administrações intraperitoneais de uma solução de hidrocloreto

de cocaína numa concentração de 15 mg/kg de peso corporal. Os grupos controlo foram

injectados com uma dose isovolumétrica de soro fisiológico segundo o mesmo

procedimento experimental. Os efeitos da exposição a cocaína na resposta adaptativa a

um estímulo ansiogénico foram avaliados 2, 5 e 10 dias após retirada da cocaína,

recorrendo-se ao elevated plus maze test (EPM). O comportamento depressivo foi

avaliado 3 dias após a última administração de cocaína, recorrendo-se ao forced swim

test (FST). O comportamento agressivo foi avaliado em ratos machos, 10 dias após a

última administração de cocaína, usando o resident-intruder paradigm. Com o objectivo

de determinar alterações nas respostas neuroquímicas e hormonais aos desafios

proporcionados pelos testes comportamentais, após cada um dos testes foram

determinados, por cromatografia de alta resolução com detecção electroquímica (HPLC-

EC), os níveis dos neurotransmissores dopamina (DA), serotonina (5-HT) e dos seus

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principais metabolitos (DOPAC, ácido 3,4-dihidroxifenilacético; HVA, ácido homovanílico;

5-HIAA, ácido 5-hidroxindolacético), em áreas cerebrais de origem do sistemas

dopaminérgico (SN-VTA, substância negra e área tegmental ventral) e serotoninérgico

(núcleo dorsal da rafe) e em áreas de projecção destes directamente envolvidas nos

efeitos da exposição a drogas de abuso (núcleo accumbens (Nac), estriado dorsal, córtex

pré-frontal, amígdala, hipocampo, hipotálamo e cerebelo). Estes doseamentos foram

também efectuados em condições basais, 30 minutos, 1, 5 e 10 dias após a última

administração de cocaína. Do mesmo modo, os níveis plasmáticos de corticosterona

foram doseados por ensaio imunoenzimático, depois dos testes comportamentais. Ainda

a nível plasmático, foram doseadas, por ensaio imunoenzimático, em ratos macho e

fêmea, as seguintes hormonas: hormona adrenocorticotrópica (ACTH), testosterona,

progesterona e corticosterona, em amostras de sangue recolhidas 30 minutos, 1, 5 e 10

dias após a última administração de cocaína. Os níveis plasmáticos de estradiol foram

também determinados em ratos fêmea nos mesmos pontos de avaliação. Os níveis de

expressão de mRNA para a hormona libertadora da corticotropina (CRH) e receptor de

androgénios (AR) foram medidos no hipotálamo; e os níveis de expressão de mRNA

para: receptor da hormona libertadora de gonadotropinas, receptor da CRH, AR, sub-

unidade β da hormona luteinizante, receptor de glucocorticoides (GR) e

proopiomelanocortina foram determinados na hipófise de ratos machos, 10 dias após o

termo da administração de cocaína, recorrendo à técnica de reacção em cadeia da

polimerase em tempo real (qRT-PCR). Para completar a avaliação de alterações na

actividade do sistema dopaminérgico foram avaliados os níveis de expressão de mRNA

para: tirosina hidroxilase (TH), monoamina oxidase-A, transportador de DA, receptores

dopaminérgicos D1, D2, D3 e de GR em vária regiões cerebrais (SN-VTA, Nac, estriado

dorsal, córtex pré-frontal, amígdala, hipocampo e hipotálamo) 10 dias após a última

administração de cocaína, recorrendo à técnica de qRT-PCR.

Os dados obtidos no estudo comportamental indicam que a exposição a cocaína

durante a adolescência afectou a capacidade de resposta adaptativa a desafios

ambientais. A análise dos resultados mostra que, em ratos macho expostos a cocaína, se

verificam diferenças na capacidade de avaliação de risco durante a execução do EPM,

que são acompanhadas por alterações na neurotransmissão dopaminérgica e

serotoninérgica no córtex pré-frontal e amígdala. Estes animais, apresentam também

sensibilização da resposta do eixo HPA ao estímulo ansiogénico proporcionado pelo

EPM. Estas alterações poderão estar implicadas na diminuição verificada na capacidade

de avaliação de risco perante o estímulo aplicado.

XIV

Na avaliação do comportamento depressivo observado no FST, não se

verificaram diferenças entre grupos. Contudo, os dados neuroquímicos indicam que a

resposta à situação stressante proporcionada pelo FST foi afectada.

Na avaliação dos dados obtidos durante o conflito social proporcionado pelo

resident-intruder paradigm verificou-se que a exposição a cocaína intensificou o

comportamento de investigação social e reduziu o comportamento defensivo, indicando

que a cocaína afectou, em ratos macho, a capacidade de interagir adequadamente com

os conspecificos, no entanto não foram observados efeitos do tratamento no

comportamento agressivo. Estas alterações comportamentais foram suportados por

alterações da actividade dopaminérgica e/ou serotonérgica em áreas cerebrais como a

amígdala, o hipocampo e o córtex pré-frontal.

A análise dos dados hormonais obtidos sugere uma adaptação à exposição

crónica a cocaína, não sendo observadas diferenças nos níveis plasmáticos de ACTH e

de corticosterona 1, 5 e 10 dias após a última administração. No entanto, a activação do

eixo hipotálamo-hipófise-supra-renais (HPA) é evidenciada, em ratos macho, 30 minutos

após a última administração de cocaína, resultando em níveis aumentados de ACTH.

Os dados hormonais obtidos indicam ainda alterações, a curto e a longo prazo, no

funcionamento do eixo hipotálamo-hipófise-gonadas (HPG), também em ratos macho,

verificando-se níveis diminuídos de testosterona 30 minutos e 10 dias após a última

administração de cocaína. Em concordância, 10 dias após a última administração de

cocaína, estes animais apresentavam diminuição do peso relativo das gónadas. Nos

ratos fêmea não foram observadas alterações nos níveis de estradiol ou progesterona,

evidenciado, mais uma vez, uma resposta diferenciada entre géneros.

O estudo neuroquímico em ratos macho, aponta para o desenvolvimento de

tolerância nas respostas dos sistemas dopaminérgico e serotoninérgico aos efeitos

agudos da cocaína após o tratamento crónico durante a adolescência, uma vez que para

a maioria das áreas cerebrais analisadas não foram observadas alterações nos níveis de

DA, 5-HT e seus metabolitos, 30 minutos após a última administração de cocaína.

Contudo, 1 dia após a última administração, verifica-se um aumento da actividade

dopaminérgica no SN-VTA e estriado dorsal, em ratos macho, e na amígdala nas fêmeas.

É proposto que este aumento da actividade dopaminérgica seja resultado do estímulo

produzido pela apresentação de pistas previamente associados à administração de

cocaína, potenciando uma resposta do sistema de recompensa desencadeada pela

expectativa da proximidade da administração. A longo prazo, os dados obtidos no estudo

neuroquímico, revelam um aumento de actividade dopaminérgica na via nigrostriatal, este

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aumento é suportado não só pelo aumento dos níveis de DOPAC na SN-VTA e dos

níveis de HVA no estriado dorsal, mas também pelo aumento dos níveis de expressão do

mRNA para a TH na SN-VTA. Nesta região cerebral foi ainda evidenciado aumento dos

níveis de 5-HT. A análise neuroquímica também evidência a resposta diferenciada entre

géneros, verificando-se nas fêmeas, 10 dias após a última administração de cocaína,

aumento de actividade dopaminérgica na amigdala e hipocampo e de actividade

serotoninérgica no hipocampo e Nac.

Os dados apresentados neste estudo mostram que a exposição a cocaína durante

a adolescência induz alterações hormonais, neuroquímicas e comportamentais, a curto e

longo prazo. A comparação dos resultados obtidos neste estudo, com dados da

bibliografia sobre os efeitos da cocaína no adulto, suporta a existência de uma resposta

diferenciada a esta droga durante a adolescência. Salientam-se, na presente dissertação,

as diferenças observadas entre ratos adolescentes macho e fêmea, após exposição

crónica a cocaína, a nível comportamental, neuroquímico e hormonal. Simultaneamente,

este trabalho mostra, pela primeira vez, que a exposição crónica a cocaína na

adolescência compromete, em ratos macho, a adequação da resposta comportamental a

um estímulo ansiogénico, o que é suportado pelos parâmetros fisiológicos avaliados. Os

dados apresentados contribuem de forma relevante para a clarificação dos factores

envolvidos na maior susceptibilidade à experimentação de drogas de abuso evidenciada

na adolescência.

XVI

Abstract

Throughout adolescence, the neurobiological systems are still undergoing

important development rearrangements, associated to continuous plastic events that

result from an integrated process of overproduction and elimination of synapses, evolution

of the neurotransmitter systems and progressive myelination. Hormonal levels are also

changed dramatically during this period as a result of the onset of puberty. Concomitant to

these changes in the brain and hormonal status, significant transitions occur in cognitive,

psychological and social competences. At this time, environmental factors may have a

strong impact on the unique neurobiological and psycho-physiological profile of

adolescents. Epidemiological data indicate that experiences with drugs of abuse and the

onset of addictive disorders are primarily concentrated in the adolescence and early

adulthood. However, studies on the effects of drugs of abuse, have primarily been

conducted in adult animal models and little is known regarding the effects that these drugs

on the adolescent. The objective of the present study was to contribute to further

characterize the effects of exposure to drugs of abuse at this key developing time, using

an animal model of exposure to cocaine. Experimental protocols were developed to

assess short and long-term neurochemical, hormonal and behavioural effects of chronic

cocaine exposure, throughout adolescence, in a rat model.

Male and female Wistar rats were daily injected intraperitoneally with 3 doses of 15

mg/kg per body weight of cocaine hydrochloride, in 0.9% NaCl solution, from postnatal

day (PND) 35 to PND 50. Controls were given isovolumetric 0.9% NaCl solution following

the same experimental protocol. The anxiety-like behaviour was evaluated using the

elevated plus maze test (EPM) in male rats, 2, 5 and 10 days after cocaine withdrawal,

and in female rats, 2 days after withdrawal. The depressive-like behaviour was evaluated

using the forced swim test (FST) both in male and female rats, 3 days after cocaine

withdrawal. The aggressive behaviour was evaluated in male rats, 10 days after

withdrawal, using the resident-intruder paradigm. The levels of neurotransmitters were

analysed after each behavioural test and at 30 minutes, 1, 5 and 10 days after the last

administration of cocaine, through measuring levels of dopamine (DA), serotonin (5-HT)

and its metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; homovanilic acid, HVA; 5-

hydroxyindoleacetic acid, 5-HIAA), by high performance liquid chromatography with

electrochemical detection (HPLC-EC), in the ventral mesencephalon (SN-VTA), the dorsal

raphe nucleus, the nucleus accumbens (Nac), the dorsal striatum, the prefrontal cortex,

the amygdala, the hippocampus, the hypothalamus and the cerebellum. The plasma

levels of adrenocorticotropic hormone (ACTH), corticosterone, testosterone and

XVII

progesterone were determined by an enzyme immunoassay, both in male and female rats

at 30 minutes, 1, 5 and 10 days after the last administration of cocaine. In females, the

levels of estradiol were also determined. Levels of corticosterone were assessed, as well,

after each behavioural challenge. Moreover, in male rats, the expression levels of mRNA

transcripts for corticotrophin-releasing hormone (CRH) and the androgen receptor (AR)

were measured in the hypothalamus; while the expression levels of mRNA transcripts for

CRH receptor 1, gonadotropin-releasing hormone receptor, AR, luteinizing hormone β-

subunit, glucocorticoid receptor (GR) and proopiomelanocortin were measured in the

pituitary, 10 days after cocaine withdrawal, by quantitative real-time polymerase chain

reaction (qRT-PCR). The expression levels of mRNA transcripts, 10 days after cocaine

withdrawal, for tyrosine hydroxylase (TH) were measured by qRT-PCR in the SN-VTA,

Nac and dorsal striatum, while the expression levels of mRNA transcripts for monoamine

oxidase-A, DA transporter, DA receptors (D1, D2, D3) and GR were measured in the SN-

VTA, Nac, dorsal striatum, prefrontal cortex, amygdala, hippocampus and hypothalamus.

The most relevant results obtained indicate that chronic cocaine exposure through

adolescence negatively interfered with the adaptive responses to environmental

challenges. Data analysis showed impaired risk-assessment during the EPM apparatus

exploration, both 2 and 10 days after withdrawal, which was supported by impaired

dopaminergic and serotonergic neurotransmission in the prefrontal cortex and impaired

dopaminergic neurotransmission in the amygdala. These neurochemical alterations may

be involved in the failure of cocaine-treated male rats to exhibit an adaptive behaviour

during the EPM. Cocaine treatment did not have a significant effect on depressive-like

behaviour in both male and female rats, as assessed 3 days after withdrawal. However,

when exposed to the stressful situation provided by the FST, an altered response was

observed in the neurochemical data. Moreover, although no long-term effects of cocaine

were observed in the aggressive behaviour of male rats, as assessed 10 days after

withdrawal, cocaine-exposed males intensified the social investigation behaviour and

reduced the defensive behaviour during the social conflict provided by the resident-

intruder paradigm, indicating that cocaine affected the ability of young adult rats to

properly interact with conspecifics. In accordance, the dopaminergic and the serotonergic

activities, in response to this social conflict, were altered in cocaine-exposed animals.

The neurochemical data also showed that cocaine exposure throughout

adolescence produced long-lasting changes upon the monoamine systems. In male rats,

this was reflected by increased dopaminergic activity in the nigrostriatal pathway,

supported by the increased levels of DOPAC in SN-VTA, increased levels of HVA in

dorsal striatum and increased expression levels of TH mRNA in the SN-VTA; and long-

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lasting increased 5-HT levels in SN-VTA. In cocaine-treated females, these alterations

were revealed through increased dopaminergic activity in amygdala and in hippocampus,

and increased serotonergic activity in hippocampus and Nac.

The analysis of the hormonal data indicates an activation of the hypothalamus-

pituitary-adrenal (HPA) axis in male rats, 30 minutes after the last administration of

cocaine, as showed by the increased ACTH levels. Despite this acute effect after chronic

cocaine treatment, the basal activity of the HPA axis did not appear to have been affected,

as no changes were observed in ACTH and corticosterone levels at 1, 5 and 10 days after

withdrawal, in both male and female rats. Nevertheless, a disruption of the HPA axis

response under anxiogenic conditions was revealed in young adult male rats, which

displayed a sensitization of the corticosterone response to the EPM when tested 10 days

after withdrawal. Importantly, this response was accompanied by an apparent loss of risk

assessment abilities.

Globally, data presented in this study showed that chronic cocaine exposure

throughout adolescence produced hormonal, neurochemical and behavioural short and

long-term alterations. The present study highlights important gender difference in the

behavioural, neurochemical and hormonal effects of chronic cocaine exposure throughout

adolescence. Simultaneously, it was shown that adolescent male rats exposed to cocaine,

fail to exhibit an adaptive behavioural response to an anxiogenic stimulus, which was

paralleled by alterations in physiologic parameters. Moreover, compared to published data

our results seem to indicate that adolescent rats respond to chronic cocaine treatment

differently than adult animals, and extensively contributes to the growing understanding of

the mechanisms involved in the increased susceptibility to drug abuse during

adolescence.

XIX

Résumé

Pendant l'adolescence, les systèmes neurobiologiques continuent à présenter des

importants réarrangements, associés à des événements plastiques résultants d'un

processus intégré de surproduction et l'élimination des synapses, évolution des systèmes

de neurotransmetteurs et progressive myélinisation. Les niveaux hormonaux change

également au cours de cette période. Concomitante de ces changements dans le cerveau

et le statut hormonal, d'importantes transitions arrivent dans les compétences cognitives,

psychologiques et sociales. À ce moment, les facteurs environnementaux peuvent avoir

un impact fort sur le profil neurobiologique et psycho-physiologique unique des

adolescents. Les données épidémiologiques indiquent que les expériences avec les

drogues et l'apparition des troubles de dépendance sont essentiellement concentrées

dans l'adolescence et au début de l'âge adulte. Toutefois, des études sur les effets de

l'abus des drogues, ont, principalement, été effectuées parmi des modèles animaux en

adultes. L'objectif de cette étude était de contribuer à caractériser les effets de l'exposition

aux drogues à ce moment du développement, en utilisant un modèle animal de

l'exposition à la cocaïne. Des protocoles expérimentaux ont été développés afin d'évaluer

à court et à long terme les effets neurochimiques, hormonaux et comportamentaux de

l'exposition chronique à la cocaïne, au cours de l'adolescence dans un modèle animal un

rat.

Des mâles et des femelles des rats Wistar ont été administrées, par jour et par

voie intrapéritonéale, 3 doses de 15 mg/kg de poids corporel de chlorhydrate de cocaïne,

dans une solution 0,9% NaCl, du jour postnatal (PND) 35 au PND 50. Les contrôles ont

reçu une solution isovolumetric NaCl 0,9% suivant le même protocole expérimental. Le

comportement d’anxiété a été évalué en utilisant le elevated plus maze test (EPM) chez

les rats mâles, 2, 5 et 10 jours après le dernier apporte de cocaïne, et chez les rats

femelles, 2 jours après. Le comportement dépressif a été évalué à l'aide du forced swim

test (FST) dans les rats mâles et femelles, 3 jours après le retrait de cocaïne. Le

comportement d’agression a été évalué chez les rats mâles, 10 jours après le retrait, en

utilisant le resident-intruder paradigm. Les niveaux de neurotransmetteurs ont été

analysés après chaque test de comportement et à 30 minutes, 1, 5 et 10 jours après la

dernière administration de cocaïne, par la mesure des niveaux de dopamine (DA),

sérotonine (5-HT) et de ses métabolites (acide 3,4-dihydroxyphenylacétique, DOPAC;

acide homovanilique, HVA; acide 5-hydroxyindole acétique, 5-HIAA), par

chromatographie liquide d’haute performance avec détection électrochimique (HPLC-EC),

dans le mesencephale ventral (VTA-SN), le noyau raphé dorsal, le nucleus accumbens

XXI

(Nac), le striatum dorsal, le cortex préfrontal, l'amygdale, l'hippocampe, l'hypothalamus et

le cervelet. Les niveaus plasmatiques de l'hormone adrénocorticotrope (ACTH), de la

corticostérone, de la testostérone et de la progestérone ont été déterminés dans les rats

mâles et femelles à 30 minutes, 1, 5 et 10 jours après la dernière administration de

cocaïne. Chez les femelles, les niveaux de l'estradiol ont également été déterminés. Les

niveaux de corticostérone ont été aussi évalués après chaque défi de comportement. En

outre, chez les rats mâles, les niveaux d'expression de l'mRNA des transcriptions pour l’

hormone de libération de la corticotrophine (CRH) et le récepteur des androgènes (AR)

ont été mesurés dans l'hypothalamus, alors que le niveau d'expression de l'mRNA des

transcriptions pour récepteur CRH-1, récepteur de la hormone de libération des

gonadotrophines, AR, hormone lutéinisante sous-unité β, ré cepteur des glucocorticoïdes

(GR) et proopiomélanocortine ont été mesurés dans l'hypophyse, 10 jours après le retrait

de cocaïne, par la réaction quantitative en chaîne de la polymérase en temps réel (qRT-

PCR). Les niveaux d'expression des transcriptions de l'mRNA, 10 jours après le retrait de

cocaïne, de la tyrosine hydroxylase (TH) ont été mesurés par qRT-PCR dans le SN-VTA,

Nac et striatum dorsal, tandis que les niveaux d'expression de l'mRNA transcrits pour la

monoamine-oxydase A, DA transporteur, DA récepteurs (D1, D2, D3) et des GR ont été

mesurés dans le SN-VTA, Nac, striatum dorsal, cortex préfrontal, l'amygdale,

l'hippocampe et l'hypothalamus.

Les résultats les plus pertinents obtenus indiquent que l'exposition chronique de

cocaïne pendant l'adolescence perturbe négativement les réponses adaptatives aux défis

environnementaux. L'analyse des données a montré facultés d'évaluation des risques lors

de l' apparat d'exploration de l’ EPM, 2 et 10 jours après le retrait, qui a été appuyée par

une altération de la neurotransmission dopaminergique et sérotoninergique dans le cortex

préfrontal et l'altération de la neurotransmission dopaminergique dans l'amygdale. Ces

modifications neurochimiques peuvent être impliqués dans la faillite des rats mâles en

afficher un comportement adaptatif au cours de l'EPM. Le traitement avec cocaïne n'a pas

eu d'effet significatif sur le comportement dépressif dans les rats mâles et femelles,

d’après évaluation 3 jours après le retrait. Toutefois, lorsqu'il est exposé à la situation de

stress par la FST, une altération de la réponse a été observée dans les données

neurochimiques. En outre, même si aucun effet à long terme de la cocaïne a été observé

dans le comportement agressif des rats mâles, d’après évaluation 10 jours après le retrait,

les mâles exposés à cocaïne ont intensifié le comportement de recherche sociale et

diminué défensive comportement au cours des conflits sociaux prévues par le resident-

intruder paradigm, ce qui indique que cocaïne a affecté la capacité des jeunes rats

adultes de bien interagir avec leurs congénères. Conformément, les activités

XXII

dopaminergiques et sérotoninergiques, en réponse à ce conflit social, ont été modifiées

dans animaux exposés à cocaïne.

Les données neurochimiques de cocaïne ont montré également que l'exposition à

la cocaïne pendant l'adolescence produit des changements durables aux systèmes

monoamine. Chez les rats mâles, ça s'est traduit par une augmentation de l'activité

dopaminergique dans la voie nigrostriatal, soutenue par l'augmentation des niveaux de

DOPAC au SN-VTA, l’augmentation des niveaux d'HVA dans le striatum dorsal et

l'augmentation des niveaux d'expression des mRNA de la TH dans le SN-VTA et niveaux

durables et augmentés de 5-HT dans le SN-VTA. Dans les femelles traitées à la cocaïne,

ces modifications ont été révélés grâce à une augmentation de l'activité dopaminergique

dans l'amygdale et l'hippocampe, et l'augmentation de l'activité sérotoninergique dans

l'hippocampe et Nac.

L'analyse des données hormonales indique une activation de l'axe de

l'hypothalamus-hypophyso-surrénalien (HPA) dans les rats mâles, 30 minutes après la

dernière administration de la cocaïne, comme l'a montré l'augmentation des niveaux

d'ACTH. Malgré ce grave effet chez les rats mâles, l'activité basale de l'axe HPA ne

semble pas avoir été touchés par le traitement avec de la cocaïne, comme aucun

changement n'a été observé dans l'ACTH et dans les niveaux de corticostérone 1, 5 et 10

jours après le retrait, chez les rats mâles et femelles. Néanmoins, une rupture de la

réponse de l'axe HPA sous des conditions anxiogéniques a été révélé chez les jeunes-

adultes rats mâles, en faisant preuve d'une sensibilisation de la réponse à la

corticostérone EPM lors d'un essai 10 jours après le retrait. Surtout, cette réponse a été

accompagnée par une apparente perte de capacités d'évaluation des risques, qui est

renforcée par la baisse des niveaux de testostérone observée aussi bien à 30 minutes et

10 jours après la dernière administration de cocaïne.

Généralement, les données présentées dans cette étude ont montré que

l'exposition chronique à la cocaïne pendant l'adolescence produit des modifications

hormonales, neurochimiques et comportamentales à court et à long terme. La présente

étude met en évidence une importante différence entre les sexes en ce qui concerne les

effets comportamentaux, hormonaux et neurochimiques de l'exposition chronique à la

cocaïne, au cours de l'adolescence. Simultanément, il a été démontré que les rats mâles

adolescents exposés à la cocaïne, n’ont pas été en mesure de présenter une réponse

comportamentale adaptative à un stimulus anxiogène, qui se traduit par des modifications

de paramètres physiologiques. De plus, nos résultats par rapport aux données publiées,

semblent indiquer que les rats adolescents répondent différemment des animaux adultes

à un traitement chronique de cocaïne. Ce travail contribue largement à la compréhension

XXIII

des mécanismes impliqués dans la susceptibilité des adolescents à l’égard de

l'expérience de la drogue.

XXIV

Este trabalho foi co-financiado pela Fundação para a Ciência e Tecnologia através de

uma bolsa de doutoramento (SFRH/BD/17195/2004) e pelo Programa de Financiamento

Plurianual do IBMC.

This work was funded by Fundação para a Ciência e Tecnologia through a PhD fellowship

(SFRH/BD/17195/2004) and by the Programa de Financiamento Plurianual do IBMC.

XXV

Contents

Chapter 1- Introduction

1.1. General 3

1.2. Neuropharmacology of cocaine 4

1.3. Addiction: the transition to compulsive drug seeking 6

1.4. Cocaine and the Dopaminergic and Serotonergic Systems 8

1.4.1. Effects of cocaine upon the dopaminergic system 8

1.4.2. Effects of cocaine upon the serotonergic system 10

1.5. Cocaine and the Neuroendocrine System 12

1.5.1. The Hypothalamus-Pituitary-Adrenal axis 12

1.5.1.1. Effects of cocaine upon the HPA axis activity 14

1.5.1.2. The HPA axis and the behavioural effects of cocaine 17

1.5.1.2.1. Stress and increased vulnerability to cocaine 18

1.5.1.2.1.1. Interaction between stress, glucocorticoids and vulnerability to drugs 20

1.5.2. The Hypothalamus-Pituitary-Gonadal axis 21

1.5.2.1. The Hypothalamus-Pituitary-Testicular axis 21

1.5.2.2. The Hypothalamus-Pituitary-Ovaries axis 23

1.5.2.2.1. Estrous cycle 25

1.5.2.3 Effects of cocaine on the HPG axis 26

1.5.2.4. Effects of sex and sex hormones in the response to cocaine 28

1.5.3. Interactions HPG/ HPA axes and the response to cocaine 31

1.6. The adolescence 34

1.6.1. Adolescence: A critical period of addiction vulnerability 37

1.7. Objectives 43

1.8. References

45

Chapter 2- Methods

2.1. Animal model 69

2.1.1. Introduction 69

XXVII

2.1.2. Animals 71

2.1.3. Chronic administration and assessment time points 72

2.2. Blood and tissue collection 73

2.3. Hormone plasma levels measures 73

2.3.1. Steroid plasma levels 73

2.3.2. ACTH plasma levels 74

2.4. Neurotransmitters determinations 75

2.5. mRNA expression levels determination 76

2.6. Behavioural tests 77

2.6.1. Elevated plus maze test 77

2.6.2. Forced swim test 77

2.6.3. Aggressiveness test 78

2.7. Statistical analysis 79

2.8. References

80

Chapter 3- Short-term effects of cocaine exposure throughout adolescence in rat

3.1. Introduction 85

3.2. Material and Methods 87

3.2.1. Animals 87

3.2.2. Behavioural tests 87

3.2.2.1. Elevated plus maze test 87

3.2.2.2. Forced swim test 87

3.2.3. Blood and tissue collection 88

3.2.4. Hormone plasma levels determinations 88

3.2.5. Neurochemical determinations 89

3.2.6. Statistical analysis 89

3.3. Results 90

3.3.1. Analyses performed 30 minutes and 1 day after the last administration of cocaine 90

XXVIII

3.3.1.1. Biometric measures 90

- Evolution of body weight 90

- Adrenal weight 90

- Gonadal weight 91

3.3.1.2. Hormone plasma levels 92

- ACTH 92

- Corticosterone 92

- Testosterone 92

- Progesterone 92

- Estradiol 92

3.3.1.3. Neurochemical determinations 95

3.3.2. Behavioural studies and associated measures 111

3.3.2.1. Anxiety-like behaviour 111

- Elevated plus maze test 111

- Corticosterone plasma levels 114

- Neurochemical determinations 115

3.3.2.2. Depressive-like behaviour 125

- Forced swim test 125



3.4. Discussion 137

3.4.1. Effects of chronic cocaine treatment during adolescence on the body weight evolution 137

3.4.2. Effects of chronic cocaine treatment during adolescence on the neuroendocrine function 137

3.4.3. Effects of chronic cocaine treatment during adolescence on the dopaminergic and serotonergic systems function 144

3.4.4. Effects of chronic cocaine treatment during adolescence on the anxiogenic-like behaviour 159

3.4.5. Effects of chronic cocaine treatment during adolescence on the depressive-like behaviour 164

3.5. Conclusion 166

XXIX

3.6. References

168

Chapter 4- Long-term effects of cocaine throughout adolescence in rat

4.1. Introduction 187

4.2. Material and Methods 189

4.2.1. Animals 189

4.2.2. Behavioural tests 189

4.2.2.1. Elevated plus maze test 189

4.2.2.2. Resident-intruder paradigm 189

4.2.3. Blood and tissue collection 190

4.2.4. Hormone plasma levels determinations 190

4.2.5. Neurochemical determinations 191

4.2.6. mRNA expression levels determination 191

4.2.7. Statistical analysis 193

4.3. Results 193

4.3.1. Basal analyses 195

- Evolution of body weight 195

- Adrenal weight 195

- Gonadal weight 196

4.3.1.2. Hormone plasma levels 197

- ACTH 197

- Corticosterone 197

- Testosterone 197

- Progesterone 197

- Estradiol 198

4.3.1.3. Neurochemical determinations 199

4.3.1.4. mRNA expression levels determination 215

XXX

4.3.1.4.1. Dopaminergic mRNA-related expression levels 215

4.3.1.4.2. HPA and HPG axes mRNA-related expression levels 222

4.3.2. Behavioural studies and associated measures 225

4.3.2.1. Anxiety-like behaviour 225

- Elevated plus maze test 225



4.3.2.2. Aggressive behaviour 238

- Resident-intruder paradigm 238



4.4. Discussion 253

4.4.1. Effects of chronic cocaine treatment during adolescence and withdrawal on the body weight evolution 253

4.4.2. Effects of chronic cocaine treatment during adolescence and withdrawal on the neuroendocrine function 253

4.4.3. Effects of chronic cocaine treatment during adolescence on the dopaminergic and serotonergic systems function 258

4.4.4. Effects of chronic cocaine treatment during adolescence on the anxiogenic-like behaviour 271

4.4.5. Effects of chronic cocaine treatment during adolescence on the aggressive behaviour 276

4.5. Conclusion 282

4.6. References

285

Chapter 5- General conclusion

5. General conclusion

303

Appendix I- Published articles 307

Article- Alves CJ

309

, Magalhães A, Summavielle T, Melo P, de Sousa L, Tavares MA, Monteiro PRR. 2008. Hormonal, neurochemical, and behavioral response to a Forced Swim Test in adolescent rats throughout cocaine withdrawal. Ann.N.Y.Acad.Sci 1139:366-373.

XXXI

Chapter 1

Introduction

1.1. General

Cocaine is a psychostimulant drug, obtained from the leaves of the plant

Erythroxylum coca. For thousand years the natives of South America have chewed the

coca leaf to increase strength and energy, but the europeans did not hear about this plant

until the Spanish colonized South America in the XVI century (Karch, 1996). In the early

1560s, Nicholas B. Monardes published a book describing the practice of the natives of

chewing coca leaves to induce “great contentment”, at the time he was already aware that

coca had also undesirable side effects (Karch, 1996).

Isolation of cocaine, the coca’s active principle, was first described by Albert

Niemann in his Ph.D. dissertation titled “On a New Organic Base in the Coca Leaves”,

which was published in 1860 (Karch, 1996). For a long time the medical community

remained unimpressed with coca and even after the purification of cocaine, interest in its

therapeutic applications remained slight. However, in 1884, with the publication of

Freud’s paper “Über Coca” and the discovery of cocaine’s local anesthetic properties by

Köller, the interest of the medical community in this alkaloid increased (Karch, 1996).

Cocaine was propelled into the limelight and physicians around the world were soon

experimenting cocaine in a wide range of conditions (Karch, 1996).

The first reports of cocaine toxicity appeared less than one year after Köller’s and

Freud’s publications, and during the 1880´s several reports were published describing

toxic reactions associated with cocaine and cocaine-related deaths (Karch, 1996).

However, none of these negative reports appeared to have much impact. The popularity

of coca was not affected and thousands of cocaine-containing patented medicines flooded

the market, some with truly enormous amounts of cocaine (Karch, 1996).

During the early years of the last century it became evident that cocaine was

addicting and could produce serious medical complications, especially with the availability

of cocaine powder for intranasal or intravenous use. In 1906 cocaine became illegal in the

United States and its use waned (Karch, 1996). Between 1924 and 1973, there was only

one reported fatality and it involved a surgical misadventure, this absence of case reports

reflected a decline in its use, probably related with the outlawing of cocaine and with the

appearance of amphetamines (Dackis and O'Brien, 2001).

In the 1960s cocaine resurfaced with a mythology of safety and glamour, and this

perception encouraged millions of first-time users, forming the pool from which cocaine-

addicted individuals emerged (Dackis and O'Brien, 2001). With the appearance of “crack”

cocaine in 1986, another order of magnitude increase in dosage occurred and cocaine-

3

related illness became a significant cause of morbidity and mortality (Dackis and O'Brien,

2001).

Presently, according to the 2008 World Drug Report of the Office of Drug and

Crime of the United Nations, about 5% of the population between the ages of 15 and 64

use illicit drugs, which represents about 200 million people (UNODC, 2008). Cocaine is

between the most used drugs, with 16 million of users (UNODC, 2008).

The 2008 World Drug Report of the Office of Drug and Crime of the United Nations

reported that the cocaine consumer market in North America contracted, however the

consumption increased in South America, Western Europe and West and Southern Africa

(UNODC, 2008). The largest numbers of cocaine users are found in North America (7.1

million people), followed by West and Central Europe (3.9 million), and South America

(including Central America and the Caribbean: 3.1 million) (UNODC, 2008).

Most of the global increase of cocaine use over the last decade can be attributed

to rapidly rising cocaine consumption in Europe (UNODC, 2008). Increases in cocaine use

in 2006 were reported by a number of South-European countries, notably Spain, Portugal,

Italy and some countries of the western Balkan, as well as France, the United Kingdom,

Ireland and several Nordic countries (UNODC, 2008). The highest prevalence rates for

cocaine use in Europe are found in Spain, where cocaine use doubled among the general

population (age 15-64), from 1.6% in 1999 to 3.0% in 2005. In fact cocaine use levels in

Spain are similar to those reported from the USA (UNODC, 2008).

A major problem in what concerns to cocaine use is its consumption by young

people. In Europe, population surveys carried out in a number of countries have recorded

a marked increase in use among young people since the mid-1990s (EMCDDA, 2008).

1.2. Neuropharmacology of cocaine

Cocaine possesses potent psychostimulant properties as measured by antifatigue

and stimulant actions (Fischman et al., 1983; Romach et al., 1999). In rodents, cocaine

increases locomotor activity (Bhattacharyya and Pradhan, 1979; Abel et al., 1989) and

decreases food intake (Balopole et al., 1979). Cocaine has also a high abuse potential,

and experimental studies have shown that cocaine induces place preference conditioning

(Spyraki et al., 1982) and readily acts as reinforcer for drug self-administration (Roberts

and Koob, 1982; Goeders et al., 1993).

4

Psychostimulants of high abuse potential, such as cocaine and amphetamine,

interact initially to block monoamine transporter proteins which are located on

monoamonergic nerve terminals (Glowinski and Axelrod, 1965; Ferris et al., 1972;

Iversen, 1973; Ritz et al., 1987). Cocaine inhibits, with about equal potency, the uptake of

the three major monoamines neurotransmitters, dopamine (DA), serotonin (5-HT) and

norepinephrine (NE) (Rothman et al., 2001), thereby, potentiating monoaminergic

transmission (Pettit and Justice, 1989; Broderick et al., 2003).

However, the primary neuropharmacological action responsible for its psychomotor

stimulant and reinforcing effects appears to be on the dopaminergic systems in the central

nervous system (CNS) (Koob, 1992a). Antidepressant drugs that block NE and/or 5-HT,

but not DA reuptake, produce neither significant reinforcement in animal models nor

euphoria in humans (Hyman, 1996). Adrenoceptor antagonists, such as

phenoxybenzamine and phentolamine also have no effects on cocaine reinforcement,

while on the other hand, DA receptor antagonists block cocaine reinforcement (De Wit

and Wise, 1977). In addiction, lesion of DA terminals within the nucleus accumbens (Nac)

or full pharmacological blockade of DA receptors inhibits cocaine self-administration

(Roberts et al., 1980).

Brain dopaminergic neurons are organized into two major pathways that originate

in the midbrain and project to numerous forebrain and cortical regions. The

mesocorticolimbic dopaminergic system, projects from the ventral tegmental area (VTA) to

the ventral forebrain, including the Nac, olfactory tubercle, frontal cortex, amygdala and

septal area. The nigrostriatal dopaminergic system arises primarily from the substantia

nigra (SN) and projects to the corpus striatum. This last dopaminergic pathway is

implicated in the focused repetitive behaviour associated with psychostimulants abuse,

called stereotyped behaviour (Creese and Iversen, 1974; Robbins and Everitt, 1996). The

mesocorticolimbic dopaminergic system has been primarily implicated in cocaine-induced

locomotion and in the reinforcing effects of cocaine (Everitt and Wolf, 2002).

The mesolimbic dopaminergic system and its forebrain targets are very old from an

evolutionary point of view and are part of the motivational system that regulates

responses to natural reinforcers such as food, drink, sex and social interaction (for review

see Nestler, 2001). Drugs of abuse affect the brain reward system with a strength and

persistence not seen in natural reinforcers (Nestler, 2001; Robinson and Berridge, 2003).

In particular, the mesoaccumbens dopaminergic pathway, extending from the VTA

to the Nac, a brain region thought to be involved in converting emotion into motivated

action and movement (for review see Mogenson et al., 1980), seems to be where cocaine

5

act to produce its acute reinforcing actions (Hyman, 1996). Blockage of cocaine induced

locomotor activation has been observed following 6-hydroxydopamine lesions of the

region of the Nac (Le Moal and Simon, 1991; Sellings et al., 2006). Lesions of the Nac

also produce disruption of cocaine self-administration (Roberts et al., 1980; Pettit et al.,

1984; Zito et al., 1985). However, 6-hydroxydopamine lesions of the frontal cortex and

caudate nucleus fail to significantly alter established cocaine self-administration (Martin-

Iverson et al., 1986). Neurochemical studies using in vivo studies demonstrate that

cocaine increases extracellular DA to a greater extent in the Nac (Carboni et al., 1989;

Pettit and Justice, 1989; Cass et al., 1992).

Figure 1.2.1- Sagittal section through a representative rodent brain illustrating the dopaminergic pathways

implicated in psychomotor stimulant and reinforcing actions of cocaine. Nac- nucleus accumbens; SN-

substantia migra; VTA- ventral tegmental area.

1.3. Addiction: the transition to compulsive drug seeking

The drug addiction process is normally accepted to be composed of four major

phases: initiation, during which drug consumption is relatively low and irregular;

maintenance, during which frequent and compulsive intake of large amounts of drugs is

observed; withdrawal, during which the individual attempts to quit drug self-administration;

and relapse, during which the individual resumes compulsive drug use (Sarnyai et al.,

2001).

The acute reinforcing effects of cocaine lead to patterns of drug use that, in

epigenetically vulnerable individuals, eventually result in addiction (Hyman, 1996). Drug

addiction is a chronically relapsing disorder characterized by the compulsion to seek and

take a drug, with loss of control in limiting intake (Nestler, 2001). Addiction is also

6

characterized by the emergence of a negative emotional state (e.g., dysphoria, anxiety,

irritability) when access to the drug is prevented (Koob and Moal, 2006).

Drug-induced neuroadaptations are thought to be critical in the transition to

addiction. Extensive studies both in animal models of a variety of species, as well as basic

clinical research in humans, using neurochemical, molecular and related behavioural

technologies, as well as a variety of imaging techniques, have documented that indeed

chronic exposure to drugs does cause alterations in specific aspects of brain function

which are persistent over varying periods of time or, in some cases, may even be

permanent (Kreek and Koob, 1998; Nestler, 2001).

As cocaine use and duration increases, the positive reinforcing effects are

diminished while the dysphoria (including agitation, anxiety and even panic attacks)

increase, suggesting acute tolerance to the arousing and positive mood effects of cocaine

(Koob and Moal, 2006). Tolerance can be defined as a given drug producing a decreasing

effect with repeated dosing or when larger doses must be administered to produce the

same effect (Koob and Moal, 2006). Tolerance to the reinforcing effects of cocaine may

be marked, leading to administration of very high drug doses (Hyman, 1996). However,

tolerance does not develop to the stereotyped behaviour and psychosis induced by

stimulants and, in fact, these behavioural effects appear to show a sensitization (Post et

al., 1992; Hyman, 1996). Sensitization being defined as the long-lasting increment in

response occurring upon repeated presentation of a stimulus (Nestler, 2001).

The neuroadaptative processes of tolerance and sensitization together with

withdrawal symptoms have been proposed as key elements in the development of

addiction (Hyman and Malenka, 2001). Withdrawal signs associated with cessation of

chronic drug administration usually are characterized by responses that are opposite to

the initial effects of the drugs. Withdrawal from chronic or high dose cocaine use in

humans is associated with relatively few overt physical signs but a number of

motivationally relevant symptoms such as dysphoria and depression, anxiety, anergia,

insomnia and craving for the drug (a compelling desire to re-experience the cocaine

experience (Koob and Caine, 1999)) (Gawin and Kleber, 1986; Weddington et al., 1990;

Satel et al., 1991; Miller et al., 1993).

There are several theoretical explanations of how drug-induced alterations in

psychological function might cause a transition to addiction. The most traditional

theoretical explanation of how drug–induced alterations might cause transition to addiction

is the hedonic view that drug pleasure and subsequent unpleasant withdrawal symptoms

are the chief causes of addiction (Robinson and Berridge, 2003). It defends that drugs are

7

taken first because they are pleasant, but with repeated drug use homeostatic

neuroadaptations lead to tolerance and addiction, such that unpleasant withdrawal

symptoms ensue upon the cessation of use, thus, compulsive drug taking is maintained,

to avoid unpleasant withdrawal symptoms (Koob and Moal, 1997). It is suggest that

repeated drug use induces tolerance or downregulation in the mesolimbic dopaminergic

system, decreasing the pleasant drug “highs”, sudden cessation of drug use causes

dopaminergic (and serotonergic) neurotransmission to further drop below normal levels, at

least for several days, resulting in the dysphoric state of withdrawal (Koob and Moal,

1997). This theoretical explanation also suggest that repeated drug use can activate brain

and hormonal stress responses (Koob and Moal, 2006). As a result, addicts who originally

take drugs to gain a positive hedonic state are spiraled into a predominantly negative

hedonic state, which causes the transition to addiction (Koob and Moal, 1997, 2006).

1.4. Cocaine and the Dopaminergic and Serotonergic Systems

1.4.1 Effects of cocaine upon dopaminergic system

As mentioned above, the increase in extracellular DA following cocaine

administration plays a major role in cocaine reinforcement. Studies using microdialysis

have shown that acute cocaine administration leads to an immediate and dose-dependent

increase in extracellular DA levels (Church et al., 1987; Carboni et al., 1989; Maisonneuve

and Kreek, 1994). Increased synaptic levels of DA will stimulate DA receptors and activate

several signal transduction pathways. Pharmacological studies with selective D1, D2 and

D3 DA receptors antagonists and knockout studies have shown that all three receptors

subtypes appear to mediate the reinforcing effects of cocaine, albeit possibly different

components of the response. Low doses of D1 DA receptor antagonist block the

reinforcing effects of intravenous cocaine self-administration (Maldonado et al., 1993;

Caine and Koob, 1994). D2 DA receptor antagonists block responding for cocaine but also

have pronounced motor response inhibitory actions (Koob and Moal, 2006). D3 DA

receptor antagonists block drug-seeking behaviour associated with cocaine in second-

order and progressive-ratio schedules (Koob and Moal, 2006).

Many studies have shown that chronic administration of psychostimulant drugs

leads to a significant changes in the dopaminergic system.

In humans, imaging studies found reduced dopaminergic function in cocaine-

addicted (Wu et al., 1997; Volkow et al., 1999). In animals studies, data from intense

continuous self-administration or repeated binge experimenter administered cocaine also

show significantly decreased extracellular basal DA (Weiss et al., 1992; Kreek and Koob,

8

1998). Although, as observed during an acute binge pattern of cocaine administration, the

extracellular DA levels rise following each dose of cocaine with much greater intensity in

the Nac than in the dorsal striatum, after chronic binge pattern cocaine, there are a

significant decrease in the extracellular DA levels at the basal time points, and a lower

elevation following each cocaine administration, both in the dorsal striatum and in Nac

(Maisonneuve et al., 1995). However, chronic administration of cocaine under intermittent

schedules rather than a binge-like or continuous pattern results in a rise in the

extracellular DA increase with repeated administration (Kalivas and Duffy, 1993; Pierce

and Kalivas, 1997a). The data described above show that the response of dopaminergic

neurons depended on the doses and pattern of exposure (Koob and Moal, 2006).

Continuous access seems to be more likely to produce decreases in firing and

transmission, and limited or intermittent access are more likely to produce later increases

in firing and DA neurotransmission (Koob and Moal, 2006).

Decreases in both D1 and D2 receptors have been observed after long-term,

heavy exposure to passive administration or self-administration of cocaine in rats

(Tsukada et al., 1996; Maggos et al., 1998), nonhuman primates (Moore et al., 1998a, b)

and humans (Volkow et al., 1993).

In in vitro studies, cocaine administration did not alter quantitatively measured DA

transporter (DAT) mRNA levels in the SN or the VTA following subacute (3 days) binge,

chronic (14 days) binge or 10 days withdrawal from a chronic binge administration pattern

(Maggos et al., 1997). However, decreases in DAT function including both binding and

mRNA levels have been observed in selective brain regions, following 10 days of

withdrawal from chronic cocaine administration (Kuhar and Pilotte, 1996).

The changes in brain dopaminergic function are likely to result in decreased

sensitivity to natural reinforcers since DA also mediates the reinforcing effects of natural

reinforcers, and on disruption of frontal cortical functions, such as the inhibitory control

and salience attribution (Volkow et al., 2003). These alterations could contribute to the

loss of control and compulsive drug intake that characterize addiction (Volkow et al.,

2003). Moreover, reduced DA-mediated reward could explain the high rates of

depression, irritability, anxiety, and suicide that have been reported in cocaine-addicted

individuals (Dackis and O'Brien, 2001).

9

1.4.2. Effects of cocaine upon the serotonergic system

Although the dopaminergic system still plays an important role in the investigation

of the behavioural effects of cocaine, it has became evident that models that involve only

DA do not fully explain the complex effects of cocaine on behaviour. The essential role of

the DAT in cocaine reinforcement has been challenged, namely with studies using

knockout mice, to show that mice lacking DAT still self-administer cocaine (Rocha et al.,

1998) and exhibit cocaine-conditioned place preference (Sora et al., 2001). However,

DAT/ Serotonin transporter (SERT) double knockout mice exhibit a lack of cocaine

reinforcement (Sora et al., 2001). Moreover, pretreatment with the 5-HT reuptake inhibitor

fluoxetine decreases intravenous cocaine self-administration (Carroll et al., 1990; Peltier

and Schenk, 1993). Thus, the focus of cocaine research expanded to include the

serotonergic system.

As already mentioned, serotonergic system is one of the main targets of cocaine in

brain. By binding to SERT, cocaine blocks 5-HT reuptake and, thus, causes an increase in

extracellular 5-HT levels (Ritz et al., 1990; Muller et al., 2003).

The increased concentration of 5-HT in the synaptic cleft causes profound

activation of 5-HT receptors. Currently, seven families of 5-HT receptors (5-HT1- 5-HT7)

with at least 14 subtypes are known to mediate the effects of 5-HT (Barnes and Sharp,

1999; Hoyer et al., 2002). In general, the serotonergic system in the brain regulates much

different behaviours, including locomotor activity, eating, drinking, and is deeply involved

in emotions and mood. Furthermore, 5-HT is an important factor in maintaining plasticity

at the cellular level, which is the basis of behavioural adaptation (Muller and Huston,

2006).

The contribution of the serotonergic system to the psychostimulant effects of

cocaine is demonstrated clearly by lesion studies and pharmacological manipulation of the

global activity of the serotonergic system in rodents and monkeys, and by transgenic

approaches in mice (Porrino et al., 1989; Loh and Roberts, 1990; Glatz et al., 2002; Muller

et al., 2003; Rothman et al., 2005; Wee et al., 2005). Several studies with selective 5-HT

receptor-ligands suggest the contribution of a large number of different 5-HT receptor

subtypes to the behavioural effects of cocaine (Muller and Huston, 2006). Stimulation of

serotonin-1A (5-HT1A) receptors facilitates the establishment of locomotor sensitization

(De La Garza and Cunningham, 2000; Carey et al., 2001). 5-HT1B receptor stimulation

appears to play a role in facilitating the reinforcing (Parsons et al., 1996b; Neumaier et al.,

2002) and discriminative properties of cocaine (Callahan and Cunningham, 1995). 5-HT2

receptor blockade attenuates cocaine-induced hyperactivity in mice (O'Neill et al., 1999;

10

Muller and Huston, 2006). While, 5-HT3 receptor antagonism have been shown to block

the behavioural expression of cocaine sensitization in rats and mice (Reith, 1990;

Kankaanpaa et al., 1996; King et al., 2000; Kankaanpaa et al., 2002).

Most of the 5-HT receptor subtypes that facilitate cocaine-induced behaviour are in

the VTA and the Nac, the core areas of the mesolimbic DA releasing system. In both

areas cocaine causes a profound increase in the extracellular concentration of the 5-HT

(Muller et al., 2003), which activates the local populations of 5-HT receptors. In fact,

several studies have shown that 5-HT receptors in these areas regulate the behavioural

effects of cocaine by modulating the dopaminergic response in the Nac (Kankaanpaa et

al., 2002; Szumlinski et al., 2004). For instance, microinfusion of 5-HT into the VTA

enhanced the release of DA in the Nac, with the same effect being observed with 5-HT3

receptor activation (Jiang et al., 1990; Chen et al., 1991; Van Bockstaele et al., 1994;

Matell and King, 1997).

Nevertheless, modulating only the serotonergic response also modulates the

behavioural effects of cocaine (Muller et al., 2002a) without influencing cocaine-induced

DA increase in the Nac (Muller et al., 2002b). Thus, there is evidence to support at least

two mechanisms by which the cocaine-induced increase in the extracellular 5-HT level

translates into behavioural changes: by direct stimulation of 5-HT receptors and by

indirect effects of 5-HT receptor stimulation that regulates the activity of other transmitter

systems (e.g. the dopaminergic system) (Muller and Huston, 2006).

Beside the role of the serotonergic system on psychostimulant action of cocaine,

long-term cocaine use induces adaptive processes in serotonergic neurons that contribute

to the emergence and persistence of a cocaine-dependent state. Withdrawal from

continuous access to cocaine self-administration was reported to decrease the

extracellular 5-HT in the Nac (Parsons et al., 1995, 1996a). This effect is consistent with

changes in 5-HT receptor function and transport which may contribute to a post-cocaine

deficiency in extracellular 5-HT (Koob and Moal, 1997; Kreek and Koob, 1998). Increased

5-HT1B mRNA in Nac shell and dorsal striatum (Hoplight et al., 2007) and increased

densities of 5-HT reuptake sites have been demonstrated in rats following repeated

cocaine exposure (Cunningham et al., 1992).

The serotonergic system has been implicated in the chronic effects of cocaine

(Pierce and Kalivas, 1997b) and alterations in 5-HT dynamics may be important in the

transition to addiction (Mash et al., 2000). In fact, many studies suggest that alterations in

serotonergic system contribute to the craving and depressed mood associated with

cocaine withdrawal (Pollack and Rosenbaum, 1991; Buydens-Branchey et al., 1997;

11

Buydens-Branchey et al., 1998; Harris et al., 2001). Moreover, these studies are

supported by the knowledge that 5-HT plays an important role in mood and impulse

control (Young et al., 1996; Lucki, 1998; Merens et al., 2007) and dysfunction in the

serotonergic neurotransmission has been linked to the etiology of a variety of clinical

disorders such as depression, anxiety and obsessive-compulsion disorder (Reilly et al.,

1997; Lucki, 1998).

1.5. Cocaine and the Neuroendocrine System

Cocaine’s interactions with the HPA (Hypothalamus-Pituitary-Adrenal) and HPG

(Hypothalamus-Pituitary-Gonads) axes system are not yet full understood. However,

cocaine related changes in these hormones have broad implications for normal

reproductive function as well as immune function (Mello and Mendelson, 1997). In

addiction, there is evidence that cocaine’s perturbation of the HPA axis may be related to

its reinforcing properties (Mello and Mendelson, 1997). An improved understanding of the

interactions between the neuroendocrine system and cocaine use and abuse will be

helpful in clarifying some aspects of the neurobiology of drug abuse.

1.5.1. The Hypothalamus-Pituitary-Adrenal axis

The HPA axis consists of a complex, well-regulated interaction between the brain,

anterior pituitary and adrenal cortex, and is the hormonal system that activates the

integrative physiological response to stress, which helps the organism to adapt to

increased demands and maintain homeostasis after challenge (Mello and Mendelson,

1997). This endocrine system is also vital for supporting normal physiological functioning,

regulates various body processes including digestion, the immune system, mood and

sexuality, and energy usage.

The synthesis and secretion of glucocorticoids (the final product of the HPA axis),

in the adrenal cortex is primarily mediated by the action of adrenocorticortipic hormone

(ACTH) on melanocortin-2 receptors integral to cortical cell membranes (Mountjoy et al.,

1992), with the adrenal sensitivity changing as a circadian function (Jasper and Engeland,

1994). Usually ACTH concentrations in the circulation are in the low picomolar range

(Dallman et al., 1987) and at these concentrations has a specific action in the adrenals,

but few extra-adrenal effects.

On the other hand, ACTH secretion from the anterior pituitary is usually entirely

controlled by the secretory activity of corticotrophin-releasing hormone (CRH) and

12

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arginine vasopressin (AVP)- containing neurons in the medial parvocellular portion of the

paraventricular nuclei of the hypothalamus (PVN). Thus, circulating concentrations of

ACTH and glucocorticoids precisely reflect the prior secretion of CRF/AVP by the

hypothalamic median eminence (Alexander et al., 1996). Although CRH is required to

stimulate ACTH synthesis and secretion from the pituitary, AVP interacts with CRH to

potentiate the secretion of ACTH from the corticotrophs (Gillies et al., 1982; Antoni, 1993).

The CRH/AVP ratio in parvocellular neurons of the PVN changes under various conditions

and in this way can control the amount of ACTH released in response to stimulation of the

PVN (Antoni, 1993).

Figure 1.5.1.1- Schematic diagram of the hypothalamic-pituitary-adrenal axis. ACTH- adrenocorticotropic

hormone; Cort- corticosterone; CRH- corticotrophin-releasing hormone; (-), negative feed-back. Adapted from

The HPA axis function has a diurnal rhythm, for example, ACTH and glucocorticoid

levels are highest in the rat (a nocturnal species) in evening and lowest in the morning

(Dallman et al., 1987). These rhythms are generated and controlled by inputs from

suprachiasmatic nucleus to the CRH systems (Watts and Swanson, 1987, 1989) and

feedback actions of the circulating concentrations glucocorticoids on the HPA axis (Akana

et al., 1992a; Akana et al., 1992b). In fact, glucocorticoids are involved in the suppression

of their own release through negative feedback systems that inhibit the release of ACTH

(Keller-Wood and Dallman, 1984; Dallman et al., 1987). Feedback systems operate

primarily at the level of the parvocellular part of the PVN and anterior pituitary (Keller-

Wood and Dallman, 1984; Dallman et al., 1987; Cole et al., 2000), although other brain

13

sites such as the hippocampus and medial prefrontal cortex are also involved in the

regulation of HPA axis activity (Kovacs et al., 1986; Jacobson and Sapolsky, 1991; Diorio

et al., 1993).

In the CNS, corticosteroids exert their effects through two corticosteroids

receptors, a high-affinity low-capacity type I or mineralocorticoid receptors (MR) that are

saturated by low basal levels of the hormone, and a lower-affinity high-capacity type II or

glucocorticoid receptor (GR) that are activated by high glucocorticoid levels such as those

observed during the circadian peak or after a stressful event (Joels and de Kloet, 1994).

These cytosolic receptors regulate gene transcription when bound to DNA response

elements or by interactions with other transcription factors (Joels et al., 2004). The

distribution of MR is more discrete than that of GR. High expression of MR in

hippocampus has been shown in rats, humans and nonhuman primates (Krozowski and

Funder, 1983; Rupprecht et al., 1993; Sanchez et al., 2000). MR are also distributed in the

amygdala, prefrontal cortex, lateral septum, and in a variety of motor neurons in the brain

stem (Birmingham et al., 1984). In contrast to MR, GR are broadly distributed throughout

the brain and pituitary (Cintra et al., 1991). There is dense GR staining in hypothalamus,

limbic brain, cortex and brain stem. Of interest, most neurons that synthesize and secrete

monoamines contain GR (Fuxe et al., 1985; Harfstrand et al., 1986; Ceccatelli et al., 1989;

Cintra et al., 1991).

In the CNS, glucocorticoids influence protein synthesis, neuronal excitability and

neurotransmitter metabolism (McEwen, 1980; Keller-Wood and Dallman, 1984). In

addition, glucocorticoids are important regulators of brain development and neuronal

plasticity; they can influence virtually all aspects of neural development, including

neurogenesis, synaptogenesis, dendritic morphology and cell death (McEwen, 2000).

1.5.1.1. Effects of cocaine upon the HPA axis activity

There is increasing empirical support for the notion that dysregulation of the HPA

axis may increase vulnerability to depression, and a number of other psychiatric disorders,

as well as immune dysfunction and cardiovascular disease (Heinrichs et al., 1995;

Chrousos and Gold, 1998; Nemeroff, 1998). Cocaine, like stress, modulates HPA axis

activity and thereby contributes to the disruption of normal neuroendocrine function.

Acute cocaine produces a robust and dose-dependent activation of the HPA axis

in rats (Rivier and Vale, 1987; Saphier et al., 1993), rhesus monkeys (Sarnyai et al., 1996;

Broadbear et al., 1999b, a) and humans (Baumann et al., 1995b; Heesch et al., 1995).

Acute administration of cocaine stimulates ACTH and cortisol secretion in humans (Sholar

14

et al., 1998; Elman et al., 1999) and in rhesus monkeys (Sarnyai et al., 1996; Broadbear

et al., 1999a), and ACTH and corticosterone release in rats (Rivier and Vale, 1987;

Borowsky and Kuhn, 1991b; Levy et al., 1991).

There is considerable evidence that the stimulatory effects of cocaine on HPA axis

are mediated by CRH (Mello and Mendelson, 1997). Cocaine stimulates CRH release

from hypothalamic tissue in vitro (Calogero et al., 1989) and alters CRH levels in different

brain areas in vivo (Sarnyai et al., 1993). Moreover, cocaine-induced increases in ACTH

and corticosterone release are prevented by passive immunization against CRH (Rivier

and Vale, 1987; Sarnyai et al., 1992). These results show that the action of cocaine on the

HPA axis is mediated via CRH receptors and depends on the release of endogenous

hypothalamic CRH.

Although the exact mechanisms underlying the effects of cocaine on the HPA axis

remain to be clarified, it is increasingly apparent that cocaine-related stimulation of ACTH,

and by inference of CRH, is modulated by several interacting neurotransmitter systems

(Mello and Mendelson, 2002). Because CRH release is regulated, in part, by DA and 5-

HT, antagonists that are selective for DA or 5-HT receptors attenuate cocaine-induced

stimulation of ACTH (Borowsky and Kuhn, 1991b; Levy et al., 1991). Moreover, both DA

and 5-HT receptor agonists stimulate ACTH release in rats (Borowsky and Kuhn, 1991b;

Levy et al., 1994a; Baumann et al., 1995a)

In contrast to the acute stimulant effects of cocaine in the HPA axis, there are

conflicting reports in the literature as to whether chronic cocaine exhibits persistent

stimulatory effects on the HPA axis. Repeated cocaine exposure has been reported to

produce tolerance (Zhou et al., 1996), sensitization (Schmidt et al., 1995) or no changes

(Borowsky and Kuhn, 1991a; Levy et al., 1992) in the HPA axis response, depending on

the experimental design used (Mantsch et al., 2003). It is suggested that the frequency

and duration of cocaine administration, among other variables, may be of critical

importance in determining the effects of cocaine on HPA axis activity (Mantsch et al.,

2003).

Intermittently cocaine exposure usually does not alter basal levels of ACTH and

cortisol or corticosterone, or the hormonal response to acute stimulation with cocaine or

synthetic CRH factor (Borowsky and Kuhn, 1991a; Levy et al., 1992; Torres and Rivier,

1992a, b; Laviola et al., 1995; Broadbear et al., 1999b). Nevertheless, decreases in CRH

receptors have been reported after intermittently cocaine (Goeders et al., 1990; Mello and

Mendelson, 1997). Overall, knowledge obtained until nowadays seems to indicate that

15

intermittently cocaine exposure does not result in tolerance or sensitization of the HPA

axis to stimulation by cocaine or stress (Mello and Mendelson, 1997).

However, tolerance to the stimulatory effects of cocaine on HPA axis function has

been reported in rats following chronic binge pattern administration (Zhou et al., 1996).

This tolerance is observed as attenuated corticosterone and ACTH responses to cocaine

(Zhou et al., 1996; Zhou et al., 2003). In rats a significant reduction in plasma

corticosterone response to cocaine was observed on day 14 of binge administration as

compared with day 1 or 3 (Zhou et al., 1996). Moreover, the development of tolerance to

the effects of cocaine on the HPA hormone response is associated with a significant

reduction in the CRH mRNA levels in the hypothalamus (Zhou et al., 1996).

During early withdrawal from chronic binge cocaine administration, an activation of

the HPA axis of the rat has been observed, as indicated by the significant elevation of

plasma ACTH and corticosterone 1 and 4 days after withdrawal (Zhou et al., 2003),

however, 10 days after withdrawal, ACTH and corticosterone were at control levels (Zhou

et al., 2003). Increased basal levels of corticosterone 1 day after withdrawal from

intermittent cocaine administration has also been reported (Levy et al., 1994b). In

contrast, when rats are permitted to self-administer a very high dose of cocaine during

long-access sessions, plasma corticosterone is not elevated during acute withdrawal but

rather is decreased (Mantsch et al., 2000).

In humans, as already mentioned, cocaine also disrupts HPA axis function. Acute

cocaine administration increases plasma ACTH and cortisol levels in cocaine abusers

(Teoh et al., 1994; Baumann et al., 1995b; Elman et al., 1999). However, the effects of a

challenge dose of cocaine on ACTH secretion, are significantly lower in cocaine-

dependent men than in occasional cocaine users (Mendelson et al., 1998). Nevertheless,

one early study has shown higher basal plasma ACTH and cortisol levels in cocaine

addicts one day after the cessation of cocaine self-administration (Vescovi et al., 1992).

However, after a brief period of abstinence, basal and CRH stimulated ACTH and cortisol

levels in cocaine-dependent patients do not show differences from healthy subjects

(Vescovi et al., 1992; Baumann et al., 1995b; Mendelson et al., 1998; Jacobsen et al.,

2001a).

Behaviourally, cocaine use in humans has been reported to produce profound

subjective feeling of well being and a decrease in anxiety (Gawin and Ellinwood, 1989).

However, some of the major symptoms observed during withdrawal from chronic cocaine

intoxication can often included severe anxiety as well as restlessness, agitation and

depression (Gawin and Ellinwood, 1989). Interestingly, CRH has been reported to be

16

involved in a variety of neuropsychiatric disorders including depression and anxiety (Gold

et al., 1984; Musselman and Nemeroff, 1996), suggesting that the anxiety associated with

cocaine use and withdrawal may depend, in part, on the effects this psychostimulant on

the CRH release and subsequent activation of the HPA axis (Goeders, 1997). Anxiogenic-

like behaviour has also been observed in non-human animals using a variety of

behavioural paradigms, following acute, during chronic administration of cocaine and

during withdrawal (Costall et al., 1989; Fontana and Commissaris, 1989; Rogerio and

Takahashi, 1992; Sarnyai et al., 1995).

1.5.1.2. The HPA axis and the behavioural effects of cocaine

The HPA axis seems to have particularly importance in drug addiction (Piazza and

Le Moal, 1996; Goeders, 1997). The activity of the HPA axis prior to, at the time of, and

subsequent to cocaine availability appears to be an important determinant of whether or

not individuals will engage in cocaine-seeking behaviour (Goeders and Guerin, 1996b;

Mantsch et al., 1998; Mantsch and Goeders, 1999). The temporal concordance between

peak plasma cocaine levels, peak levels of ACTH, and reports of subjective high have

prompted speculation that perturbation of the HPA axis may contribute to cocaine’s

abuse-related effects (Goeders, 1997; Goeders, 2002b; Mendelson et al., 2002).

In fact, preclinical studies suggest that the reinforcing properties of cocaine may be

influenced by the HPA axis activity (Piazza and Le Moal, 1996; Goeders, 1997; Goeders,

2002a). Drug-naive rats that were surgically adrenalectomized did not learn to self-

administer cocaine (Goeders and Guerin, 1996b). Moreover, metyrapone, which blocks

corticosterone synthesis, significantly decreased cocaine-induced locomotion and relapse

of cocaine self-administration (Piazza et al., 1994; Goeders and Guerin, 1996a; Goeders

and Guerin, 1996b). Pretreatment with CRH receptor antagonist also produced dose-

related decreases in cocaine self-administration in rats (Goeders and Guerin, 2000). On

the other hand, elevated plasma corticosterone facilitates the initiation of cocaine self-

administration (Goeders and Guerin, 1996b; Mantsch et al., 1998). Dose-response studies

have shown that adrenalectomy induces a vertical downward shift to the dose-response

curve to cocaine during the maintenance phase (Deroche et al., 1997). These data

converge to suggest a role for the HPA axis in the modulation of cocaine reinforcement

(Goeders, 1997; Goeders, 2002a).

Although high levels of corticosterone are necessary to maintain self-

administration behaviour, drug-induced increases in hormone levels are not critical

(Marinelli and Piazza, 2002). Thus, self-administration of psychostimulants can

17

dramatically increase glucocorticoid secretion (Baumann et al., 1995b; Broadbear et al.,

1999b; Galici et al., 2000), but blocking this drug-induced increase in hormone levels does

not modify response to cocaine (Deroche et al., 1997). In addition, animals can

consistently self-administer cocaine at doses that do not increase hormone levels

(Broadbear et al., 1999a), further supporting the notion that drug-induced glucocorticoid

secretion is not essential to maintain self-administration behaviour (Marinelli and Piazza,

2002).

Although corticosterone plays a facilitatory role on many behavioural responses to

psychostimulants, its role in relapse is a little more controversial. Suppression of

glucocorticoids does not seem to have important effects on relapse induced by drug

priming. Thus, cocaine-induced reinstatement of cocaine self-administration is only

minimally decreased by adrenalectomy (Erb et al., 1998) and is not modified by

ketoconazole, which reduces circulating levels of corticosterone (Mantsch and Goeders,

1999). Instead, corticosterone may play a significant role in stress and cue-induced

reinstatement of drug seeking (Goeders, 2002a; Goeders and Clampitt, 2002).

1.5.1.2.1. Stress and increased vulnerability to cocaine

Scientists are ware of the existence of a complex relationship between stress, the

subsequent activation of the HPA axis and the neurobehavioural effects of cocaine for

many years now (Goeders, 1997; Piazza and Le Moal, 1998; Koob, 1999).

It has been hypothesized that exposure to physically or psychologically stressful

events may render an individual more sensitive to the reinforcing effects of drugs such as

cocaine (Piazza and Le Moal, 1996; Goeders, 1997; Piazza and Le Moal, 1998). This

hypothesis is supported by studies where prior exposure to stressful stimuli results in an

enhancement of behaviours pertaining to acquisition (Piazza et al., 1990; Haney et al.,

1995; Goeders and Guerin, 1996b), maintenance of cocaine self-administration (Miczek

and Mutschler, 1996), and reinstatement of cocaine-seeking in rats following extinction of

operant behaviour (Erb et al., 1998).

A faster acquisition of cocaine self-administration has been found in rats exposed

to social stress, such as exposure to a resident aggressive animal (Haney et al., 1995;

Tidey and Miczek, 1997). Physical stressors can also enhance the propensity to develop

cocaine self-administration. This has been shown for repeated tail pinch (Piazza et al.,

1990), food restriction (Papasava and Singer, 1985; Carr et al., 2000), and electric foot

shocks (Goeders and Guerin, 1996b; Xi et al., 2004). Very early life events, such as

prenatal stress, can also increase vulnerability to psychostimulants (Kippin et al., 2008).

18

The reinstatement of cocaine seeking behaviour can be elicited by exposure to

brief periods of intermittent electric footshock or food restriction stress in rats (Shaham et

al., 2000; Shalev et al., 2003). However, cocaine reinstatement was prevented in

adrenalectomized rats but not in adrenaloctomized animals with corticosterone

replacement. In addition, CRH receptor antagonists were also found to block (Erb et al.,

1998) or attenuate (Shaham et al., 1998) footshock-induced reinstatement in intact

animals and in adreneloctomized animals with corticosterone replacement (Erb et al.,

1998). This last example supports the view that CRH acting at extra-hypothalamic sites in

the brain also plays an important role in stress-induced reinstatement of cocaine seeking

(Shaham et al., 2000). In fact, acute cocaine has been reported to affect CRH-like

immunoreactivity in a number of brain regions that are not thought to be directly linked to

pituitary-adrenal activity, including basal forebrain and amygdala (Sarnyai et al., 1993;

Gardi et al., 1997). Extrahypothalamic CRH systems appear to be involved in certain

symptoms of acute drug withdrawal, such as anxiety, and in stress-induced relapse to

drug seeking (Sarnyai et al., 2001).

In contrast to the effects observed during acquisition or reinstatement, neither

exposure to footshock (Goeders and Guerin, 1996b) nor exogenous injections of

corticosterone affect ongoing cocaine self-administration. This inability to affect

maintenance of drug use is probably related to the fact that plasma corticosterone is

significantly elevated in a dose-related manner during cocaine self-administration, and

further increases in corticosterone are without effect, since a threshold critical for reward

has already been crossed (Goeders and Clampitt, 2002). However, low-dose cocaine self-

administration can be attenuated by drugs that inhibit the synthesis and/or release of

corticosterone, since in this case plasma concentrations of corticosterone is probably

reduce below the critical reward threshold (Goeders, 2002a).

In humans, there is also a growing literature that suggests a possible linkage

between stress and addiction. A number of studies have identified individuals with dual

diagnosis of post traumatic stress disorders (PTSD) and drug addiction (e.g. Zaslav,

1994; Donovan et al., 2001; reviewed by Jacobsen et al., 2001b). Although a causal

relationship between exposure to stress and drug addiction has not been clearly

established, prevalence estimates suggest that rates of drug abuse among individuals

with PTSD may be as high as 60-80%, suggesting relationship between stress and

increased drug addiction in some cases (Donovan et al., 2001).

Exposure to stress or simply the presentation of stress-related imagery have also

been identified as potent events for provoking relapse to drug seeking in humans (Lamon

and Alonzo, 1997; Sinha et al., 2000; Sinha, 2001). Clinical studies have demonstrated

19

that simple exposure to environmental stimuli or cues previously associated with drug

taking can produce intense drug craving (Robbins et al., 1999).

Preclinical investigations have also demonstrated cue-induced reinstatement (Meil

and See, 1996; See, 2002). Once again, several studies have suggested an important

role for the HPA axis in addiction, showing a link between HPA axis and the ability of

environmental cues to stimulate cocaine-seeking behaviour in rats. Pretreatment with, the

corticosterone synthesis inhibitor, ketoconazole, reversed the cue-induced reinstatement

of extinguished cocaine-seeking behaviour and also attenuated the conditioned increases

in plasma corticosterone observed during reinstatement (Goeders and Clampitt, 2002).

Pretreatment with a CRH receptor antagonist also resulted in a similar decrease in the

ability of environmental cues to stimulate cocaine-seeking behaviour (Goeders and

Clampitt, 2002).

It is not inherently intuitive how exposure to a stressor can increase vulnerability to

drug self-administration (Goeders and Clampitt, 2002). The preclinical literature suggests

that stress increases reinforcement associated with psychomotor stimulants, possibly

through a process similar to sensitization (Piazza and Le Moal, 1998; Goeders, 2002b),

whereby repeated intermittent injections of cocaine increase the behavioural and

neurochemical responses to subsequent exposure to the drug. Interestingly, exposure to

stressors or administration of corticosterone can also result in sensitization to the

behavioural and neurochemical (e.g., Nac DA) response to cocaine (Rouge-Pont et al.,

1995; Prasad et al., 1998). These effects are attenuated in adrenalectomized rats (Prasad

et al., 1998; Przegalinski et al., 2000) or when corticosterone synthesis is inhibited

(Rouge-Pont et al., 1995). The ability of stressors to facilitate the acquisition of drug self-

administration may, therefore, result from a similar sensitization phenomenon, perhaps

involving DA (Goeders, 1997; Piazza and Le Moal, 1998).

1.5.1.2.1.1. Interaction between stress, glucocorticoids and vulnerability to drugs

Stress, through activation of the HPA axis and the release of glucocorticoids,

influences various regions of the brain including dopaminergic neurons (Piazza and Le

Moal, 1996; Piazza and Le Moal, 1997; Piazza and Le Moal, 1998), that express

corticosteroid receptors (Harfstrand et al., 1986). In normal situations, glucocorticoids

state-dependently increase dopaminergic function, especially in mesocorticolimbic

regions, during various consummatory behaviours exhibited in rodent’s active period of

the light/dark cycle (Piazza and Le Moal, 1996). The interaction of glucocorticoids with

mesocorticolimbic dopaminergic system may have a significant impact on vulnerability to

20

self-administer psychostimulant drugs, since increased dopaminergic transmission in

these pathways is critical for the reinforcing properties of abusive drugs, rendering the

individuals more susceptible to the drug reinforcement (Roberts et al., 1980; Koob and

Moal, 1997).

GR have been identified in rodent brain on dopaminergic neurons in the VTA and

SN as well as in dopaminergic terminal regions including the Nac and prefrontal cortex

(Harfstrand et al., 1986; Diorio et al., 1993; Cintra et al., 1994). Stress-induced elevations

in corticosterone may play a critical role in activation of dopaminergic transmission, with

corticosterone treatment increasing, and adrenalectomy decreasing extracellular DA

levels in Nac and prefrontal cortex of rodents (Imperato et al., 1989; Piazza et al., 1996)

This suggest that stress-induced increases in glucocorticoids may interact with

mesocorticolimbic brain regions to facilitate drug taking behaviour .

1.5.2. The Hypothalamus-Pituitary-Gonadal axis

The HPG axis, also known as reproductive axis, is a critical part in the

development and regulation of the reproductive function.

1.5.2.1. The Hypothalamus-Pituitary-Testicular axis

Regulation of the hypothalamus-pituitary-testicular (HPT) axis begins with at the

level of the hypothalamus, where neurosecretory cells synthesize and release

gonadotropin-releasing hormone (GnRH) in a pulsatile fashion into the hypothalamic-

hypophysial-portal circulation. In response gonadotropes in the anterior pituitary

synthesize and release the gonadotropins follicle-stimulating hormone (FSH) and

luteinizing hormone (LH), both of which ultimately control gonadal function (Swerdloff et

al., 2002).

In males, LH is the primary stimulus for the secretion of testosterone and FSH

stimulates spermatogenesis (Swerdloff et al., 2002). Testosterone is secreted by the

testicular Leydig cells, but only when they are stimulated by LH from the anterior pituitary

gland (Swerdloff et al., 2002).

FSH seems to mediate its effects on the testes predominantly through its action on

the Sertoli cells. FSH is known to bind a specific receptor on these cells and stimulate the

production of a large number of proteins including the inhibin-related peptides, androgen-

binding protein, transferring, androgen receptor, and γ-glutamyl transpeptidase (Swerdloff

et al., 2002).

21

Figure1.5.2.1.1- Schematic diagram of the male hypothalamic-pituitary-testicular axis. E2- estradiol; FSH-

follicle-stimulating hormone; GnRH- gonadrotopin-releasing hormone; LH- luteinizing hormone; T-

testosterone; (-)- negative feed-back. Adapted from (Norman and Litwack, 1998).

Beside the stimulation by the GnRH, gonadal steroids and testicular peptide

substances, such as activin and inhibin action on the gonadotropes provides additional

modulatory effects (Swerdloff et al., 2002). The primary regulator of LH secretion is the

serum level of testosterone (Swerdloff et al., 2002). It appears that the inhibitory effects

are largely accomplished at both the hypothalamic and pituitary levels (Swerdloff et al.,

2002).

Several inhibitory peptides (e.g., activin and follistatin) have been postulated as

selecting FSH inhibitors, but evidence to relate changing serum levels of either peptide

with increased FSH have not been shown in men. Thus, the understanding of FSH

regulation is incomplete (Swerdloff et al., 2002).

The secretion of GnRH is regulated in a complex fashion by neuronal input from

higher cognitive and sensory centers and by the circulating levels of sex steroids and

peptide hormones such as prolactin, activin, inhibin and leptin (Swerdloff et al., 2002). The

local effectors of GnRH synthesis and release include a number of neuropeptides,

opioids, catecholamines, indolamines, nitric oxide and excitatory amino acids, γ-

aminobutyric acid (GABA), neuropeptide Y, vasoactive intestinal peptide, and CRH. In

general, the catecholamines, excitatory amino acids, and nitric oxide in physiologic

22

amounts are stimulatory, whereas opioid peptides and β-endorphin are inhibitory

(Swerdloff et al., 2002). Testosterone, either directly or through its metabolic products,

estradiol and dihydrotestosterone (DHT) has predominantly inhibitory effects on the

secretion and release of GnRH (Swerdloff et al., 2002).

1.5.2.2. The Hypothalamus-Pituitary-Ovaries axis

The female hormonal system, like that of the male, consists of three hierarchies of

hormones, as follows: A hypothalamic releasing hormone, the GnRH, that like in males

stimulates the anterior pituitary to release FSH and LH, that in turn will stimulate the

ovarian to produce estrogens and progestins (Guyton and Hall, 2006).

Figure 1.5.2.2.1- Schematic diagram of the female hypothalamic-pituitary-ovarian axis. Depending upon

hormone concentration, time course, and prior hormonal status, E2 can either inhibit or stimulate LH secretion

through effects at either the hypothalamus or pituitary. E2- estradiol; FSH- follicle-stimulating hormone; GnRH-

gonadrotopin-releasing hormone; LH- luteinizing hormone; P- progesterone; (-)- negative feed-back; (+),-

positive feedback. Adapted from (Norman and Litwack, 1998).

By far the most important of the estrogens is estradiol, and the most important

progestin is progesterone (Guyton and Hall, 2006). Estradiol is produced by the ovarian

follicles, and progesterone is secreted from the ovarian follicle and from corpus luteum

after ovulation. These hormones are not secreted in constant amounts throughout the

female monthly sexual cycle, but drastically differing rates during different parts of the

cycle (Guyton and Hall, 2006). In fact, this changes in levels of gonadotropins and

gonadal steroid hormones define the functional distinct phases of the monthly sexual

23

cycle: the follicular phase, the ovulatory phase, and the luteal phase (Guyton and Hall,

2006).

Figure 1.5.2.2.2- Schematic diagram of the pattern of changes in the gonadotropins (LH and FSH) and the

ovarian steroids hormones (estradiol and progesterone) across a typically menstrual cycle in human females.

Adapted from (Guyton and Hall, 2006).

During each month of the female sexual cycle, there is a cyclical increase and

decrease of both FSH and LH. During the first few days of each monthly female sexual

cycle, the concentrations of both FSH and LH secreted by the anterior pituitary gland

increase slightly to moderately, with a increase in FSH slightly greater than that of LH and

preceding it in a few days. Adequate FSH levels are necessary for normal development

and maturation of the ovarian follicles (Goodman and Hodgen, 1983) and the early growth

of the primary follicle is stimulated mainly by FSH alone, LH is necessary for final follicular

growth and ovulation (Guyton and Hall, 2006). About 2 days before ovulation (for reasons

that are not completely understood) the rate of secretion of LH by the anterior pituitary

gland increases markedly, FSH also increases at the same time, and the FSH and LH act

synergistically to cause rapid swelling of the follicle during the last few days before

ovulation, leading to decrease in the rate of secretion of estrogen and a increase in

amounts of secretion of progesterone (Guyton and Hall, 2006). During the periovulatory

phase, the preovulatory LH and FSH surge is stimulated by increases in estradiol levels,

and an abrupt increase in progesterone precedes the onset of the LH surge. LH levels

decrease after ovulation and the luteal phase of the menstrual cycle begins (Nippoldt et

al., 1989). Progesterone levels gradually increase and remain elevated until the

24

postovulatory corpus luteum regresses. Throughout the luteal phase the levels of FSH are

low.

Gonadotropin and ovarian steroid hormone levels are controlled by a complex and

changing pattern of reciprocal stimulation and inhibition across the monthly sexual cycle

(Nippoldt et al., 1989). Estrogen in small amounts has a strong effect to inhibit the

production of both LH and FSH. Also, when progesterone is available, the inhibitory effect

of estrogen is multiplied, even though progesterone by itself has little effect (Nippoldt et

al., 1989). In addition to the feedback effects of estrogen and progesterone, other

hormones seem to be involved, especially inhibin. This hormone inhibits the secretion of

FSH and, to a lesser extent, the secretion of LH by the anterior pituitary gland (Guyton

and Hall, 2006).

Multiple neuronal centers in the higher brain’s limbic system transmit signals into

the arcuate nuclei to modify both the intensity of GnRH release and the frequency of the

pulses (Guyton and Hall, 2006). It has been suggested that gonadal steroids as well as

opioid peptides are important in the inhibitory regulation of GnRH release (Yen et al.,

1985). In addiction, the system is modulated further in a complex fashion by the combined

action of inhibin, activin, and follistatin (Norman and Litwack, 1998).

1.5.2.2.1. Estrous cycle

Although the estrous cycle of the rat is shorter than the reproductive cycle of

humans (4 vs. 28-35 days), the endocrinological changes closely resemble those seen in

humans (Quinones-Jenab et al., 2001). The 4-5 day cycle is composed of proestrus,

estrus, metestrus and diestrus (Barbacka-Surowiak et al., 2003).

Ovarian follicles and oocytes grow, mature and differentiate to reach the ability to

ovulate during estrus, these processes are controlled first by FSH and then by LH

(Barbacka-Surowiak et al., 2003). Gradually increasing FSH level stimulates the

proliferation of follicular cells and synthesis of estradiol, the LH determines the

differentiation of follicles (Barbacka-Surowiak et al., 2003). The maximum level of estradiol

turns on a positive feedback mechanism resulting in the preovulatory LH surge (Chappell

et al., 2000), the LH surge usually occurs on the day of proestrus and later during

proestrus, the preovulatory peak level of progesterone occurs (Barbacka-Surowiak et al.,

2003). Estrus is the time of ovulation, mating and oocyte fertilization, while a short

postovulatory metestrus is the time of corpus luteum formation. Diestrus can be

significantly shortened or even skipped during increased reproductive activity in spring,

whilst it can be markedly prolonged in late fall or winter. Diestrus is characterized by the

25

onset of follicular activity and estrogen surges, while progesterone levels remain low

(Barbacka-Surowiak et al., 2003).

Figure 1.5.2.2.1.1- Relative concentrations of ovarian hormones during the rat estrous cycle. Estrogen and progesterone fluctuate during the rat estrous cycle. In diestrus, estrogen levels begin to rise and

progesterone levels are low. In proestrus, estrogen levels peak and descend while progesterone levels rapidly

peak and decline by the end of the phase. Estrogen and progesterone decline during estrus and begin to rise

by the end of metestrus. Adapted from (Quinones-Jenab et al., 2001).

1.5.2.3 Effects of cocaine on the HPG axis

A number of studies have demonstrated cocaine’s modulation of the HPG axis.

Clinical studies indicate that women who abuse cocaine may have a variety of menstrual

cycle disorders, including amenorrhea and luteal phase dysfunction, which compromise

fertility (Smith et al., 1984; Mello and Mendelson, 1997). However, clinical evidence of

cocaine’s disruptive effects on reproductive function is often complicated by the fact that

many human cocaine abusers are polydrug abusers (Teoh et al., 1992; Mello and

Mendelson, 1997). Nevertheless, cocaine effects on different components of the

reproductive cycle have been demonstrated by preclinical studies. Chronic cocaine self-

administration and cocaine withdrawal were both associated with disruptions of menstrual

cycle regularity compared with control conditions in rhesus monkeys (Mello et al., 1997).

And chronic exposure to high doses of cocaine (10-20 mg/kg/day) disrupted estrous

cyclicity in rats and decreased rates of ovulation (King et al., 1990; King et al., 1993). In

the case of the estrous cycle it was demonstrated that the disruption of the cyclicity

depends on the cocaine treatment regimen (Booze et al., 1999; Sell et al., 2000), namely

it was found to be cocaine dose-dependent (King et al., 1993).

26

The mechanisms through which cocaine disrupts reproductive function are poorly

understood (Mello et al., 1997). It may be through cocaine’s direct stimulation of LH

release, or indirect through putative changes in ovarian hormone concentrations that

could alter feedback control of gonadotropin release (Mello et al., 1990; Mello et al.,

1993). In fact, acute administration of cocaine changes basal levels of anterior pituitary

hormones that are important for reproductive cycle normalcy. Frequent repetition of these

acute hormonal effects of cocaine might contribute to the reproductive cycle abnormalities

observed during chronic cocaine self-administration (Mello et al., 1997).

Acute administration of cocaine stimulates the release of LH in men and women

(Mendelson et al., 1989; Heesch et al., 1996; Mendelson et al., 2001; Mendelson et al.,

2002), as well as in male and female rhesus monkeys (Mello et al., 1990). This effect was

not predictable from the pharmacological actions as an indirect DA agonist of cocaine or

from the clinical literature on DA-gonadotropin interactions (Judd et al., 1979; Mutiara et

al., 2006). It has been suggested that cocaine’s stimulation of LH reflects a cocaine

action upon the GnRH release and not a direct action on the pituitary (Mello and

Mendelson, 1997; King et al., 2001). Acute administration also increases FSH in men

(Heesch et al., 1996). Acute cocaine increases plasma estradiol levels in female rhesus

monkeys during the follicular phase but has no effects on estradiol levels during the luteal

phase (Mello et al., 2000) and do not alter progesterone levels during either the follicular

or the luteal phase of the menstrual cycle (Mello et al., 2000). Acute cocaine treatment

also did not alter serum levels of testosterone in men (Mendelson et al., 2003). In addition,

in men, chronic cocaine abuse had no demonstrable effects on pulsatile release patterns

of LH or testosterone (Mendelson et al., 1989).

In rodents, acute cocaine transiently increased LH, but not FSH, in female rats

(King et al., 2001). However, in contrast to acute stimulatory effects of cocaine on LH, this

hormone levels measure at proestrus and diestrus were significantly lower after chronic

high-dose cocaine treatment (King et al., 1990). Progesterone plasma levels were

increased after acute and chronic cocaine administration in males and female rats

(Quinones-Jenab et al., 2000a; Walker et al., 2001a; Chin et al., 2002). In female rats, the

cocaine-induced levels of progesterone fluctuated within the estrous cycle and reached

their highest levels during proestrus (Quinones-Jenab et al., 2000b). Acute cocaine

administration did not affect plasma levels of estrogen, however, cocaine-stimulated

progesterone release was greatest when estrogen levels were at their highest (Walker et

al., 2001a). Cocaine has been shown to decrease testosterone levels in male rats after

acute and chronic treatment (Chin et al., 2002; Festa et al., 2003), whereas others

27

showed no changes in testosterone levels after an acute cocaine injection (Walker et al.,

2001b).

As described earlier, the normal reproductive function depends on the correct

interaction between GnRH, anterior pituitary and gonadal steroids hormones, thus

cocaine-induced alterations in the HPG axis seems to be, at least, one of the causes for

the clinical evidences of cocaine’s disruptive effects on the reproductive function. For

example, although the functional implications of a cocaine-related increase in LH are

unclear, repeated episodes of cocaine intoxication accompanied by elevated LH levels

could compromise folliculogenesis and prompt early ovulation (Zeleznik, 1981; Dierschke

et al., 1985; Mello and Mendelson, 1997). On the other hand, due to the profound effects

of the gonadal hormones on the modulation of CNS plasticity, cocaine-induced alterations

in the HPG axis may play a key role in modulating CNS neuronal functions and thus be an

important component in the cascade of events that follows the administration of cocaine

(Quinones-Jenab et al., 2001).

1.5.2.4. Effects of sex and sex hormones in the response to cocaine

Clinical and preclinical studies have invalidated the long-standing view of cocaine

as a predominately male problem. The current literature suggests that women are more

sensitive than men to the addictive properties of cocaine and could be more vulnerable to

its powerful psychostimulanting effects (Kosten et al., 1993; Chen and Kandel, 2002).

More severe cocaine use patterns are observed in females at the time of intake (Robbins

et al., 1999). Moreover, women consume cocaine by more addictive routes and progress

to dependence more rapidly than men (McCance-Katz et al., 1999). In fact, prevalence of

cocaine addiction was actually shown to be higher in adolescent females than adolescent

males (Kandel et al., 1997). Women also report shorter abstinence periods between

cocaine use than men (Kosten et al., 1996) together with stronger cravings in response to

cocaine cues as compared to men (Robbins et al., 1999).

It is not likely that these effects in women are due to differences in cocaine

metabolism as it has been demonstrated that blood levels of cocaine, after intravenous

administration are comparable in men and women, and do not vary significantly across

the menstrual cycle (Mendelson et al., 1999a). Also, no sex differences were found in

cocaine pharmacokinetics after intranasal cocaine administration (McCance-Katz et al.,

2005). Similarly, in rhesus monkeys, cocaine pharmacokinetics are similar in males and

females after intravenous administration (Mendelson et al., 1999b).

28

Preclinical data also has demonstrated sex differences in cocaine response. For

example, female rats do acquire cocaine self-administration more rapidly and at lower

cocaine doses than males, and after acquisition female rats also self-administer more

cocaine than did males (Lynch and Carroll, 1999; Elman et al., 2001), indicating that

females are more sensitive to the reinforcing effects of cocaine (Russo et al., 2003a).

Reinstatement of extinguished cocaine-reinforced responding is also greater in female

rats (Lynch and Carroll, 2000). Adding to that, female rats exhibit greater locomotor and

stereotypic behaviour than male rats after acute cocaine administration (Sell et al., 2000;

Chin et al., 2002; Festa et al., 2004). Regardless of dose, injection frequency, or length of

cocaine administration, female rats consistently demonstrate behavioural sensitization

after fewer cocaine injections as compared to male rats (Glick et al., 1983; Chin et al.,

2002). However, no sex differences in behaviour stereotypy were observed after chronic

cocaine administration, thus suggesting that locomotor and motor stereotypy respond

differentially to chronic cocaine (Chin et al., 2002).

The dynamic endocrinological profile of females has also been shown to affect

cocaine-induced responses in both clinical and preclinical studies.

In a human study, female cocaine users in the luteal phase had an attenuated

subjective response to smoked cocaine and less desire to smoke cocaine than did those

in the follicular phase (Sofuoglu et al., 1999). Also, a greater “high or good feeling” was

reported during the follicular phase after cocaine intake (Evans et al., 2002). Because the

luteal phase of the cycle is characterized by high levels of progesterone, these studies

suggest that progesterone attenuates the subjective effects of cocaine (Festa and

Quinones-Jenab, 2004).

Several preclinical studies have shown that the estrous cycle also influence the

behavioural response to acute cocaine administration in rats. Rats in estrus demonstrate

a higher incidence of cocaine-induced stereotypic and locomotor activity than do rats

during diestrus (Quinones-Jenab et al., 1999; Sell et al., 2000). Unlike studies of acute

cocaine administration, results from studies examining estrous cycle effects during chronic

cocaine administration have not yielded consistent results (Festa and Quinones-Jenab,

2004).

A clear picture is emerging which suggests that the biological basis of sex-specific

differences in cocaine effects resides in the disparate regulation of the CNS by male and

female gonadal hormones (Quinones-Jenab et al., 2001; Lynch et al., 2002).

For instance, gonadectomy of female rats decreased locomotor activity in

response to acute cocaine (Chin et al., 2002). However, gonadectomized female rats

29

treated with estrogen, or estrogen and progesterone showed enhanced locomotor activity

responsiveness to cocaine when they were compared to gonadectomized female rats

treated with progesterone alone or vehicle (Sell et al., 2000). Also chemical blockade or

surgical removal of estrogen do reduce cocaine self-administration during acquisition,

when compared to estrogen producing rats (Lynch et al., 2001).

Gonadectomy of male rats either increase or have no effect on behavioural

responses to acute cocaine (van Luijtelaar et al., 1996; Walker et al., 2001b; Chin et al.,

2002). Discrepancies in the results may be due to the cocaine dose or the strain of rat

used. Chen et al. (2003) showed that in gonadectomized male rats testosterone

replacement attenuated cocaine-induced behavioural stereotypy and locomotion. It was

suggested that testosterone reduced cocaine-induced sensitization of behavioural

stereotypy by increasing reuptake of striatal DA (Chen et al., 2003).

Overall, acute and chronic cocaine administration studies have demonstrated that

estrogen increases, testosterone reduces, and progesterone reduces or has no effect on

cocaine-induced activity in rodents (Festa and Quinones-Jenab, 2004). Although the

number of clinical studies is limited, the inhibitory effects of progesterone have been

consistent in both clinical and rodent studies (Festa and Quinones-Jenab, 2004).

Given the increasing evidence that there are sex differences in response to

cocaine, and that these differences might be related to fluctuations in gonadal hormone

levels, studies have attempted to determine whether there are pharmacokinetic

differences between males and females and whether hormonal fluctuations in females

alter the pharmacokinetics of cocaine. Most studies in rodents have reported no

differences in cocaine plasma levels between males and females (Bowman et al., 1999;

Festa et al., 2004). Further, while some studies have shown sex differences in cocaine

metabolite levels, the results have been inconsistent (Bowman et al., 1999; Chin et al.,

2001; Festa et al., 2004). Moreover, based on the data available in both monkeys and

humans, differences in the pharmacokinetics of cocaine across the menstrual cycle or

between males and females appear to be minimal (Kosten et al., 1996; Mendelson et al.,

1999a; Sofuoglu et al., 1999; Evans et al., 2002; Evans and Foltin, 2006). Thus, the

evidence that females and male have comparable cocaine levels in both plasma and brain

suggests that reported sex differences in the response to cocaine are not due to

substantial differences in brain drug levels (Bowman et al., 1999).

Still, there is considerable preclinical evidence that sex hormones modulate the

dopaminergic transmission. For instance, the rates of release and uptake of DA in the

dorsal striatum are greater in females than in males and the density of striatal DA

30

receptors is higher in females (Andersen and Teicher, 2000; Walker et al., 2000).

Moreover, dopaminergic neurotransmission varies throughout the estrous cycle,

demonstrating that dopaminergic activity is sensitive to endogenous fluctuations of

ovarian hormones (Davis et al., 1977; Becker and Ramirez, 1981; Thompson and Moss,

1994).

Ovarian hormones modulate the dopaminergic system at different levels. Estrogen

acts directly on the striatum to affect DA release (Becker, 1990a), turnover rates (Becker,

1990b) and reuptake (Thompson et al., 2000). Additionally, direct infusions of estrogen

into Nac result in increased DA levels that are significantly higher than those observed

following vehicle administration (Thompson and Moss, 1994). Mesocorticolimbic DA

release and reuptake are also controlled by progesterone (Cabrera et al., 1993). However,

the modulation by progesterone seems to be bimodal, where according to progesterone

plasma levels, DA release is either synergized or inhibited (Cabrera et al., 1993). Similarly

to the dopaminergic system, estrogen and progesterone can alter the function of the

serotonergic neuronal system at various levels, including secretion, receptor binding sites

and transporters (Luine, 1993; Bethea et al., 1998; Flugge et al., 1999). In male rats,

testosterone enhances turnover of both DA and 5-HT and also affects 5-HT binding sites

as well as uptake sites (McQueen et al., 1999).

The data presented above seems to indicate that sex hormones may influence

cocaine reward by altering monoamine neurotransmitter systems in the striatum and

mesolimbic areas (Becker, 1999; Russo et al., 2003b).

Given that cocaine remains a major public health problem and appears to be

increasing among women, understanding the interaction between cocaine, hormones and

behaviour will facilitate the development of effective treatment strategies for cocaine

abusers that may need to be gender-related.

1.5.3. Interactions HPG/ HPA axes and the response to cocaine

Countless studies have shown that stress and HPA axis activation inhibits

reproductive function and behaviour. The first line of evidence derives from the

observation that the administration of CRH results in an immediate decrease in pulsatile

GnRH and LH release (Olster and Ferin, 1987; Petraglia et al., 1987). The suppression of

the GnRH pulse generator by a variety of stressful stimuli can be blocked by CRH

antagonists (Rivier et al., 1986; Maeda et al., 1994). The suppressive effects of the HPA

system on the HPG function are also manifested at least in part as an inhibition of

gonadotropin release through effects of glucocorticoid hormones on the hypothalamus

31

and/or anterior pituitary gland. Treatment of adult male rats with corticosterone decreases

GnRH mRNA levels (Gore et al., 2006). Moreover, glucocorticoids suppress GnRH

release both in hypothalamic tissue in vitro (Calogero et al., 1999) and in GT1-7 cells

(Attardi et al., 1997). Decreases in circulating LH levels may also be due, at least in part,

to direct actions of corticosterone on pituitary gonadotropes. These cells have been

shown to express GRs in several studies (Kononen et al., 1993; Teitsma et al., 1999;

Breen et al., 2004), and they are also likely to be influenced by other pituitary cells

expressing GRs (e.g. corticotropes). In addition, the pituitary GnRH receptor may be

involved, as glucocorticoids can reduce GnRH-stimulated LH release directly from

pituitary cells (Briski and Sylvester, 1991).

There is also evidence that stress-activated inputs, including catecholaminergic

inputs to the hypothalamic PVN suppress LH pulses via CRH (Maeda et al., 1994).

In addition to the direct and indirect effects of cocaine on gonadotropins, cocaine

abuse may also disrupt the reproductive function by stimulation of the HPA axis. It is

possible that the acute stimulatory effects of cocaine on the HPA axis contribute to the

reproductive dysfunction observed during cocaine abuse (Mello et al., 1997).

This relationship between HPA and HPG axes is by no means unidirectional. The

reproductive status and gender impact heavily on both basal and stress-induced HPA axis

activity (Dallman et al., 2002).

Adult female rats have higher basal and stress levels of ACTH and corticosterone

than do males (Lesniewska et al., 1990b). The higher basal levels of corticosterone in

females are partly buffered by their higher levels of corticosteroid binding globulin

(McCormick et al., 2002) as only unbound corticosterone is generally considered to be

biologically active (Rosner, 1990). However, females have higher levels of free

corticosterone in response to stressors (Lesniewska et al., 1990b; Tinnikov, 1999; Beiko

et al., 2004). Females also have higher levels of CRH (Hiroshige et al., 1973) and

increased levels of CRH mRNA in the PVN (Watts and Swanson, 1989).

The sex differences in HPA axis function are largely due to sex hormonal

regulation of the HPA axis (Kudielka and Kirschbaum, 2005; McCormick and Mathews,

2007). Female rats have higher CRH, ACTH and corticosterone levels during proestrus,

when estradiol levels are high, than during other phases of the estrous cycle (Hiroshige et

al., 1973; Atkinson and Waddell, 1997; Walker et al., 2001a). Furthermore, plasma ACTH

and corticosterone levels in response to stress are higher during proestrus than during

other phases of the cycle (Viau and Meaney, 1991; Carey et al., 1995). Gonadectomy of

adult female rats reduces ACTH and corticosterone levels (Viau and Meaney, 1991;

32

McCormick et al., 2002; Seale et al., 2004b). Animal studies consistently reveal a strong

stimulatory influence of estradiol on HPA axis function (Lesniewska et al., 1990b; Viau

and Meaney, 1991; Norman et al., 1992; McCormick and Mathews, 2007), with

modulatory effects on MR and GR (Burgess and Handa, 1992; Carey et al., 1995).

Estradiol decreases GR binding and mRNA levels in various brain areas (Burgess and

Handa, 1992; Carey et al., 1995). Moreover, estradiol may directly enhance CRH gene

transcription in the hypothalamus through binding to estrogen-responsive elements on the

CRH gene transcription (Vamvakopoulos and Chrousos, 1993). Estradiol also directly

stimulate corticosterone secretion in dispersed adrenocortical cells (Nowak et al., 1995).

Thus, estrogen may influence adrenal corticosterone secretion directly and indirectly.

Although estradiol-mediated effects seem to be the most potent modulators of

HPA axis, stress regulation by progesterone might also contribute. In animal studies, it

was showed that progesterone can function as a glucocorticoid antagonist; can bind to

GR and MR receptors; can increase the rate of dissociation of glucocorticoids from the

receptor; and can diminish the effectiveness of corticosterone feedback on stress

responsiveness (Ahima et al., 1992; Carey et al., 1995).

Androgens exert specific and in part contrasting effects (inhibitory) at different

levels of HPA axis regulation as shown in several animal studies (Bingaman et al., 1994;

Handa et al., 1994; Viau and Meaney, 1996). Studies on gonadectomized males as adults

submitted to androgen replacement indicate that androgens typically inhibit the HPA axis

response to stress (Handa et al., 1994; Seale et al., 2004a), although there are some

reports of no effect of adult male gonadectomy on corticosterone levels (Lesniewska et

al., 1990b; Lesniewska et al., 1990a; Chen and Herbert, 1995).

Testosterone influences adrenal steroidogenesis leading to decreased

corticosterone levels in the adrenal gland (Malendowicz and Mlynarczyk, 1982). At central

levels androgen inhibits hypothalamic CRH (Bingaman et al., 1994), and replacement of

androgens limited to the medial preoptic area decrease corticosterone release in

response to stress in gonadectomized rats, while increasing GR levels (Viau and Meaney,

1996; McCormick et al., 2002). It also should be noted that testosterone can also exert

estrogenic effects after being metabolized to estrogen by aromatization in both brain and

peripheral tissues (Finkelstein et al., 1991; Bagatell et al., 1994).

Cocaine, like other stressors, is known to exert sexual-dimorphic HPA axis effects

in rats. Female have greater ACTH and corticosterone plasma levels after acute cocaine

exposure than do males (Kuhn and Francis, 1997). Moreover, the HPA axis response to

cocaine is strongly influenced by activational effects of ovarian hormones (Walker et al.,

33

2001a). Gonadectomy decreased cocaine-induced ACTH and corticosterone plasma

levels response in females, whereas castration had no effect on males (Kuhn and Francis,

1997). The estrous cycle stage has also been shown to influence cocaine-induced

secretion of ACTH, that are greatest in rats in proestrus (Walker et al., 2001a). The

variation in cocaine-stimulated HPA axis activation with the estrous cycle stage and

gonadectomy, support a role for ovarian steroids in the increased HPA axis responses of

females to cocaine (Walker et al., 2001a). This sexually dimorphic HPA axis responses to

cocaine may mediate some aspects of behavioural responses to cocaine including

cocaine self-administration (Walker et al., 2001a).

1.6. The adolescence

Adolescence is defined operationally as the transition period that characterizes the

passage from childhood to adulthood (Spear, 2000). This period encompasses not only

reproductive maturation, but also cognitive, emotional and social maturation (Sisk and

Foster, 2004).

The adolescent brain is a brain in transition, and differs anatomically and

neurochemically from that of the adult (Spear, 2000). This remodeling of brain appears

highly conserved across species and promotes behaviours supporting the evolutionary

fitness theme of optimal species reproduction (Ernst and Mueller, 2008). Adolescence is

likewise characterized by expression of a number of typical behavioural features, such as

increased novelty seeking and risk taking (Irwin, 1989), and a shift in social affiliation

towards more peer-based social interactions (La Greca et al., 2001). This is the time when

individuals begin to move away from the familiar cell and acquire independence in order to

fulfill adult roles in terms of reproductively mature and socially competent behaviour. This

impetus to move away from the protective nest is necessary for preventing genetic

inbreeding and for enabling genetic diversity, which serves the overall evolutionary fitness

goal (Ernst and Mueller, 2008). Behaviours of novelty-seeking and risk-taking facilitate this

transition (Spear, 2000; Ernst and Mueller, 2008). In the human adolescent, these

propensities may be expressed however, in drug use, as well as a variety of other deviant

behaviours (Spear, 2000).

Observation suggests enhanced mood liability and emotional intensity during

adolescence (Buchanan et al., 1992). Affective processes and response to

reward/punishment, which contribute substantially to decision-making, may present

adolescence specific features that facilitate risk-taking behaviour in this age group (Martin

et al., 2002). The cognitive function also develops throughout adolescence. Although this

34

function improves dramatically, qualitatively as well as quantitatively, from infancy into

childhood, it continues to be refined across adolescence (Ernst and Mueller, 2008).

Among the transitional processes seen at some point during adolescence are

puberty and its concomitant hormonal and physiological changes, along with considerable

growth spurt (Spear et al., 2002; Spear, 2007). The initiation of puberty is determined by

an increase, after the prepubertal quiescence period, in the pulsatile pattern of

hypothalamic GnRH secretion, and activation of HPG axis, it culminates in gonadal

maturation and in total capacity to express reproductive behaviour (Payne et al., 1977;

Wiemann et al., 1989; Sisk and Foster, 2004).

It is recognized that in addition to the well-known perinatal period of steroid-

dependent organization of neural circuits and behaviour, adolescence is another period of

development during which steroid hormones organize the nervous system (Sisk and Zehr,

2005). As brain-hormone interactions during the adolescent brain “growth spurt” are

integral to behavioural maturation, temporal dissociations between gonadal maturation

and adolescent brain development are likely to have consequences for adult behaviour

(Sisk and Foster, 2004). In fact, the absence of gonadal hormones or pharmacological

blockade of their action during adolescence prohibits adult-typical expression of behaviour

that are organized during adolescence, even if hormones are replaced in adulthood,

demonstrating irreversible and adverse consequences for behaviour if gonadal hormones

are absent during adolescence (Sisk and Foster, 2004).

Despite the importance of gonadal hormones, it is clear that some important

aspects of behavioural maturation are not driven solely by the appearance of steroid

hormones at the time of puberty (Sisk and Foster, 2004). Structural remodeling of the

brain during adolescence occurs through both steroid-dependent and steroid-independent

mechanisms (Sisk and Foster, 2004).

Both the imaging work in humans and the often more detailed neuroanatomical

assessments emerging from studies with laboratory animals have revealed a surprising

degree of neural sculpting during adolescence, with notable similarities in the brain

regions affected often emerging across a variety of species (Spear, 2007).

During this period there is a massive loss of synapses in neocortical brain regions

(Huttenlocher, 1984; Spear, 2000). Among the synapses undergoing particularly notable

pruning during adolescence are those providing excitatory input to the neocortex

(Bourgeois et al., 1994), as well as synapses contributing to reverberating circuits within

particular cortical regions (Woo et al., 1997). This ontogenetic reduction in excitatory input

35

to cortex and connectivity within reverberating circuitry could contribute to the reduction

and refinement of brain effort during adolescence (Spear, 2007).

Progressive myelination of axons results in considerable developmental increases

in cortical white matter through adolescence and into adulthood (Sowell et al., 2003),

providing faster communication between regions, and has been presumed to increase

overall speed of information processing within the brain. Although less prominent and

consistent than developmental increases in white matter, ontogenetic declines in volume

of gray matter are seen in regions such as the frontal cortex (Giedd et al., 1999; Rapoport

et al., 1999).

Among the brain regions showing particularly marked ontogenetic alterations

during adolescence is the prefrontal cortex, with this region showing considerable synaptic

culling and a prominent loss of excitatory input (Bourgeois et al., 1994). In contrast to the

adolescent-associated loss of excitatory drive to cortex (Zecevic et al., 1989), DA input to

prefrontal cortex increases during adolescence (Kalsbeek et al., 1988; Rosenberg and

Lewis, 1994).

During adolescence certain subcortical regions also undergo considerable

remodeling, especially those regions that form part of an interconnecting network of

circuitry with prefrontal cortex (Spear, 2000). For instance, projections from the amygdala

to prefrontal cortex continue to be elaborated throughout adolescence (Woo et al., 1997;

Cunningham et al., 2002). The amygdala of the adolescent also shows a different pattern

of stress-induced activation compared to the adult (Kellogg et al., 1998).

Among the numerous alterations seen in the mesocorticolimbic brain regions

during adolescence are regionally specific ontogenetic alterations in the patterns of DA

production and utilization. Estimates of DA synthesis and turnover on prefrontal cortex are

higher early in adolescence than later in adolescence and in adulthood, whereas DA

synthesis and/or turnover estimates in Nac and striatum conversely are lower earlier than

late in adolescence (Teicher et al., 1993; Andersen et al., 1997).

The HPA axis, known to be, as already mentionated in this chapter, one of the

main systems regulating physiological responses to stressors, is also in transition during

adolescence. For example, plasma concentrations of corticosterone remains elevated

longer after exposure to a stressor in adolescent rats compared to adults, and sex

steroids regulate HPA axis function in adults but not in adolescents (reviewed in

McCormick et al., 2008). Glucocorticoids affect ongoing behavioural and

neurophysiological processes and can shape future function by altering synaptogenesis,

dendritic morphology and neurogenesis (Joels et al., 2004). Altered HPA axis function in

36

adolescence may make adolescents particularly susceptible to the negative

consequences of stressors.

1.6.1. Adolescence: A critical period of addiction vulnerability

As mentioned above, evidences suggest that human adolescents are more

involved in reckless behaviours than adults and are motivated to seek novel sensations

(Irwin, 1989; La Greca et al., 2001; Martin et al., 2002). Even when considering an animal

model of adolescence in rat, animals undergoing this transition period exhibit more risk

taking, novelty-seeking, and peer-directed social interactions than adults (Douglas et al.,

2003, 2004; Stansfield and Kirstein, 2006). Studies have shown that high levels of

novelty/sensation-seeking are powerful predictors of risky activities such as hazardous

driving, unprotected sex and drug use (Andrucci et al., 1989; Wills et al., 1994). Hence,

along with increased sensation and novelty seeking during adolescence, an increase in

drug use is seen as well (Spear, 2000).

In fact, epidemiological data indicate that experimentation with addictive drugs and

the onset of addictive disorders are primarily concentrated in the adolescence and early

adulthood (Chen and Kandel, 1995; Chambers et al., 2003). Furthermore, adolescents are

more likely to progress from use to addiction state than adults (Chen et al., 1997) and

demonstrate a more precipitous progression of drug use (Estroff et al., 1989; Warner et

al., 1995). There is growing evidence suggesting that adolescence represents a period of

heightened biological vulnerability to the addictive properties of drugs of abuse (Chambers

et al., 2003).

Indeed, adolescents of a variety of species differ in their sensitivity to various drugs

relative to their adult and sometimes younger counterparts (Spear, 2000). Adolescent rats

and mice are less sensitive than younger animals or adult to the effects of psychomotor

stimulants such as amphetamine and cocaine when indexed in terms of acute locomotor

stimulation and stereotypy (Laviola et al., 1995; Bolanos et al., 1998; Laviola et al., 1999)

Adolescent male rats also are more likely to acquire self-administration of nicotine plus

acetaldehyde than adult male rats (Belluzzi et al., 2005). In addition to higher levels of

self-administration compared to adults, adolescent rats also exhibit reduced evidence of

aversive drug effects. The conditioned taste aversion to amphetamine is reduced in

adolescent rats (Infurna and Spear, 1979). Adolescence is also a time of relative

insensitivity to the aversive properties of cocaine (Schramm-Sapyta et al., 2006).

Adolescents require close to 75% more extinction trials than adults to extinguish cocaine

place-preferences compared with adults (Brenhouse and Andersen, 2008). These

37

examples, demonstrate that adolescents are less sensitive to use-limiting effects of drugs,

and therefore may tolerate higher levels than adults which may facilitate development of

addiction (Schramm-Sapyta et al., 2006).

As already mentioned, the brain of the adolescent differs considerably from the

adult in a number of neural systems, namely in systems implicated in the action of drugs

(Spear, 2000). In fact, the mesocorticolimbic dopaminergic brain region, known as taking

part of the circuitry implicated in drug reward processes (Koob, 1992b; Robinson and

Berridge, 2003) is among the neural systems known to undergo developmental alterations

during adolescence (Spear, 2000).

Adolescence is a critical period of transition from a more emotional regulation of

the structures that mediate substance abuse, to a more mature cortical regulatory

mechanism (Spear, 2000). During adolescence, the primary dopaminergic projections to

the Nac extend from the VTA and are predominately modulated by the amygdala (Oades

and Halliday, 1987). However, in adulthood, this previously amygdaloid-modulated system

receives projections from the medial prefrontal cortex, and this developmental transition is

critical in the functional nature of the system (Cunningham et al., 2002).The development

of this system allows for a transition from more emotionally directed behaviour to more

contextually regulated behaviour. Because adolescents lack sufficient cortical regulation,

provided by the medial prefrontal cortex, their behaviour tends to be more impulsive and

guided by emotion than adults, increasing the chances of risky behaviours, including

initiating drug use (Chambers et al., 2003).

Most of what it is known about the consequences of developmental exposure to

drugs and other chemicals is based on exposures during prenatal and early postnatal

period, with little emphasis on exposure periods that include adolescence (Catlow and

Kirstein, 2007; Spear, 2007; Lubman and Yucel, 2008). Given that drug use frequently

occurs during adolescence, and with the increasing recognition that adolescence is a time

of considerable neural restructuring and sculpting of the brain (Spear, 2000), there is,

likewise, a growing interest in assessing whether this developmental transition represents

an unusually sensitive period to disruption by drugs, or whether this remodeling reflects a

window of opportunity for unusual plasticity and recovery (Spear, 2000; Catlow and

Kirstein, 2007; Spear, 2007). Nevertheless, growing evidence from the developmental

neuroscience field suggests that early onset of drug use may adversely affect ongoing

brain development and the critical maturational processes that are occurring during the

adolescent period (Lubman et al., 2007). The following table (table 1.6.1.1) summarizes

the most significant effects of cocaine exposure during adolescence on the rat animal

model.

38

Table 1.6.1.1- Summary of the most common effects of cocaine exposure during adolescence in the rat.

Report Route of administration

Daily dose Exposure period

Evaluation period

Observed effects

(Philpot and Kirstein, 1999)

i.p. 2 or 20 mg/kg/day

PND 21-24

PND 25 (after challenge injection)

↑ followed by suppression in DA overflow in the Nac.

(Collins and Izenwasser, 2002)

i.p. 50 mg/kg/day

PND 28-35

PND45 (after challenge injection)

No sensitization to locomotor activity;

No effects on either DAT or SERT densities.

(Catlow and Kirstein, 2007)

i.p. 20 mg/kg/day

PND 35-44

PND 70-79

PND 65

PND 100

↑DA levels in response to sucrose intake compared to saline and adult.

(Frantz et al., 2007)

i.p.

i.v. catheter

10 or 20 mg/kg; four injections spaced 5 days apart

0.37, 0.74 and 2.92 mg/kg; under a fixed ratio 1 lever-response contingency- 2 hour session

PND 37-52

PND 75-90

PND 37-52

PND75-90

PND 37-52

PND 75-90

PND 37-52

PND 75-90

↓ sensitivity to acute cocaine compared to adult;

↓ overall activity compared to adults;

No effects on cocaine-stimulated Nac DA.

(Marin et al., 2008)

i.p. 10 mg/kg/day

PND 30-34 PND 37, 64 and 97(All preceded by a challenge injection )

Behavioral sensitization on PND37 e 64;

↑ GluR1 in mPFC on PND37 in sensitized rats;

No effects on TH or NR1 in Nac or mPFC.

(Walker and Kuhn, 2008)

ip 15 mg/kg/day

PND 28

PND 65

PND 28

PND 65

↑ DA release in caudate nucleus compared to adult rats.

(Catlow and Kirstein, 2005)

i.p 20 mg/kg/day

PND 35

PND 60

PND 35

PND 60

↑ locomotor activity in the adolescent females compared to adults

(Lepsch et al., 2005)

i.p. 10 mg/kg/day

PND 38 PND 38 Pre-exposure to both chronic restraint and variable stress ↑ cocaine-induced locomotor activity

(Maldonado and Kirstein, 2005)

i.p. 30 mg/kg/day

PND 45

PND 60

PND 45

PND 60

Prior handling ↑ cocaine-induced locomotor activity, but not in adults.

DA- dopamine; DAT- dopamine transporter; GluR1- GluR1 glutamate receptor subunit; i.p.- intraperitoneal injection; i.v.- intravenous injection; mPFC- medial prefrontal cortex; Nac- nucleus accumbens; NR1- NR1 glutamate receptor subunit; PND- postnatal day; SERT- serotonin transporter; TH- tyrosine hydroxilase; ↑ increase; ↓ decrease.

39

Table 1.6.1.1- Summary of the most common effects of cocaine exposure during adolescence in the rat (continuation).



Evaluation period

Observed effects

(Caster et al., 2007)

i.p. 40 mg/kg/day

PND 28

PND 42

PND 65

PND 28-29

PND 42-43

PND 65-66

Behavioral sensitization to non-ambulatory horizontal activity, sniffing behaviors, and stereotypies in animals of all ages;

Ambulatory sensitization and greater increases in stereotypies observed in the youngest adolescents;

(Badanich et al., 2008)

i.p. 5, 10 or 20 mg/kg/day

PND 35

PND 45

PND 60

PND 35

PND 45

PND 60

↑ acute cocaine-induced locomotor activity in early adolescence comparison to late adolescent and adult rats.

(Badanich et al., 2006)

i.p. 5 or 20 mg/kg/day

PND 31-34

PND 41-44

PND 56-59

PND 35

PND 45

PND 60

Cocaine CPP were found for all ages at the high dose;

PND 35 was the only age group to have a preference at the low dose.

(Schramm-Sapyta et al., 2006)

i.p. 10-40 mg/kg/day

PND 29

PND 66

PND 30

PND 67

↓ susceptibility to cocaine CTA compared to adult.

(Perry et al., 2007)

i.v. catheter

0.4 mg/kg under fixed ration 1 lever-response contingency

PND 29- until acquisition , or PND 59

PND 154- until acquisition or, PND 184

PND 29-59

PND 154-184

↑ avidity for sweets in adolescents;

↑ vulnerability to initiate drug abuse in adolescents.

(Brenhouse and Andersen, 2008)

i.p. 10 mg/kg/day

20 mg/kg/day

PND 39-40

PND 78-79

PND 41 until CPP extinction

PND 80 until CPP extinction

↑ extinction trials than adults to extinguish CPP;

Once extinguished, adolescents display a greater preference for a previously cocaine-paired environment.

CPP-conditioned place preference; CTA- conditioned aversive properties; i.p.- intraperitoneal injection; i.v.- intravenous injection; PND- postnatal day; ↑ increase; ↓ decrease.

40




Evaluation period

Observed effects

(Lynch, 2008) i.v. catheter

0.75 mg/kg under a fixed ratio 1 lever-response contingency

PND 30- until acquisition or PND 50

PND 30- until acquisition or PND 50

Females acquired cocaine self-administration more rapid than males;

A greater % of females acquired self-administration;

Response was positively associated with levels of estradiol.

(Zakharova et al., 2009)

i.p. 3, 5, 7.5, 10 or 20 mg/kg/day

PND 35-37

PND 67-69

PND 38

PND 70

Adolescent and adult male rats developed CPP;

The dose-effect curve for cocaine CPP was shifted to the left in adolescents compared to adults.

(Kerstetter and Kantak, 2007)

i.v. catheter

1 mg/kg under a fixed ratio 5 lever-response contingency- 2h sessions

Starting PND 37- 18 sessions

Starting PND 74-79- 18 sessions

18 days later

Normal learning in the conditioned cue preference task in adolescents;

Adults present deficit learning.

(Black et al., 2006)

i.p. 3x5 mg/kg/day from PND35-36;

3x10 mg/kg/day from PND37-39;

2 days of abstinence

3x15 mg/kg/day from PND 42-46;

PND 35-46 PND 56-57

PND 70-71

↑ responsiveness to the stimulant effects of cocaine;

Abnormally rapid shifts in attention in an attentional set-shifting task;

Acute alterations in the expression of genes in the PFC.

(Santucci et al., 2004)

s.c. 10 mg/kg/day

20 mg/kg/day

PND 26-33 PND 44

PND142-150

PND 158-166

PND 410-416

Residual and temporary deleterious effects on memory.

(Santucci, 2008)

s.c. 20 mg/kg/day PND 28-35 About 5 and 9 weeks later

Residual impairs in working memory;

↑ fear memory.

CPP- cocaine place-preferences; i.p.- intraperitoneal; i.v.- intravenous; PFC- prefrontal cortex; PND - postnatal day; s.c.-

subcutaneous injection;; ↑- increase.

41




Evaluation period

Observed effects

(Stansfield and Kirstein, 2005)

i.p. 20 mg/kg/day

PND 35

PND 60

PND 35

PND 60

Rapid approach to novel object compared to adult.

(Stansfield and Kirstein, 2007)

i.p. 20 mg/kg/day

PND 30-50 PND 66-69

↑locomotor response in novel environment;

↓time in the center of the novel open field

↓time with a novel object;

Anxiogenic response to novelty.

(Santucci and Madeira, 2008)

s.c. 10 mg/kg/day

PND 30-37 PND 92-97

PND 110-119

Anxiogenic response persists 12 weeks beyond cessation of drug treatment.

(Raap et al., 2000)

s.c. 20 mg/kg/day

PND 21-60 PND 25-80 No effects on onset of puberty;

No effects on date of first ovulation;

↑ number of cycles with no clear proestrus-estrus transition.

i.p. - intraperitoneal; PND - postnatal day; s.c.- subcutaneous; ↑ - increase; ↓ - decrease.

The data presented in the table 1.6.1.1 seems to indicate that adolescence

represents a period of heightened biological vulnerability to the addictive properties of

cocaine, also providing information that cocaine use during adolescence may have

neurobehavioural long-lasting effects.

In addition, it is evident in the overview of the literature references, and as

mentioned by several authors (e.g. Donovan et al., 2001; Catlow and Kirstein, 2007;

Spear, 2007; Lubman and Yucel, 2008) that the characterization of the effects of cocaine

upon the developing adolescent require further investigation in order to the full

understanding of the processes underlying the greater vulnerability to the addictive

properties of drugs during this developmental period.

42

1.7. Objectives

The objective of the present study was to contribute to the further characterization

of the effects of cocaine during adolescence, knows to be a sensitive period in what

concerns to drug problems. To achieve this aim, a series of experimental protocols were

developed in order to characterize short and long-term neurochemical, hormonal and

behavioural effects of chronic cocaine exposure during adolescence in a rat animal model.

The specific objectives of this thesis were:

- To evaluate the short and long-term effects of chronic cocaine exposure during the

adolescence period on the activity of the HPA and the HPG axes of both male and female

wistar rats, by assessing the hormonal plasma level 30 minutes, 1 day, 5 days and 10

days after last administration of cocaine, and by studying, 10 days after last

administration, the expression of genes known to be involved in the activity of the axes;

- To evaluate the short and long-term effects of chronic cocaine exposure during

adolescence period on the dopaminergic and serotonergic systems of both male and

female wistar rats, by assessing the monoamines levels, in areas known to be involved

with drug addiction, 30 minutes, 1 day, 5 days and 10 days after last administration of

cocaine, and by studying, 10 days after last administration, the expression of genes

related with monoaminergic system;

- To evaluate the short and long-term effects of chronic cocaine exposure during

adolescence period, on behavioural strategies adopted to cope with an environmental

anxiogenic situation, by submitting the animals, 2, 5 and 10 days after last administration,

to the elevated plus maze test (EPM), and related the behavioural data with the HPA axis

and monoaminergic system activity, by measuring hormonal and neurochemical

alterations induced by the test;

- To evaluate the presence of depressive-like symptoms during acute withdrawal from

chronic cocaine exposure during adolescence, both in male and female adolescent rats,

by submitting the animals, 2 days after last administration, to the forced-swim test (FST),

and related the behavioural data obtained with the HPA axis and monoaminergic system

activity, by measuring hormonal and neurochemical alterations induced by the test;

- To evaluate the long-term effects of chronic cocaine exposure during adolescence on

the aggressive behaviour of male rats, using a resident-intruder paradigm, and relate the

behavioural data with the HPA axis and monoaminergic system activity, by measuring

hormonal and neurochemical alterations induced by the test;

43

- To determine the relevance of gender-related differences in the responsiveness to the

chronic cocaine exposure during adolescence.

44

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Chapter 2

Methods

2.1. Animal Model

2.1.1. Introduction

The selection of an appropriate animal model is crucial to obtain conclusive data

out of the experimental design (Vorhees, 1995). As discussed in the first chapter the

effects of cocaine exposure have been studied in a variety of animal models.

Clinical studies exhibit a series of limitations in what concerns to the assessment

of drug abuse effects. In human studies it is difficult to exclude the influence of other

factors such as the use of other illegal and legal drugs, co-morbid psychiatric conditions,

and differences in lifestyle (Porrino et al., 2007). Because of these and many other

problems, it is virtually impossible to isolate and address the issue of the consequences of

chronic exposure in the human population (Porrino et al., 2007). One approach to

addressing this question is the use of animal models. Animal models enable to predict the

effects of drug exposure in humans by systematically manipulating variables and

controlling for many of the problems encountered in human studies (Porrino et al., 2007).

The usefulness of animal models depends in large part on their validity as

simulations of human psychopathology or the human behaviour (Willner, 1997). In the

case of drugs abuse, like humans, other animals voluntarily self-administer drugs, and

their reinforcing properties have been demonstrated in animals such as rodents and non-

human primates (Mello et al., 1997; Tidey and Miczek, 1997; Carroll et al., 2002).

Moreover, in general, there is close agreement between the drugs self-administered by

animals and those which are abuse by people (Willner, 1997). The possibility of studying

these behaviours in animals has helped to understand the neurobiological basis of drug

taking (Koob and Moal, 1997; Nestler and Aghajanian, 1997; Everitt and Wolf, 2002) and,

more generally, the brain systems for reward (Wise, 2002).

In the present study, the effects of chronic cocaine exposure during adolescence

period were analyzed. This evaluation was achieved using an animal model of exposure

to cocaine during adolescence in Wistar rats.

The developmental stage of adolescence is not uniquely human. Developing

organisms from other species likewise undergo adolescent-typical transitions that include

pubertal changes and a growth-spurt, along with expression of certain adolescent-typical

behavioural patterns. For instance, even when considering a simple animal model of

adolescence in the rat, animals undergoing this transition exhibit more risk taking, novelty-

seeking, and peer-directed social interactions than adults (Douglas et al., 2003, 2004;

Stansfield and Kirstein, 2006). In fact, similarities across species in adolescent-typical

physiological and behavioural characteristics, including a marked remodelation of the

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brain, are consistent with the notion that adolescence is a highly conserved

developmental stage during evolution, with a number of specific adolescent typical

behaviour patterns postulated to confer adaptive significance (Spear, 2007). Across-

species similarities in physiological and behavioural attributes of adolescence provide

reasonable face and construct validity for the use of animal models to study potential

neurotoxic effects during adolescence (Spear, 2007).

A critical issue when assessing consequences of drug exposure during

adolescence is the timing of the exposure period (Spear, 2007). Adolescence lacks clear

markers of onset and offset (McCormick and Mathews, 2007). In rats, the age range from

28 to 42 days postnatal has been conservatively classified as adolescence, based on

timing of age-specific behavioural changes, neural changes in brain, puberty, and the

growth spurt (Spear, 2007). Yet, some harbingers of adolescence may begin in females

as early as postnatal day (PND) 23 or so, with some residual signs perhaps lasting until

55 days or so in males (Spear, 2007). Use of this conservative age range is not meant to

imply, however, that animals slightly younger or older than this prototypic age range might

not also be undergoing adolescent transitions (Spear, 2000). Thus, to ensure that an

exposure period blankets the entire adolescence period in rats of both sex, it might be

necessary to begin shortly after the conventional age of weaning and continue until at

least 55 days (Spear, 2007). Day 60 onward is commonly agreed upon as adulthood in

that the animal has achieved physical and sexual maturity (McCormick and Mathews,

2007).

For our animal model design we choose a more restricted exposure period. We

decided for a period that comprises the reproductive maturation and the pubertal

development, since organizational effects of gonadal steroids during puberty may be

particularly relevant to drug abuse problems (Laviola et al., 1999; Spear, 2000; Chambers

et al., 2003). Thus, we decided to coincide the beginning of the administration of cocaine

with the onset of the puberty. Physical markers have been used as estimates of puberty

onset (McCormick and Mathews, 2007). In male rats, a marker is balanopreputial

separation (separation of the prepuce from the glans penis) which occurs at approximately

40 days of age (Korenbrot et al., 1977). In females, a marker is vaginal opening which

occurs at approximately 35 days of age (Evans, 1986). However, there is a significant

variation in the literature for days of balanopreputial separation (32 to 46 days) and day of

vaginal opening (32 to 38 days) (McCormick and Mathews, 2007). Based on these data

the PND 35 was chosen for the beginning of the chronic administration of cocaine that

proceeded until PND 50. This period of exposure allows starting the evaluation of the

chronic exposure effects within the transition period to the adulthood.

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When selecting an adequate animal model to assess the impact of a drug of

abuse, it is crucial to determine which dose, route of administration and frequency of

administration would be more satisfactory to the study in perspective (Spear, 1996). In the

design of our animal model was selected a binge pattern of cocaine administration,

consisting in three daily 15mg/kg intraperitoneal (i.p.) injections administered 1hour apart,

followed by no cocaine administration for 22 hours. The daily i.p. dose of 3x15mg/kg of

cocaine was selected based on previous studies in which this dose has was demonstrated

to induced neurobiological and behavioural alterations (Tsukada et al., 1996; Sarnyai et

al., 1998; Zhou et al., 2001; Schlussman et al., 2002; Zhou et al., 2003; Zhou et al., 2005;

Black et al., 2006). This is an elevated dose still, not enough to induce seizures

convulsion, or more several lethal consequences (Schlussman et al., 2005). The dosing

schedule was chosen to mimic the pattern of consumption often seen in human cocaine

addicts with respect to repeated administrations over a short time period, followed by a

period of abstinence (Gawin, 1991). For many drugs, a pattern of self-administration

develops under unlimited access conditions, consisting of periods of drug intake

alternating with periods of abstinence, during which other activities such as eating,

drinking and sleeping occur (Willner, 1997). This binge pattern of drug consumption has

been described in animals self-administering a wide variety of psychostimulants, and with

alcohol, and is also characteristic of the problematic use of both these drug classes in

humans (Willner, 1997).

2.1.2. Animals

Animals born from nulliparous three-month old female Wistar rats acquired from

Charles River Laboratories España S.A. (Barcelona, Spain) were used in this study.

Animals were housed in animal facility room of our laboratory at the Institute for Molecular

and Cell Biology, Porto, Portugal. Animals were maintained under stable conditions,

constant temperature (20-22 ºC) and humidity (60%) with a 12 hour light/dark cycle. Water

and appropriate food were supplied ad libitum. Institutional guidelines regarding animal

experimentation were followed. Rats were handled daily and regularly weighed.

Cylindrical plastic tubes and soft paper for nest construction were made available to

reduce stress. All procedures were approved by the Portuguese Agency for Animal

Welfare (General Board of Veterinary Medicine in compliance with the Institutional

Guidelines and the European Convention).

In the day after birth, here designated as postnatal day (PND) 1, litters were

standardized to 10 pups and balanced to 5 males and 5 females whenever possible.

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Animals were weaned on PND 21 and on PND 30 animals housed individually in order to

avoid variable social influences that could play a role in cocaine behavioural,

neurochemical and hormonal effects (de Jong et al., 2007) and were randomly assigned

to the different experimental groups. Only one male and one female from each litter were

used in each experimental group. At least 6 animals were used in each experimental

group.

2.1.3 Chronic administration and assessment time points

Rats were treated following a binge pattern administration of cocaine. Animals

were daily injected 3 times i.p. with 15mg/kg of body weight cocaine hydrochloride, in

0.9% NaCl solution from PND 35 to PND 50. The daily injections were administered at

hourly intervals, between 9:00 and 11:00 a.m., in a volume of 1mL/kg. Controls were

given isovolumetric 0.9% NaCl solution following the same experimental protocol. Cocaine

hydrochloride was obtained from Sigma Chemicals (St. Louis, MO, USA) and prepared in

0.9% NaCl solution. During the entire period of drug administration animals were daily

weighed.

Figure 2.1.3.1- Schematic diagram representing the cocaine exposure period and the assessment time points

estasblished to evaluate the effects of cocaine administration throughout adolescence. PND- postnatal day.

The assessments were done at different time points after the end of the exposure period:

- at PND 50, 30 minutes after last binge cocaine administration of cocaine. This time point

of assessment allowed the study of the hormonal and neurochemical effects of the acute

binge cocaine administration during a chronic exposure;

- at PND 51 and 52, 1 and 2 days, after the end of the exposure period, respectively.

These time points of assessment allowed to obtain hormonal, neurochemical and

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behavioural data of the short-term effects of the chronic cocaine administration during

adolescence period;

- at PND 55, 5 days after the end of the exposure period. This time point of assessment

allowed to evaluate, within the adolescence period, hormonal, neurochemical and

behavioural long-term effects of the chronic cocaine administration during adolescence;

- at PND 60, 10 days after the end of the exposure period. This time point of assessment

allowed to evaluate, in the early adulthood, the long-term effects of cocaine exposure

during the adolescence period, measured as neurochemical, hormonal, behavioural and

mRNA expression data.

2.2. Blood and tissue collection

At the end of the experimental periods animals were decapitated and brains were

rapidly removed. Brain areas related with the response to cocaine, such as substantia

nigra and ventral tegmental area (SN-VTA), Nac, dorsal striatum, prefrontal cortex,

amygdala, hippocampus, dorsal raphe nucleus, cerebellum and a hypothalamus section

that include suprachiasmatic, pre-optic and paraventricular nuclei were dissected on ice.

Dissection was processed according to the atlas by Paxinos and Watson (1998). Pituitary

glands were also collected. Tissue samples were frozen by immersion in 2-methylbutane

cooled over dry ice and stored at -70ºC until used for further analyses. Trunk blood was

collected in lithium heparin cover tubes and centrifuged at 1600g for 15 minutes, obtained

plasma was kept at –70ºC until hormonal assays. Gonads and adrenal glands were also

collected and weighed, and the data recorded.

2.3. Hormone plasma levels measures

All hormone plasma levels were measured by enzyme immunoassay, using

commercially available assays.

2.3.1. Steroid plasma levels

The progesterone, estradiol, testosterone and corticosterone plasma levels were

measured by enzyme immunoassay, using commercially available kit provided by

Cayman Chemical Company (Ann Arbor, MI, USA).

For progesterone, assay sensitivity was 0.012ng/mL, and the intra- and inter-assay

coefficients of variation were less 8% and 12%, respectively. For estradiol, assay

sensitivity was 0.004ng/mL, and the intra- and inter-assay coefficients of variation were

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less 10% and 12%, respectively. For testosterone, assay sensitivity was 0.004ng/mL, and

the intra- and inter-assay coefficients of variation were less 10% and 15%, respectively.

For corticosterone assay sensitivity was 0.04ng/mL, and the intra- and inter-assay

coefficients of variation were less than 7% and 9%, respectively.

These assays are based on the competition between the sterois and an steroid-

acetylcholinesterase (AChE) conjugate for a limited number of steroid-specific rabbit

antiserum binding sites. Because the concentration of the steroid AChE conjugate is held

constant while the concentration of steroid varies, the amount of steroid AChE conjugate

that is able to bind to the rabit antiserum will be inversely proportional to the concentration

of the steroid present. A rabbit antiserum-steroid (either free or AChE conjugate) complex

binds to the mouse monoclonal anti-rabbit antibody. After removing the unbound reagent,

a colour reaction is developed using a substrate to the AChE. The intensity of this color,

determined spectrophotometrical, is proportional to the amount of the steroid AChE

conjugate, which is inversely proportional to the amount of free steroid present. Sample

concentrations were read from a calibration curve.

Corticosterone plasma levels after behavioural tests were measured by enzyme

immunoassay, using commercially available kit provided by Immunodiagnostic Systems

Ltd (Boldon, UK). Assay sensitivity was 0.23 ng/mL. The intra- and inter-assay coefficients

of variation were less than 3.1% and 7.2%, respectively. This assay is also a competitive

enzyme immunoassay. Samples are incubated with enzyme (horseradish peroxidase)

labelled corticosterone in the presence of the corticosterone antibody. The corticosterone

present in the samples competes with the horseradish peroxidase labelled corticosterone

to the corticosterone antibody. After removing the unbound reagent, a colour reaction is

developed using a chromogenic substrate to the horseradish peroxidase.The intensity of

this colour, determined spectrophotometrical, is inversely proportional to the concentration

of corticosterone. Sample concentrations were read from a calibration curve.

2.3.2. ACTH plasma levels

ACTH plasma levels were measured by enzyme immunoassay, using a

commercially available kit provided by Phoenix Pharmaceuticals, Inc. (Belmont, CA,

USA). For the ACTH assay the sensitivity was 0.18 ng/mL, and the intra- and inter-assay

coefficients of variation were less than 5 % and 14%, respectively.

The assay is based on the presence of a secondary antibody that can bind to the

fragment cristallizable (Fc) region of the primary antibody whose fragment, antigen

binding (Fab) region will be competitively bound by both biotinylated peptide or targeted

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peptide in samples. The biotinylated peptide is able to interact with streptavidin-

horseradish peroxidase (SA-HRP) which catalyzes a substrate solution. The intensity of

the colour developed, determined spectrophotometrical, is directly proportional to the

amount of biotinylated peptide-SA-HRP complex but inversely proportional to the amount

of the peptide in samples. Sample concentrations were read from a calibration curve.

2.4. Neurotransmitters determinations

Levels of DA and 5-HT and their metabolites, 3,4-dihydroxyphenylacetic acid

(DOPAC), homovanillic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) were

quantified by a modified method of high-performance liquid chromatography with

electrochemical detection (HPLC-EC) (Ali et al., 1993), using a Gilson Medical Electronics

system (Gilson Inc., Middleton, WI, USA). The HPLC-EC system was composed of a

LC307 delivery pump, LC142 electrochemical detector, LC234 auto-injector, and 712

HPLC controller software version 1.30. The analytic column was a Supelcosil LC-18 3μM

(Supelco Inc., Bellefonte, PA, USA).

Prior to neurochemical determinations, 200 µl of 0.2 N perchloric acid were added

to the dissected brain regions. Samples were then disrupted by ultrasonication and

centrifuged (15,000 g) at 4ºC, during 5 minutes. Supernatant was collected and filtered

through a Costar micro centrifuge filter (0.2 µm nylon filter) (Corning Incorporated,

Corning, NY, USA). Aliquots of 50 µl were injected directly onto the HPLC-EC system for

the quantification of DA, 5-HT and their metabolites.

The mobile phase solution was composed of 70 mM potassium phosphate

monobasic buffer (pH was adjusted to 3.0 by adding with phosphoric acid), 10% (v/v)

methanol, 1mM 1-Heptanesulfonic acid and 107.5 μM sodium ethylenediaminetetraacetic

acid. The mobile phase was filtered through a 0.2 μm filter, degassed, and delivered at a

flow rate of 1.0 mL/min.

Concentrations of DA, 5-HT, DOPAC, HVA and 5-HIAA were calculated using

standard curves generated using 5 known amounts of standard amines purchased from

Sigma Chemicals (ST. Louis, MO, USA). Concentrations are expressed in ng/mg protein.

The pellet of each sample was ressuspended in 0.2 M phosphate buffer and, after

sonication, the total content of protein was assayed in duplicate using a colorimetric assay

from Bio-Rad (Munchen, Germany) based on the Bradford assay (Bradford, 1976).

Bovine serum albumin was used as a standard protein.

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The ratios of DOPAC to DA, HVA to DA and DOPAC and HVA to DA were

determined for each animal and used as indexes of DA turnover rate, whereas the ratio of

5-HIAA to 5-HT was used as an index of 5-HT turnover rate.

2.5. mRNA expression levels determination

Total RNA was extracted from brain regions related with cocaine’s response using

the RNeasy® Lipid Tissue Mini kit (Qiagen, Austin, Texas, USA) and following the

instructions of the manufacturer. Purity was estimated from the ratio of absorbance

readings at 260 and 280 nm and only ratio between 1.8 e 2 were accepted. RNA quality

was checked in agarose gel. RNA concentration was determined in a NanoDrop

spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA). One µg of RNA was

reverse transcribed, using the SuperScriptTM First-Strand Synthesis System for RT-PCR

(Invitrogen, Carlsbad, CA, USA), using oligo(dt) primers and following the instructions of

the manufacturer.

The expression levels of mRNA transcripts for the different genes studied were

measured by quantitative real-time polymerase chain reaction (qRT-PCR). The reference

gene, glyceraldahyde-3-phosphate-dehydrogenase (GAPDH) was used as internal

standard for normalization. The qRT-PCR reactions, using the equal amounts of total RNA

from each sample, were performed on the iQ5 Multicolor Real-time PCR Detection

System from Bio-Rad (Hercules, CA, USA), using the iQTM SYBR® Green Supermix (Bio-

Rad Laboratories, Hercules, CA, USA). Product fluorescence was detected at the end of

the elongation cycle. All melting curves exhibited a single sharp peak at a temperature

characteristic of the primer used. Primer design was performed using the software Beacon

Designer (Premier Biosoft International, Palo Alto, CA, USA).

Linearity and efficiency of PCR amplification reactions were assessed for all pair of

primers using standard curves generated by increasing amounts of cDNA. The

relationship between the threshold cycle (Ct) and logarithm of the cDNA concentrations

were studied according to correlation coefficient and the slopes, calculated by Bio-Rad

iQ5 Optical System Software, version 2.0 (Bio-Rad Laboratories, Inc, 2006). For all primer

sets, standard curves using four points, diluted over a 100-fold range, always led to a high

linearity (correlation coefficients>I- 0.990I).

The PCR efficiency (Ex) was calculated using the equation Ex= 10-1/slope. Efficiency

was presented as a percentage of the template that was amplified in each cycle,

calculated by the following equation %Ex= (EX-1) x 100. Efficiency close to 100% is the

best indicator of a robust, reproducible assay. Efficiency between 90-105% was always

76

used. These amplification efficiencies of PCR assays allow the quantification of mRNA

with the comparative Ct quantification method (ΔCt method) using a reference gene.

Following this method, the relative expression of a gene was calculated by the expression:

2 (Ct(referencia) – Ct(target) ). This method assumes that both target and reference genes are

amplified with efficiencies near 100% and within 5% of each other.

2.6. Behavioural tests

2.6.1. Elevated plus maze test

The anxiety-like behaviour was assessed using the elevated plus maze test

(EPM), which exploits the conflict between the animal's innate tendency to explore novel

areas and their aversion for heights and open spaces (Montgomery, 1955; Handley and

Mithani, 1984).

The EPM apparatus consisted of four arms 46 cm long and 14 cm wide with two

arms open and two arms with 22 cm high walls. The maze was mounted 74 cm above the

floor under fluorescent lighting (300 lux).

At the time of the test, rats were placed individually on the central portion of the

EPM apparatus, facing an open arm. They were allowed to explore the apparatus for 5

minutes and were recorded using a video camera placed 1m above the apparatus.

Videotapes were analysed using the Noldus Observer software (Observer 4.1;

Noldus Information Technology, Wageningen, Netherlands). Spatiotemporal and

behavioural parameters were recorded. Spatiotemporal measures comprised the

frequencies of total, open and closed arm entries; central platform entries; the percentage

of open-arm entries (open/total x 100); and the time spent in the open, closed or central

parts of the maze. Behavioural measures comprised frequency and duration scores for

rearing (vertical movements, animal sustained in posterior paws with anterior paws

against or not to the walls), head dipping (exploratory movement of head and shoulders

over the edge of the maze) and grooming (licking the paws, washing movements over the

head, fur licking, and tail and genitals cleaning).

2.6.2 Forced swim test

The forced swim test (FST) is a behavioural paradigm that is widely used as a

predictor of antidepressant activity in rodents (Porsolt et al., 1977). In this paradigm rats

are forced to swim in water for 15 minutes on the first day of testing, and for 5 minutes on

77

the second day of testing. On the second day of testing behavioural monitoring reveals

that the animal quickly adopts an immobile posture, and this is said to reflect a state of

“behavioural despair”. Subchronic and chronic antidepressant treatment largely attenuates

FSH induced immobility (Borsini and Meli, 1988).

The FST procedure used was similar to the one described by Porsolt et al. (1977).

At the time of test rats were placed into a glass cylinder (19 cm diameter, 40 cm deep) of

22±1ºC water filled to a depth of 30 cm for 15 min of pre-test, under fluorescent lighting.

Twenty-four hours later rats were again placed into the water for 5 min. Activity during pre-

test and second swim test was video-recorded for subsequent behavioural analysis.

Videotapes analysed using the Noldus Observer software (Observer 4.1 Noldus

Information Technology, Wageningen, Netherlands) and the following behaviour

categories were analyzed: immobility, struggling, diving, as well as right and left rotation.

The data was collected for frequency, duration and latency. Immobility was defined as

lack of movement of three paws or lack of movement of all four paws. Struggling was

defined as vigorous movement of all four paws, with the rat in a relatively upright position.

Diving was defined as under water swim.

2.6.3. Aggressiveness test

The aggressiveness test was conducted during the dark phase of the diurnal cycle,

utilizing the procedures from the resident-intruder paradigm as described by (Miczek,

1979). The resident-intruder paradigm is used to evaluate the aggressive behaviour of

the resident rat face to an intruder rat, that strongly resembles the natural patterns of wild

rat to establish and defend their territory (Koolhaas et al., 1980).

The experimental animal was always the resident and the intruder was an

unfamiliar, non-experimental age-matched male rat and with similar weight. The resident

rat, in its home cage, was allowed to habituate to the room test for 40 minutes, following

which an unfamiliar intruder was placed in its home cage and their behaviour was

videotaped for 10 minutes. The recording area was illuminated by red light illumination.

Behaviour of the resident was recorded and later analysed. Frequency, duration and

latency data were collected for the following behaviours divided in to three categories: (1)

offensive behaviours that included attack, bite, pinning, wrestling, chasing and aggressive

grooming; (2) defensive behaviours that included flight, avoidance of contact and

submissive-supine posture; (3) social investigation behaviours including sniffing,

anogenital sniffing, and approach behaviour. Attack was considered when the

experimental subject lunges at the partner with its forepaws extended outward; pinning

78

was considered when the resident subject stands over the exposed ventral area of the

partner pressing it against the floor; wrestling was considered when the two animals roll

and tumble with one another; chasing was considered when the experimental animal

rapidly pursues the intruder; aggressive grooming was consider when vigorous grooming

of the immobile partner using the teeth and the forepaws; flight was considered when the

experimental animal were pursued by the intruder; supine posture was considered when

the intruder stands over the exposed ventral areas of the resident animals pressing it

against the floor; avoidance of contact was considered when the resident move away from

the intruder; anogenital sniffing was considered when the resident sniffs the anogenital

region of the intruder; approach behaviour was considered when the resident move in to

the intruder direction.

2.7. Statistical analysis

Data were analyzed by appropriate statistical test (reported in more detail in the

following chapters). Statistical analyses were performed using the software SPSS 14.0

(SPSS INC. Chicago, Illinois, USA). Differences were considered to be statistically

significant when p<0.05. All data are expressed as the mean and standard error of the

mean (S.E.M.).

79

2.8. References

Ali SF, David SN, Newport GD (1993) Age-related susceptibility to MPTP-induced neurotoxicity in mice. Neurotoxicology 14:29-34.

Black YD, Maclaren FR, Naydenov AV, Carlezon WA, Jr., Baxter MG, Konradi C (2006) Altered attention and prefrontal cortex gene expression in rats after binge-like exposure to cocaine during adolescence. J Neurosci 26:9656-9665.

Borsini F, Meli A (1988) Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology (Berl) 94:147-160.

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254.

Carroll ME, Morgan AD, Lynch WJ, Campbell UC, Dess NK (2002) Intravenous cocaine and heroin self-administration in rats selectively bred for differential saccharin intake: phenotype and sex differences. Psychopharmacology (Berl) 161:304-313.


de Jong IE, Oitzl MS, de Kloet ER (2007) Adrenalectomy prevents behavioural sensitisation of mice to cocaine in a genotype-dependent manner. Behav Brain Res 177:329-339.

Douglas LA, Varlinskaya EI, Spear LP (2003) Novel-object place conditioning in adolescent and adult male and female rats: effects of social isolation. Physiol Behav 80:317-325.

Douglas LA, Varlinskaya EI, Spear LP (2004) Rewarding properties of social interactions in adolescent and adult male and female rats: impact of social versus isolate housing of subjects and partners. Dev Psychobiol 45:153-162.

Evans AM (1986) Age at puberty and first litter size in early and late paired rats. Biol Reprod 34:322-326.


Gawin FH (1991) Cocaine addiction: psychology and neurophysiology. Science 251:1580-1586.

Handley SL, Mithani S (1984) Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of 'fear'-motivated behaviour. Naunyn Schmiedebergs Arch Pharmacol 327:1-5.

Koob GF, Moal ML (1997) Drug abuse: hedonic homeostatic dysregulation. Science 278:52-58.

Koolhaas JM, Schuurman T, Wiepkema PR (1980) The organization of intraspecific agonistic behaviour in the rat. Prog Neurobiol 15:247-268.

Korenbrot CC, Huhtaniemi IT, Weiner RI (1977) Preputial separation as an external sign of pubertal development in the male rat. Biol Reprod 17:298-303.




Miczek KA (1979) A new test for aggression in rats without aversive stimulation: differential effects of d-amphetamine and cocaine. Psychopharmacology (Berl) 60:253-259.

Montgomery KC (1955) The relation between fear induced by novel stimulation and exploratory behavior. J Comp Physiol Psychol 48:254-260.

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Nestler EJ, Aghajanian GK (1997) Molecular and cellular basis of addiction. Science 278:58-63.

Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, Fourth Edition. Academic Press, San Diego.

Porrino LJ, Smith HR, Nader MA, Beveridge TJ (2007) The effects of cocaine: a shifting target over the course of addiction. Prog Neuropsychopharmacol Biol Psychiatry 31:1593-1600.

Porsolt RD, Le Pichon M, Jalfre M (1977) Depression: a new animal model sensitive to antidepressant treatments. Nature 266:730-732.

Sarnyai Z, Dhabhar FS, McEwen BS, Kreek MJ (1998) Neuroendocrine-related effects of long-term, 'binge' cocaine administration: Diminished individual differences in stress-induced corticosterone response. Neuroendocrinology 68:334-344.

Schlussman S, Nyberg F, Kreek M (2002) The effects of drug abuse on the stress responsive hypothalamic-pituitary-adrenal axis and the dopaminergic and endogenous opioid systems. Acta Psychiatr Scand Suppl:121-124.

Schlussman S, Zhou Y, Bailey A, Ho A, Kreek M (2005) Steady-dose and escalating-dose "binge" administration of cocaine alter expression of behavioral stereotypy and striatal preprodynorphin mRNA levels in rats. Brain Research Bulletin 67:169-175.


Spear LP (1996) Assessment of the effects of developmental toxicants: pharmacological and stress vulnerability of offspring. NIDA Res Monogr 164:125-145.


Stansfield KH, Kirstein CL (2006) Effects of novelty on behavior in the adolescent and adult rat. Dev Psychobiol 48:10-15.

Tidey JW, Miczek KA (1997) Acquisition of cocaine self-administration after social stress: role of accumbens dopamine. Psychopharmacology (Berl) 130:203-212.


Vorhees CV (1995) A review of developmental exposure models for CNS stimulants: cocaine In: Mothers, babies and cocaine: the role of toxins in development (Lewis, Bendersky, eds), pp 71-94. Hillsdale: Lawrence Eribaum Associates.

Willner P (1997) Animal models of addiction. Human Psychopharmacology-Clinical and Experimental 12:S59-S68.

Wise RA (2002) Brain reward circuitry: insights from unsensed incentives. Neuron 36:229-240.

Zhou Y, Spangler R, Ho A, Jeanne Kreek M (2001) Hypothalamic CRH mRNA levels are differentially modulated by repeated 'binge' cocaine with or without D(1) dopamine receptor blockade. Brain Res Mol Brain Res 94:112-118.


Zhou Y, Bendor J, Yuferov V, Schlussman S, Ho A, Kreek M (2005) Amygdalar vasopressin mRNA increases in acute cocaine withdrawal: Evidence for opioid receptor modulation. Neuroscience 134:1391-1397.

81

Chapter 3

Short-term Effects of Cocaine Exposure throughout Adolescence in the Rat

3.1. Introduction

Although adolescents represent a risk group regarding drug abuse and addiction,

experimental research evaluating the effects of psychostimulants has been mostly

focused on the assessment of adult animals. In fact, previous studies have shown that

most elicit drug use begins in adolescence (Estroff et al., 1989; Chen and Kandel, 1995),

that adolescents are more likely to progress from use to dependence than adults (Chen et

al., 1997) and do so much faster (Clark et al., 1998).

Adolescence is an ontogenetic transition to adulthood during which the brain and

hormonal systems are still undergoing many complex changes (Spear, 2000). Disruption

of the development occurring at this stage may increase the probability of developing

psychopathologies such as drug addiction (reviewed by Spear, 2000).Therefore, there is a

growing interest in assessing the influence that drug use can have on the developing

adolescent.

It have been reported that adolescents differ from adults in their hormonal,

neurochemical and behavioural response to psychostimulants (e.g., Adriani and Laviola,

2000; Collins and Izenwasser, 2002).

Based on evidence from behavioural models of drug abuse and addiction, such as

locomotor sensitization, conditioned place preference, and self-administration,

adolescents appear to be more vulnerable to the rewarding properties and detrimental

effects on the nervous system of various drugs (see reviews by Tirelli et al., 2003; Leslie

et al., 2004; Barron et al., 2005).

The work described in this chapter was performed to evaluate the short-term

effects of chronic cocaine exposure during the adolescence period upon the activity of the

HPA and the HPG axes of both male and female Wistar rats, by assessing the hormonal

plasma levels 30 minutes and 1 day after the last administration of cocaine. The effects of

cocaine exposure were also evaluated upon the dopaminergic and serotonergic systems

by assessing the monoamines levels in brain regions known to be involved in the drug

addiction processes.

It was shown that cocaine abuse and withdrawal is linked to the development of

depressive and/or anxiety-like symptoms (Gawin, 1991; Markou et al., 1992). Moreover,

severe anxiety and depression provide part of the negative reinforcement associated with

cocaine dependence and are important motivational factors for relapse and maintenance

of repetitive cycles of cocaine abuse (Gawin et al., 1989; Markou et al., 1992; Goeders et

al., 1993; Shaham et al., 2000; Shalev et al., 2002).

85

Therefore, it was also our goal to evaluate the short-term effects of chronic cocaine

exposure during adolescence period on the behavioural strategies adopted to cope with

an environmental anxiogenic situation, by submitting the animals, 2 days after the last

administration, to the EPM. The presence of depressive-like symptoms were also verified

3 days after withdrawal from chronic cocaine exposure during adolescence, by assessing

the behaviour of both male and female adolescent rats in a FST. The effects of cocaine

treatment on the subsequent alterations on brain neurotransmitters and in the HPA axis

activity induced by the EPM or the FST were analysed.

86

3.2. Material and Methods

3.2.1. Animals

Using the animal paradigm described in Chapter 2, we have obtained animals

chronically exposed to cocaine during adolescence that were used in the following

experiments. Briefly, on PND 30 animals were housed individually, and were randomly

assigned to the different experimental groups. Only one male and one female from each

litter were used in each experimental group. Rats were treated following a binge pattern

administration of cocaine, each day being daily injected 3 times intraperitonealy with

15mg/kg of body weight cocaine hydrochloride dissolved in 0.9% NaCl solution from PND

35 to PND 50. The daily injections were administered at hourly intervals, between 9:00

and 11:00 a.m., in a volume of 1 mL/kg. Controls were given an isovolumetric 0.9% NaCl

solution following the same experimental protocol. During the entire period of drug

administration animals were daily weighed. At least 6 animals were used in each

experimental group.

3.2.2. Behavioural tests

3.2.2.1. Elevated plus maze test

On PND 52, 2 days after cocaine withdrawal, both male and female Wistar rats

were tested in an EPM apparatus as described in Chapter 2. Spatiotemporal and

behavioural parameters were registered at the time of behaviour analyses. Spatiotemporal

measures comprised the frequencies of total, open and closed arm entries and central

platform entries, as well as the time spent in the open, closed and central part of the

maze. Behavioural measures comprised frequency and duration scores for rearing and

head dipping and grooming.

3.2.2.2. Forced swim test

Starting on PND 52, 2 days after withdrawal, both male and female Wistar rats

performed the FST as described in Chapter 2. Behavioural activity was analysed and data

collected for frequency, duration and latency of immobility, struggling and diving, and

frequency and latency of right and left rotation.

87

3.2.3. Blood and tissue collection

Thirty minutes and 1 day after the last administration of cocaine, and after the

behavioural tests, animals were sacrificed by decapitation and trunk blood was collected

in lithium heparin cover tubes and centrifuged at 1600g for 15 minutes; the obtained

plasma was kept at -70ºC until hormonal assays were conducted. Brains were also rapidly

removed and SN-VTA, Nac, dorsal striatum, prefrontal cortex, amygdala, hippocampus,

dorsal raphe nucleus, cerebellum and a hypothalamic section containing the

suprachiasmatic, pre-optic and paraventricular nuclei were dissected on ice. Tissue

samples were frozen by immersion in 2-methylbutane cooled over dry ice and stored at -

70ºC until used for further analyses. Thirty minutes and 1 day after the last administration

of cocaine gonads and adrenal glands were collected and weighed, and the data obtained

was recorded.

3.2.4. Hormone plasma levels determinations

The levels of progesterone, estradiol, testosterone, corticosterone, and ACTH

were measured on plasma obtained 30 minutes and 1 day after the last administration.

The levels of corticosterone were also measured on the plasma obtained after the

behavioural tests. All hormones plasma levels were measured by enzyme immunoassay,

using commercially available assays.

The progesterone, estradiol, testosterone and corticosterone basal plasma levels

were measured using assays provided by Cayman Chemical Company (Ann Arbor, MI,

USA). For progesterone, the assay sensitivity was 0.012 ng/mL, and the intra- and inter-

assay coefficients of variation were less 8% and 12%, respectively. For estradiol, the

assay sensitivity was 0.004 ng/mL, and the intra- and inter-assay coefficients of variation

were less 10% and 12%, respectively. For testosterone, the assay sensitivity was 0.004

ng/mL, and the intra- and inter-assay coefficients of variation were less 10% and 15%,

respectively. For corticosterone, the assay sensitivity was 0.04 ng/mL, and the intra- and

inter-assay coefficients of variation were less than 7% and 9%, respectively. The ACTH

plasma levels were measured using an assay provided by Phoenix Pharmaceuticals, Inc.

(Belmont, California, USA). The assay sensitivity was 0.18 ng/mL, and the intra- and inter-

assay coefficients of variation were less than 5 % and 14%, respectively.

Corticosterone plasma levels after the behavioural tests were measured using a kit

provided by Immunodiagnostic Systems Ltd (Boldon, UK). Assay sensitivity was 0.23

ng/mL. The intra- and inter-assay coefficients of variation were less than 3.1% and 7.2%,

respectively.

88

3.2.5. Neurochemical determinations

Concentrations of DA and 5-HT and their metabolites, 5-HIAA, DOPAC, and HVA ,

30 min. and 1 day after last administration of cocaine, were quantified by HPLC-EC, in the

dissected brain regions (SN-VTA, Nac, dorsal striatum, prefrontal cortex, amygdala,

hippocampus, dorsal raphe nucleus, cerebellum and hypothalamus), as described in

Chapter 2. After each behavioural test, levels of DA and 5-HT and their metabolites were

determined in the SN-VTA, dorsal raphe nucleus, amygdala, hippocampus, prefrontal

cortex and hypothalamus. Concentrations are expressed in ng/mg protein. The ratios of

DOPAC to DA, HVA to DA and DOPAC and HVA to DA were determined for each animal

and used as indexes of DA turnover rate, whereas the ratio of 5-HIAA to 5-HT was used

as an index of 5-HT turnover rate.

3.2.6. Statistical analysis

A 2-way analysis of variance (ANOVA) with treatment (cocaine or saline) and sex

as between-subject fixed-factors with repeated measures was used to evaluate body

weight evolution during the experimental period until acute withdrawal. Student’s t-test

was used as a post-hoc test to analyse statistical significant differences.

Adrenal and gonadal weights were converted in percentage of body weight, and

adrenal data was analyzed by a 3-way ANOVA with time point of assessment (30 minutes

and 1 day), treatment and sex as between-subject fixed-factors. Gonadal data was

analyzed by a 2-way ANOVA (time point of assessment (30 minutes and 1 day) x

treatment), gender separately.

For the hormonal and neurochemical data obtained 30 minutes and 1 day after the

last administration of cocaine and after each behavioural test, statistical significance

between groups was assessed using the non-parametric Mann-Whitney U Test.

The EPM and FST behaviour data were analysed by a 2-way ANOVA with

treatment (cocaine or saline) and sex as between-subject fixed-factors and behavioural

measurements as the dependent variable. Student’s t-test was used as a post-hoc test to

analyse statistical significant differences. Whenever data did not pass normality and

homogeneity of variance tests the non-parametric Mann-Whitney U Test was used.

Differences were considered to be statistically significant when p<0.05. All data

were expressed as the mean plus S.E.M.. Statistical analyses were performed using the

software SPSS 14.0 (SPSS INC. Chicago, Illinois, USA).

89

3.3. Results

3.3.1. Analyses performed 30 minutes and 1 day after the last administration of cocaine

3.3.1.1. Biometric measures

Evolution of body weight. Cocaine-treated and control animals were weight

everyday from PND 35 to PND 52, and data was analyzed by a 2-way ANOVA (treatment

x sex) with repeated measures. Weight gain was statistically different between male and

female animals [F(1,37)= 155.627; p<0.001]), with post-hoc comparisons using Student’s

t-test showing that, except for PND 36, the daily weight gain was higher in males than in

females (figure 3.3.1.1.1). The statistical analysis revealed no significant effects of the

cocaine treatment on the weight of male and male.

0

30

60

90

120

150

35 37 39 41 43 45 47 49 51

PND

Wei

ght g

ain

(g)

saline male cocaine male saline female cocaine female

Figure 3.3.1.1.1- Effects of chronic cocaine exposure on body weight evolution during experimental perio until

acute withdrawal. Values are mean ± S.E.M.. Saline male, n=11; cocaine male, n=11; saline female, n=12;

cocaine female, n=8. PND- postnatal day. &, sex effect; p<0.001.

Adrenal weight. Adrenals were collected 30 minutes and 1 day after the last

administration of cocaine and their weight were recorded. The weight data was converted

in percentage of body weight and was analyzed by a 3-way ANOVA (time point of

assessment (30 minutes or 1 day) x treatment x sex). The statistical analysis revealed a

main effect of sex [F(1,37)= 67.195; p<0.001] in the adrenal weight (figure 3.3.1.1.2); with

female rats showing higher values than male ones. No treatment effects were found in

both male and female rats and the adrenal weight also did not differ 30 minutes and 1 day

after the last administration.

&

90

Gonadal weight. Testes and ovaries were collected 30 minutes and 1 day after

the last administration of cocaine and their weight were recorded. The percentage of body

weight was calculated and data was analyzed by a 2-way ANOVA (age x treatment). The

statistical analysis revealed no significant effects of the cocaine treatment on the weight of

male and female gonads, both when assessed 30 min or 1 day after the last

administration (figure 3.3.1.1.3). There were also no differences between time points of

assessment in both male and female gonadal weights.

Adrenal glands

0

0.02

0.04

0.06

30 min. 1 day

% o

f bod

y w

eigh

t


Figure 3.3.1.1.2- Effects of chronic cocaine exposure throughout adolescence on adrenal weight of male and

female rats assessed 30 minutes and 1 day after the last administration. Each column represents the mean +

S.E.M. for a 6 animals per group.

A Male gonads B Female gonads

0

0.4

0.8

1.2

30 min. 1 day

% o

f bod

y w

eigh

t

saline male cocaine male

0

0.03

0.06

0.09

30 min. 1 day

% o

f bod

y w

eigh

t

saline female cocaine female 3.3.1.1.3- Effects of chronic cocaine exposure throughout adolescence on (A) male gonads and (B) females

gonads weights assessed 30 minutes and 1 day after the last administration of cocaine. Each column

represents the mean + S.E.M. for a 6 animals per group.

91

3.3.1.2. Hormones plasma levels

The levels of hormones were measured on plasma obtained 30 minutes and 1 day

after the last administration of cocaine. The data was analyzed by the non-parametric

Mann-Whitney U Test.

ACTH. Cocaine-treated males presented increased levels of ACTH 30 minutes

after the last administration, when compared with controls [Z=-2.402 (2-tailed) p<0.02]

(figure 3.3.1.2.1-A). This difference was no longer observed at 1 day after withdrawal.

Both 30 minutes and 1 day after the last administration there were no significant

differences in ACTH levels between cocaine-treated and control females. Between 30

minutes and 1 day after the last administration, control males presented an increase in

ACTH plasma levels [Z=-2.242 (2-tailed) p<0.05].

Corticosterone. No significant differences were found on the plasma levels of

corticosterone between the experimental groups (figure 3.1.2.1-B).

Testosterone. In the male group, 30 minutes after the last administration, cocaine-

treated males presented lower levels of testosterone [Z=-2.242 (2-tailed) p<0.05] (figure

3.3.1.2.2-A). This difference was no longer observed 1 day after withdrawal. In the female

group (figure 3.3.1.1.5-B), there were no differences in the levels of testosterone between

cocaine-treated and control females both at 30 minutes and 1 day after the last

administration. However, there was a decrease in testosterone levels in cocaine-treated

females between the two assessed time points [Z=-2.402 (2-tailed) p<0.02].

Progesterone. No significant differences were found on the plasma levels of

progesterone between cocaine-treated rats and their respective controls and between

assessment time points. However, a trend for increased progesterone levels was found in

cocaine-treated males 30 minutes after the last administration [Z=-1.922 (2-tailed)

p=0.055] (figure 3.3.1.2.2- C).

Estradiol. There were no significant differences in plasma levels of estradiol

between cocaine-treated and control females (figure 3.3.1.1.5-E) at the two assessed time

points, and there were also no differences in the plasma levels between 30 minutes and 1

day after the last administration, both in cocaine-treated and control animals.

92

A

0

2

4

6

8

30 min. 1 day

AC

TH le

vels

(ng/

mL)


B

0

40

80

120

160

30 min. 1 day

Cor

ticos

tero

ne le

vels

(ng/

mL)


Figure 3.3.1.2.1- Effects of chronic cocaine exposure throughout adolescence on (A) ACTH and (B)

corticosterone plasma levels assessed 30 minutes and 1 day after the last administration of cocaine. Each

column represents the mean + S.E.M. for a 6 animals per group. ACTH- Adrenocorticotropic hormone.

Columns with the same letter are significantly different from each other. a,b p<0.05.

a,b

b a

93

A B

0.0

0.6

1.2

1.8

30 min. 1 dayTest

oste

rone

leve

ls

(ng/

mL)


0.0

0.1

0.2

0.3

30 min. 1 dayTest

oste

rone

leve

ls

(ng/

mL)

saline female cocaine female

C D

0.0

0.2

0.4

0.6

30 min. 1 dayProg

este

rone

leve

ls

(ng/

mL)


0

50

100

150

30 min. 1 dayProg

este

rone

leve

ls

(ng/

mL)


E

0

15

30

45

30 min. 1 day

Estra

diol

leve

ls

(pg/

mL)


Figure 3.3.1.2.2- Effects of chronic cocaine exposure throughout adolescence on plasma levels of

testosterone in (A) males and (B) females, progesterone in (C) males and (D) females and estradiol in (E)

females, assessed 30 minutes and 1 day after the last administration of cocaine. Each column represents the

mean + S.E.M. for a 6 animals per group. Columns with the same letter are significantly different from each

other. a p<0.05, b p<0.02.

a

a

b

b

94

3.3.1.3. Neurochemical determinations

The levels of DA, 5-HT and their metabolites, DOPAC, HVA and 5-HIAA were

measured by HPLC-EC 30 minutes and 1 day after the end of the exposure period, in the

following brain regions: SN-VTA, Nac, dorsal striatum, prefrontal cortex, hippocampus,

amygdala, hypothalamus, dorsal raphe nucleus and cerebellum. These different brain

regions were selected based on their involvement in cocaine response. The data obtained

was analyzed by the non-parametric Mann-Whitney U Test.

Within the SN-VTA, 30 minutes after the last administration of cocaine, no

differences were found on the levels of DA, 5-HT and their metabolites between cocaine-

treated and control male rats, or between cocaine-treated and control female rats (figure

3.3.1.3.1). However, 30 minutes after the last administration, statistical analysis revealed

that DOPAC levels were higher in cocaine-treated males than in cocaine-treated females

[Z=-2.008 (2-tailed) p<0.05], and this effect was not observed in the control group.

Moreover, in cocaine exposed females the levels of DOPAC increased from 30 minutes to

1 day after the last administration of cocaine [Z=-2.082 (2-tailed) p<0.05]. At 1 day after

the end of the exposure period, the levels of DA, DOPAC and 5-HT in SN-VTA were

increased in cocaine-treated males as compared to control males (DA: [Z=-2.315 (2-

tailed) p<0.05]; DOPAC: [Z=-2.430 (2-tailed) p<0.02]; 5-HT: [Z=-2.083 (2-tailed) p<0.05]).

The DA turnover in the SN-VTA was analyzed, as given by the ratios of DOPAC+HVA to

DA and each one of its metabolites to DA (DOPAC/DA and HVA/DA), between cocaine-

treated and control males or females both 30 min and 1day after the end of the exposure

period and no statistical differences were found (table 3.3.1.3.1). However, in males the

DOPAC/DA ratio increased from 30 minutes to 1 day after the last administration in

cocaine-treated rats but not in controls [Z=-2.030 (2-tailed) p<0.05]. The 5-HT turnover

was assessed as the ratio of 5-HIAA to 5-HT and statistical analysis revealed no

differences between groups.

Within the Nac, no differences were detected in the levels of DA and 5-HT and

their metabolites between cocaine-treated rats and their respective controls at both time

points of analyses (figure 3.3.1.3.2). Also, no differences were found on both DA and 5-HT

turnover rates between the experimental groups (table 3.3.1.3.2).

95

SN-VTA

A

0

200400

600800

10001200

1400

DA DOPAC HVA DA DOPAC HVA

Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


30 min. 1 day

B

0

200

400

600

800

1000

1200

5-HT 5-HIAA 5-HT 5-HIAA

Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


30 min. 1 day

Figure 3.3.1.3.1- Effects of chronic cocaine exposure throughout adolescence on the (A) levels of DA, and its

metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the SN-VTA,

determined by HPLC-EC for male and female rats, 30 minutes and 1 day after the last administration. Each

column represents the mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for

at least 5 animals per group. Columns with the same letter are significantly different from each other. a,b,c,d,e

p<0.05.

a a,d

b b

c c

e e

d

96

Nucleus accumbens

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metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the Nac, determined

by HPLC-EC for male and female rats, 30 minutes and 1 day after the last administration. Each column

represents the mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for at least

6 animals per group.

97

Table 3.3.1.3.1- Turnover of DA and 5-HT, expressed as [DOPAC]/[DA], [HVA]/[DA], [DOPAC+HVA]/[DA] and

[5-HIAA]/[5-HT], in the SN-VTA, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day

Male Female Male Female

saline cocaine saline cocaine saline cocaine saline cocaine

DOPAC/DA 0.22±0.05

(n=6)

0.13±0.01

(n=5)

0.20±0.06

(n=6)

0.25±0.12

(n=6)

0.16±0.02

(n=8)

0.17±0.02

(n=7)

0.22±0.08

(n=6)

0.21±0.07

(n=6)

HVA/DA 0.06±0.02

(n=6)

0.06±0.01

(n=5)

0.14±0.10

(n=6)

0.15±0.09

(n=6)

0.08±0.02

(n=8)

0.07±0.02

(n=7)

0.05±0.01

(n=6)

0.06±0.01

(n=6)

(DOPAC+HVA)/DA 0.28±0.05

(n=6)

0.19±0.01

(n=5)

0.34±0.15

(n=6)

0.40±0.21

(n=6)

0.24±0.02

(n=8)

0.24±0.02

(n=7)

0.27±0.08

(n=6)

0.27±0.06

(n=6)

5-HIAA/5-HT 0.25±0.02

(n=6)

0.18±0.02

(n=5)

0.25±0.02

(n=6)

0.27±0.06

(n=6)

0.23±0.03

(n=8)

0.22±0.02

(n=7)

0.23±0.02

(n=6)

0.25±0.04

(n=6)

Data expressed as the mean value ± S.E.M.; n, number of animals per group. In the same line, values with

same letter are significantly different. DA- dopamine; DOPAC- 3,4-dihydroxyphenylacetic acid; HVA-

homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a p<0.05.


[5-HIAA]/[5-HT], in the Nac, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day



DOPAC/DA 0.28±0.06

(n=6)

0.24±0.03

(n=5)

0.25±0.04

(n=6)

0.19±0.02

(n=6)

0.24±0.02

(n=8)

0.29±0.04

(n=6)

0.33±0.04

(n=6)

0.27±0.05

(n=6)

HVA/DA 0.07±0.02

(n=6)

0.08±0.02

(n=6)

0.06±0.01

(n=6)

0.05±0.01

(n=6)

0.08±0.01

(n=8)

0.08±0.01

(n=6)

0.06±0.00

(n=6)

0.07±0.01

(n=6)


(n=6)

0.32±0.05

(n=6)

0.30±0.05

(n=6)

0.24±0.02

(n=6)

0.32±0.02

(n=8)

0.37±0.04

(n=6)

0.39±0.04

(n=6)

0.34±0.06

(n=6)

5-HIAA/5-HT 0.50±0.21

(n=6)

0.40±0.19

(n=6)

0.54±0.20

(n=6)

0.40±0.18

(n=6)

0.32±0.12

(n=8)

0.73±0.26

(n=6)

0.79±0.32

(n=6)

0.52±0.21

(n=6)

Data expressed as the mean value ± S.E.M.; n, number of animals per group. DA- dopamine; DOPAC- 3,4-

dihydroxyphenylacetic acid; HVA- homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid.

Within the dorsal striatum, no significant differences were detected in the levels of

DA and 5-HT and their metabolites between cocaine-treated animals and their respective

controls at both time points of analyses (figure 3.3.1.3.3). However, 1 day after the end of

the exposure period, the (DOPAC+HVA)/DA ratio in the dorsal striatum were higher in

cocaine-treated males than in control males [Z=-2.083 (2-tailed) p<0.05] (table 3.3.1.3.3).

Also, at this time point, the HVA/DA and (DOPAC+HVA)/DA ratios were higher in cocaine-

treated males than in cocaine-treated females, [Z=-2.286 (2-tailed) p<0.05] and [Z=-2.143

a a

98

(2-tailed) p<0.05], respectively. This sex effects was not observed in the control group. No

differences were found on 5-HT turnover between the experimental groups.

Dorsal striatum

A

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metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the dorsal striatum,



at least 6 animals per group.

99


[5-HIAA]/[5-HT], in the dorsal striatum, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day


saline cocaine saline cocaine saline cocaine saline Cocaine

DOPAC/DA 0.18±0.01

(n=7)

0.24±0.02

(n=6)

0.21±0.03

(n=6)

0.20±0.02

(n=6)

0.19±0.01

(n=8)

0.30±0.06

(n=7)

0.17±0.02

(n=6)

0.18±0.01

(n=6)

HVA/DA 0.09±0.01

(n=7)

0.12±0.02

(n=6)

0.08±0.01

(n=6)

0.11±0.01

(n=6)

0.09±0.01

(n=8)

0.12±0.01

(n=7)

0.07±0.01

(n=6)

0.07±0.01

(n=6)


(n=7)

0.35±0.04

(n=6)

0.29±0.04

(n=6)

0.30±0.02

(n=6)

0.28±0.01

(n=8)

0.42±0.07

(n=7)

0.24±0.03

(n=6)

0.26±0.02

(n=6)

5-HIAA/5-HT 2.14±1.05

(n=7)

1.73±1.10

(n=5)

1.62±1.15

(n=6)

2.17±1.11

(n=6)

1.16±0.76

(n=7)

1.21±0.83

(n=5)

1.57±1.11

(n=5)

2.52±1.13

(n=6)



homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a,b,c p<0.05.

Within the prefrontal cortex, no statistical significant differences were found on the

levels of monoamines and their metabolites or their turnover rates (figure 3.3.1.3.4 and

table 3.3.1.3.4).

Within the hippocampus, no statistical significant differences were observed on the

levels of DA and its metabolite DOPAC, and on the levels of 5-HT and its metabolite 5-

HIAA between cocaine exposed and control males, as well as between cocaine exposed

and control females, both 30 minutes and 1 day after the last administration (figure

3.3.1.3.5). Also, no differences were found on DA and 5-HT turnover rates between the

experimental groups (table 3.3.1.3.5).

Hippocampal levels of HVA were not detectable by HPLC-EC in a considerable

number of samples. Therefore, in this brain region, only the levels of the DA metabolite

DOPAC was considered for the analysis.

a a,c

b b

c

100

Prefrontal cortex

A

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Figure 3.3.1.3.4- Effects of chronic cocaine exposure throughout adolescence on (A) the levels of DA, and its

metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the prefrontal cortex,




101

Hippocampus

A

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5

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metabolites, DOPAC and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hippocampus determined

by HPLC-EC for male and female rats, 30 minutes and 1 day after the last administration. Each column



102


[5-HIAA]/[5-HT], in the prefrontal cortex, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day



DOPAC/DA 0.30±0.04

(n=7)

0.40±0.11

(n=5)

0.34±0.05

(n=6)

0.35±0.13

(n=5)

0.37±0.10

(n=8)

0.33±0.05

(n=7)

0.36±0.08

(n=6)

0.28±0.07

(n=6)

HVA/DA 0.61±0.09

(n=7)

0.54±0.14

(n=6)

0.51±0.08

(n=6)

0.58±0.12

(n=5)

0.80±0.14

(n=8)

0.58±0.09

(n=7)

0.45±0.09

(n=6)

0.47±0.07

(n=6)


(n=7)

0.94±0.24

(n=6)

0.86±0.11

(n=6)

0.93±0.25

(n=5)

1.16±0.21

(n=8)

0.91±0.11

(n=7)

0.82±0.14

(n=6)

0.74±0.12

(n=6)

5-HIAA/5-HT 0.11±0.03

(n=7)

0.12±0.03

(n=6)

0.08±0.02

(n=6)

0.11±0.03

(n=5)

0.11±0.03

(n=8)

0.10±0.02

(n=7)

0.11±0.03

(n=6)

0.07±0.02

(n=6)



Table 3.3.1.3.5- Turnover of DA and 5-HT, expressed as [DOPAC]/[DA] and [5-HIAA]/[5-HT], in the

hippocampus, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day



DOPAC/DA 0.54±0.14

(n=6)

0.83±0.27

(n=6)

1.09±0.44

(n=6)

0.67±0.12

(n=6)

0.97±0.50

(n=8)

0.75±0.23

(n=7)

0.59±0.11

(n=6)

1.10±0.29

(n=6)

5-HIAA/5-HT 0.25±0.07

(n=6)

0.21±0.05

(n=6)

0.38±0.14

(n=6)

0.26±0.08

(n=6)

0.30±0.05

(n=8)

0.31±0.04

(n=7)

0.26±0.03

(n=6)

0.25±0.07

(n=6)


dihydroxyphenylacetic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid.

Within the amygdala, 1 day after withdrawal, the levels of DA were higher in

cocaine exposed females as compared to control females [Z=-2.082 (2-tailed) p<0.05]

(figure 3.3.1.3.6). At 30 minutes after the last administration, control males displayed

higher DOPAC/DA ratio as compared to control females [Z=-2.082 (2-tailed) p<0.05]

(table 3.3.1.3.6). Furthermore, at this time point of analysis, control males also displayed

higher levels of DOPAC as compared to control females that almost reached significance

[Z=-1.922 (2-tailed) p=0.055]. The statistical analysis also revealed, in control males, a

significant decrease on the levels of HVA from 30 minutes to 1 day after the last

administration [Z=-2.000 (2-tailed) p<0.05], and a decrease on the levels of DOPAC that

almost reached the statistical significance [Z=-1.936 (2-tailed) p=0.053], that were not

103

observed in treated animals. No differences were found on the levels of 5-HT and its

metabolite or its turnover between the experimental groups.

Amygdala

A

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metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the amygdala,



at least 6 animals per group. Columns with the same letter are significantly different from each other. a,b

p<0.05.

b

b

a a

104


[5-HIAA]/[5-HT], in the amygdala, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day



DOPAC/DA 0.37±0.06

(n=6)

0.26±0.06

(n=5)

0.19±0.05

(n=6)

0.33±0.10

(n=6)

0.33±0.06

(n=8)

0.31±0.08

(n=7)

0.38±0.09

(n=6)

0.23±0.06

(n=6)

HVA/DA 0.18±0.06

(n=6)

0.26±0.13

(n=6)

0.14±0.04

(n=6)

0.08±0.03

(n=6)

0.06±0.10

(n=7)

0.11±0.05

(n=7)

0.08±0.03

(n=6)

0.06±0.02

(n=6)


(n=6)

0.47±0.13

(n=6)

0.33±0.08

(n=6)

0.41±0.10

(n=6)

0.39±0.07

(n=7)

0.42±0.11

(n=7)

0.45±0.12

(n=6)

0.30±0.06

(n=6)

5-HIAA/5-HT 0.64±0.22

(n=6)

0.48±0.15

(n=6)

0.38±0.14

(n=6)

0.68±0.30

(n=6)

0.39±0.09

(n=8)

0.54±0.19

(n=7)

0.39±0.09

(n=6)

0.44±0.10

(n=6)




Within the hypothalamus, the levels of 5-HIAA in cocaine exposed males

increased from 30 minutes to 1 day after the last administration [Z=-2.082 (2-tailed)

p=0.037] (figure 3.3.1.3.7). Moreover, 30 minutes after the last administration, cocaine

exposed males displayed lower 5-HIAA/5-HT ratio [Z=-2.714 (2-tailed) p<0.01] and an

increase in this ratio from 30 minutes to 1 day after the last administration [Z=-2.562 (2-

tailed) p=0.01] (table 3.3.1.3.7). No differences were found in this brain region on DA, its

metabolites or its turnover, between the experimental groups.

a a

105

Hypothalamus

A


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metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hypothalamus,



at least 5 animals per group. Columns with the same letter are significantly different from each other. a p<0.05

a a

30 min. 1 day


106


[5-HIAA]/[5-HT], in the hypothalamus, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day



DOPAC/DA 0.24±0.07

(n=7)

0.26±0.05

(n=6)

0.32±0.06

(n=6)

0.26±0.06

(n=6)

0.28±0.06

(n=7)

0.28±0.04

(n=6)

0.23±0.05

(n=5)

0.33±0.09

(n=5)

HVA/DA 0.03±0.01

(n=7)

0.03±0.01

(n=6)

0.03±0.01

(n=6)

0.04±0.01

(n=6)

0.03±0.01

(n=7)

0.06±0.03

(n=6)

0.01±0.00

(n=5)

0.03±0.02

(n=5)


(n=7)

0.30±0.05

(n=6)

0.35±0.06

(n=6)

0.30±0.06

(n=6)

0.30±0.07

(n=7)

0.34±0.06

(n=6)

0.24±0.05

(n=5)

0.36±0.09

(n=5)

5-HIAA/5-HT 0.27±0.06

(n=7)

0.14±0.01

(n=6)

0.34±0.07

(n=6)

0.25±0.10

(n=6)

0.23±0.04

(n=7)

0.36±0.06

(n=6)

0.23±0.04

(n=5)

0.41±0.24

(n=5)



homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a p<0.01, b p<0.02.

Within the dorsal raphe nucleus, statistical analysis revealed that there were no

significant differences on the levels of DA and its metabolites, DOPAC and HVA between

cocaine exposed and control male and female rats, both 30 minutes and 1 day after the

last administration (figure 3.3.1.3.8). At 30 minutes after the last administration, cocaine

exposed females displayed higher levels of 5-HT as compared to control females that

almost reached significance [Z=-1.922 (2-tailed) p=0.055]. And the levels of 5-HT

decreased in cocaine exposed females from 30 minutes to 1 day after the last

administration of cocaine [Z=-2.242 (2-tailed) p<0.05]. Also, in cocaine exposed females it

was observed an increase in the 5-HIAA/5-HT ratio, from 30 minutes to 1 day after the last

administration of cocaine [Z=-2.402 (2-tailed) p<0.02] (table 3.3.1.3.8). Statistical analysis

also revealed that 30 minutes after the last administration, the 5-HIAA/5-HT ratio was

lower in cocaine-treated males as compared to control males [Z=-2.402 (2-tailed) p<0.02]

(table 3.1.3.8). And, although not statistically significant, cocaine exposed male rats

presented an increase on the levels of 5-HIAA from 30 minutes to 1 day after the last

administration of cocaine [Z=-1.922 (2-tailed) p=0.055]. No differences were found in the

DA turnover in this brain area.

a a,b b

107

Dorsal raphe nucleus

A

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metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the dorsal raphe

nucleus, determined by HPLC-EC for male and female rats, 30 minutes and 1 day after the last administration.

Each column represents the mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of

protein, for at least 6 animals per group. Columns with the same letter are significantly different from each

other. a p<0.05.

a

a

108


[5-HIAA]/[5-HT], in the dorsal raphe nucleus, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day



DOPAC/DA 0.85±0.23

(n=6)

0.70±0.22

(n=6)

1.03±0.13

(n=6)

1.12±0.27

(n=6)

1.16±0.26

(n=6)

0.75±0.17

(n=6)

1.10±0.24

(n=6)

1.00±0.26

(n=6)

HVA/DA 0.16±0.06

(n=6)

0.08±0.02

(n=6)

0.12±0.03

(n=6)

0.09±0.02

(n=6)

0.10±0.03

(n=6)

0.10±0.02

(n=6)

0.06±0.02

(n=6)

0.15±0.05

(n=6)


(n=6)

0.78±0.22

(n=6)

1.15±0.11

(n=6)

1.21±0.27

(n=6)

1.23±0.28

(n=6)

0.85±0.18

(n=6)

1.17±0.25

(n=6)

1.15±0.28

(n=6)

5-HIAA/5-HT 0.28±0.02

(n=6)

0.20±0.02

(n=6)

0.26±0.02

(n=6)

0.20±0.02

(n=6)

0.34±0.05

(n=6)

0.27±0.02

(n=6)

0.26±0.02

(n=6)

3.40±3.11

(n=6)



homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a,b p<0.02.

Within the cerebellum, statistical analysis revealed that there were no significant

difference on the levels of DA and its metabolites, DOPAC and HVA, and on the levels of

5-HT and 5-HIAA between cocaine exposed and control male and female rats, both at 30

minutes or 1 day after the last administration (figure 3.3.1.3.9). However, 1 day after the

last administration, the levels of 5-HT were lower in cocaine-treated males than in

cocaine-treated females [Z=-2.000 (2-tailed) p<0.05], and, although not statistically

significant, there was a trend to this same sex effect in saline-treated animals [Z=-1.936

(2-tailed) p=0.053]. In cocaine exposed males the DA and HVA levels decreased from 30

minutes to 1 day after the last administration (DA: [Z=-2.030 (2-tailed) p<0.05]; HVA: [Z=-

2.030 (2-tailed) p<0.05]). No differences were found both on DA and 5-HT turnover rates

between the experimental groups (table 3.3.1.3.9).

a a b b

109

Cerebellum

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30 min. 1 day


metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the cerebellum,



at least 5 animals per group. Columns with the same letter are significantly different from each other. a,b,c

p<0.05.

c

c

a

a b b

110


[5-HIAA]/[5-HT], in the cerebellum, 30 minutes and 1 day after the end of cocaine exposure period.

30 min. 1 day



DOPAC/DA 0.83±0.24

(n=7)

0.62±0.20

(n=5)

0.75±0.16

(n=6)

0.72±0.13

(n=6)

0.70±0.18

(n=8)

0.90±0.23

(n=7)

0.77±0.14

(n=6)

0.79±0.22

(n=6)

HVA/DA 1.05±0.21

(n=7)

0.74±0.17

(n=5)

0.62±0.19

(n=6)

0.82±0.19

(n=6)

0.68±0.13

(n=8)

0.95±0.22

(n=7)

0.90±0.22

(n=6)

0.93±0.26

(n=6)


(n=7)

1.36±0.23

(n=5)

1.38±0.22

(n=6)

1.54±0.26

(n=6)

1.38±0.14

(n=8)

1.85±0.33

(n=7)

1.67±0.23

(n=6)

1.72±0.34

(n=6)

5-HIAA/5-HT 0.23±0.03

(n=7)

0.18±0.05

(n=5)

0.18±0.02

(n=6)

0.18±0.01

(n=6)

0.22±0.03

(n=8)

0.48±0.25

(n=7)

0.15±0.01

(n=6)

0.15±0.02

(n=6)



3.3.2. Behavioural studies and associated measures

3.3.2.1. Anxiety-like behaviour

Elevated plus maze test. On the PND 52, 2 days after the last administration of

cocaine, animals were tested in the EPM apparatus, in a five minute session. EPM

behavioural data was analysed by a 2-way ANOVA (sex x treatment). Differences

between specific groups were determined by post-hoc analyses using the Student’s t-test.

The behavioural category, risk-like behaviour, was analysed by the non-parametric Mann-

Whitney U Test.

Statistical analyses revealed a main effect of sex [F(1,29)=5.488; p<0.05], and an

interaction between sex and treatment [F(1,29)=4.810; p<0.05] in the number of entries

into the open arms. Males presented lower frequency of open arms entries than females

and post-hoc comparisons indicate that, within the cocaine group, males had lower

frequency than females [t(15)=-3.447; p<0.005] (figure 3.3.2.1.1 A). ANOVA results also

revealed an interaction between sex and treatment for the number of closed arms entries

[F(1,29)=5.517; p<0.03], with cocaine-treated males showing a trend towards decreased

entries into the closed arms as compared with cocaine-treated females [t(15)=-2.112;

p=0.052] (figure 3.3.2.1.1 C). For the total arm entries, statistical analysis revealed an

interaction between sex and treatment [F(1,29)=6.955; p<0.02], with a post-hoc

comparison showing that there was a decrease in the number of entries of cocaine-

treated males when compared with cocaine-treated females [t(15)=-3.468; p<0.005]

(figure 3.3.2.1.1 D).

111

No significant differences were found in the time spent in open and closed arms

between both cocaine- and saline-treated male and female rats (table 3.3.2.2.1).

ANOVA revealed an interaction between sex and treatment in the frequency of

entries into central platform [F(1,29)=7.340; p<0.02]. Within the cocaine-exposed group

there was a decrease in entries in males [t(15)=-3.570; p<0.005] (figure 3.3.2.1.3 A).

Within the male group, cocaine treatment decrease, although no significantly, the

frequency of central platform entries, [t(15)=-2.052; p=0.058] (figure 3.3.2.1.3 A).

Statistical analysis also revealed a interaction between sex and treatment in the time

spent in the central platform [F(1,29)= 9.606; p<0.005], a post hoc analysis using

student’s t-test shown that within the male group, cocaine significantly decreased the time

spent in the central platform [t(15)=-3.433; p<0.005], and that in cocaine exposed group

males spent less time in central platform than females [t(15)=-3.037; p<0.01] (figure

3.3.2.1.3 B).

A B

02468

10

male female

Num

ber o

f ent

ries

saline cocaine

0

20

40

60

male female

% o

f ent

ries

saline cocaine

C D

02468

10

male female

Num

ber o

f ent

ries

saline cocaine

05

101520

male female

Num

ber o

f ent

ries

saline cocaine

Figure 3.3.2.1.1- Effects of chronic cocaine exposure throughout adolescence in the number of arm entries

during the elevated plus maze test, performed 2 days after the last administration of cocaine, both by male

and female rats. A- Number of open arm entries; B- % of open arm entries; C- Number of closed arm entries;

D- Number of total arm entries. Each column represents the mean + S.E.M.. Saline males, n=8; Cocaine

males, n=9; saline females, n=8; cocaine females, n=8. a,b p<0.005.

Open arms Open arms

Closed arms Total arm entries

a

a

b

b

112

A B

0

50

100

150

male female

Dur

atio

n (s

ec.)

saline cocaine

0

80

160

240

male female

Dur

atio

n (s

ec.)

saline cocaine

Figure 3.3.2.1.2- Effects of chronic cocaine exposure throughout adolescence on the time spent (A) in the

open arms and (B) in the closed arms during the elevated plus maze test performed 2 days after the last

administration of cocaine both by male and female rats. Each column represents the mean + S.E.M.. Saline

males, n=8; cocaine males, n=9; saline females, n=8; cocaine females, n=8.

A B

05

101520

male female

Num

ber o

f ent

ries

saline cocaine

0

20

40

60

male female

Dur

atio

n (s

ec.)

saline cocaine

Figure 3.3.2.1.3- Effects of chronic cocaine exposure throughout adolescence (A) on the number of central

platform entries and (B) on the time spent in the central platform during the elevated plus maze test performed

2 days after the last administration of cocaine, both by male and female rats. Each column represents the

mean + S.E.M. Saline males, n=8; cocaine males, n=9; saline females, n=8; cocaine females, n=8. Columns

with the same letter are significantly different from each other. a,b, p<0.005, c p<0.01.

Importantly, non-parametric statistical analysis revealed that within the male group

cocaine enhanced the risk-like behaviour (jump of the apparatus and walk on top of

closed arms) [Z=-2.785 (2-tailed) p<0.005] (table 3.3.2.1.1). This behavioural category

was only observed in cocaine-treated males. ANOVA also revealed a treatment effect in

the frequency [F(1,29)=8.295; p<0.01] and in the time spent [F(1,29)=5.455; p<0.05]

performing head-dipping behaviour. Cocaine administration increased the frequency and

the time spent in the head dipping behaviour (table 3.3.2.1.1). No group differences were

detected in frequency and duration of rearing and grooming during the EPM.

Central platform

Open arms Closed arms

Central platform

a

a b

b,c

c

113

Table 3.3.2.1.1- Frequency and duration of behavioural categories like risk-like behaviour, rearing, head

dipping and grooming, during the 5 minutes of the elevated plus maze test, in saline- and cocaine-treated

male and female rats.

Male Female

saline

(n=8) cocaine

(n=9) saline (n=8)

cocaine (n=8)

Risk-like Behaviour frequency 0.00±0.00 0.67±0.17 0.00±0.00 0.00±0.00

duration (sec.) - - - -

Rearing frequency 16.38±1.21 15.56±2.61 17.38±1.92 21.00±2.67

duration (sec.) 39.50±5.33 39.04±7.98 47.02±5.47 56.30±8.23

Head-dipping frequency 2.50±0.76 5.22±1.40 3.75±0.92 8.50±2.25

duration (sec.) 1.68±0.51 9.48±6.61 2.77±0.89 8.07±2.74

Grooming frequency 2.00±0.68 2.56±0.82 2.63±0.65 1.75±0.56

duration (sec.) 14.50±6.92 13.57±4.93 14.88±5.68 6.27±2.74


same letter are significantly different. a,b p<0.02.

Corticosterone plasma levels. After performing the EPM animals were decapitated

and corticosterone levels were measured in the obtained plasma. Statistical significance

between groups was assessed using the non-parametric Mann-Whitney U Test.

No significant differences were observed on plasma levels of corticosterone

between cocaine-treated and saline control males, and between cocaine-treated and

saline control females, after the EPM. However, nonparametric Mann-Whitney U test

showed that cocaine-treated males had lower levels of plasma corticosterone than

cocaine-treated females [Z=-2.556 (2-tailed) p<0.02] (figure 3.3.2.1.4).

0100200300400

male female

Cor

ticos

tero

ne

leve

ls (n

g/m

L)

saline cocaine

Figure 3.3.2.1.4- Effects of chronic cocaine exposure throughout adolescence on the alterations of the

corticosterone plasma levels of corticosterone induced by the elevated plus maze test, both in male and

female rats. Each column represents the mean + S.E.M for 6 animals per group. Columns with the same letter

are significantly different from each other. a p<0.02.

a

a

a a,b b

114

Neurochemical determinations. The levels of DA, 5-HT and their metabolites,

DOPAC, HVA and 5-HIAA were measured by HPLC-EC, in the prefrontal cortex,

hippocampus, amygdala, hypothalamus, SN-VTA and in the dorsal raphe nucleus. The

obtained data were analyzed by the non-parametric Mann-Whitney U Test.

Within the SN-VTA, no significant differences were detected on the levels of DA

and 5-HT, their metabolites and their turnover between the experimental groups (figure

3.3.2.1.9 and table 3.3.2.1.6).

SN-VTA

A

0

200

400

600

800

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

150

300

450

600

750

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the SN-VTA after the

elevated plus maze test, determined by HPLC-EC for male and female rats. Each column represents the

mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for at least 5 animals per

group.

115


[5-HIAA]/[5-HT], in the SN-VTA, in cocaine- and saline-treated male and female rats, after the elevated plus

maze test.

Male Female

saline cocaine saline cocaine

DOPAC/DA 0.32±0.10

(n=5)

0.31±0.05

(n=6)

0.35±0.08

(n=6)

0.34±0.07

(n=6)

HVA/DA 0.10±0.03

(n=5)

0.11±0.03

(n=6)

0.11±0.02

(n=6)

0.11±0.01

(n=6)


(n=5)

0.42±0.08

(n=6)

0.45±0.09

(n=6)

0.45±0.08

(n=6)

5-HIAA/5-HT 0.43±0.28

(n=5)

0.27±0.11

(n=6)

0.19±0.03

(n=6)

0.21±0.02

(n=6)



Within the dorsal raphe nucleus no differences were observed on the levels of DA

and 5-HT and their metabolites, and on DA and 5-HT turnover rates between the

experimental groups (figure 3.3.2.1.10 and table 3.3.2.1.7).

Within the prefrontal cortex there were no significant differences on the levels of

DA and 5-HT and their metabolites between the experimental groups (figure 3.3.2.1.6).

However, the HVA/DA turnover rate was increased in cocaine-treated males as compared

to control males [Z=-2.191 (2-tailed) p<0.05] and to cocaine-treated females [Z=-2.191 (2-

tailed) p<0.05]. The 5-HT turnover rate was also increased in cocaine-treated males as

compared to control males [Z=-2.008 (2-tailed) p=0.045] (table 3.3.2.1.3).

116


A

0

30

60

90

120

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

250

500

750

1000

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


Figure 3.3.2.1.10- Effects of chronic cocaine exposure throughout adolescence on (A) the levels of DA, and

its metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the dorsal raphe

nucleus after the elevated plus maze test, determined by HPLC-EC for male and female rats. Each column



117

Prefrontal cortex

A

0

15

30

45

60

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

100

200

300

400

500

5-HT 5-HIAANeu

rotr

ansm

ittte

rs c

once

ntra

tion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the prefrontal cortex

after the elevated plus maze test, determined by HPLC-EC for male and female rats. Each column represents

the mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for at least 5 animals

per group.

118


[5-HIAA]/[5-HT], in the dorsal raphe nucleus, in cocaine- and saline-treated male and female rats, after the

elevated plus maze test.

Male Female


DOPAC/DA 0.64±0.07

(n=5)

0.63±0.07

(n=6)

0.83±0.18

(n=6)

0.88±0.13

(n=6)

HVA/DA 0.09±0.03

(n=5)

0.10±0.03

(n=6)

0.14±0.03

(n=6)

0.12±0.03

(n=6)


(n=5)

0.74±0.09

(n=6)

0.97±0.20

(n=6)

1.00±0.14

(n=6)

5-HIAA/5-HT 0.20±0.03

(n=5)

0.18±0.02

(n=6)

0.26±0.04

(n=6)

0.27±0.06

(n=6)




[5-HIAA]/[5-HT], in the prefrontal cortex, in cocaine- and saline-treated male and female rats, after the

elevated plus maze test.

Male Female


DOPAC/DA 1.57±0.11

(n=6)

1.86±0.48

(n=5)

1.57±0.31

(n=6)

1.29±0.13

(n=6)

HVA/DA 0.61±0.07

(n=6)

0.80±0.03

(n=5)

0.51±0.10

(n=6)

0.68±0.04

(n=6)


(n=6)

2.67±0.50

(n=5)

2.08±0.27

(n=6)

1.97±0.16

(n=6)

5-HIAA/5-HT 0.06±0.00

(n=6)

0.09±0.01

(n=5)

0.11±0.04

(n=6)

0.07±0.02

(n=6)


same letter are significantly different. DA- dopamine; DOPAC- 3,4-Dihydroxyphenylacetic acid; HVA-

Homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a,b,c p<0.05.

In the hippocampus there were no differences on the levels of DA and 5-HT and

their metabolites between the experimental groups (figure 3.3.2.1.7). However, the

DOPAC/DA turnover rate was decreased in cocaine-treated females as compared to

control females [Z=-2.008 (2-tailed) p<0.05] (table 3.2.1.4). Cocaine-treated females also

displayed lower DOPAC/DA turnover rate as compared to cocaine-treated males that

almost reached significance [Z=-1.925 (2-tailed) p=0.054]. No differences were found on

5-HT turnover rate between the experimental groups (table 3.3.2.1.4).

a a,b

c c

b

119

Hippocampus

A

0

10

20

30

40

DA DOPACNeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

100

200

300

400

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolite DOPAC and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hippocampus after the

elevated plus maze test, determined by HPLC-EC for male and female rats. Each column represents the

mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for at least 5 animals per

group.

120


hippocampus, in cocaine- and saline-treated male and female rats, after the elevated plus maze test.

Male Female


DOPAC/DA 2.57±0.37

(n=6)

2.31±0.22

(n=6)

2.73±0.53

(n=5)

1.76±0.12

(n=6)

5-HIAA/5-HT 0.19±0.03

(n=6)

0.20±0.03

(n=6)

0.21±0.04

(n=5)

0.27±0.05

(n=6)


same letter are significantly different. DA- dopamine; DOPAC- 3,4-dihydroxyphenylacetic acid; 5-HT-

serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a p<0.05.

Within the amygdala, statistical analysis revealed that the levels of HVA in

cocaine-treated males were higher than in control males [Z=-2.242 (2-tailed) p<0.05]. No

significant difference were found on the levels of DA and its metabolite DOPAC, and on

the levels of 5-HT and 5-HIAA between cocaine exposed and control males, and between

cocaine exposed and control females after the elevated plus maze test (figure 3.3.2.1.5).

No differences were found both on DA and 5-HT turnover rates between the experimental

groups (table 3.3.2.1.2).

Within the hypothalamus, although there were no significant differences on the

levels of DA and 5-HT and their metabolites between the experimental groups (figure

3.3.2.1.8), cocaine-treated males displayed higher levels of DA than control males that

almost reached significance [Z=-1.924 (2-tailed) p=0.055]. No significant differences were

found on DA and 5-HT turnover rates between the experimental groups (table 3.3.2.1.5).

a a

121

Amygdala

A

0

50

100

150

200

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

150

300

450

600

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the amygdala after

the elevated plus maze test, determined by HPLC-EC for male and female rats. Each column represents the

mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for 6 animals per group.

Columns with the same letter are significantly different from each other. a p<0.05

a a

122

Hypothalamus

A

0

100

200

300

400

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

100

200

300

400

500

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hypothalamus

after the elevated plus maze test, determined by HPLC-EC for male and female rats. Each column represents

the mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for 6 animals per

group.

123


[5-HIAA]/[5-HT], in the amygdala, in saline- and cocaine-treated male and female rats, after the elevated plus

maze test.

Male Female


DOPAC/DA 0.86±0.27

(n=6)

0.57±0.14

(n=6)

0.73±0.09

(n=6)

0.56±0.06

(n=6)

HVA/DA 0.17±0.10

(n=6)

0.14±0.02

(n=6)

0.10±0.02

(n=6)

0.15±0.02

(n=6)


(n=6)

0.71±0.16

(n=6)

0.84±0.08

(n=6)

0.71±0.08

(n=6)

5-HIAA/5-HT 0.45±0.20

(n=6)

0.44±0.30

(n=6)

0.19±0.04

(n=6)

0.16±0.02

(n=6)




[5-HIAA]/[5-HT], in the hypothalamus, in cocaine- and saline-treated male and female rats, after the elevated

plus maze test.

Male Female


DOPAC/DA 0.38±0.07

(n=6)

0.43±0.09

(n=6)

0.54±0.09

(n=6)

0.50±0.09

(n=6)

HVA/DA 0.06±0.03

(n=6)

0.04±0.01

(n=6)

0.05±0.01

(n=6)

0.06±0.01

(n=6)


(n=6)

0.47±0.10

(n=6)

0.60±0.09

(n=6)

0.55±0.10

(n=6)

5-HIAA/5-HT 0.17±0.04

(n=6)

0.20±0.03

(n=6)

0.19±0.06

(n=6)

0.24±0.06

(n=6)



124

3.3.2.2. Depressive-like behaviour

Forced swim test. Starting 2 days after the last administration of cocaine, both

male and female Wistar rats performed a FST. Behavioural data was analysed by a 2-way

ANOVA with treatment (cocaine or saline) and sex as between-subject fixed-factors, and

behavioural measurements as the dependent variables.

Statistical analyses revealed an main effect of sex in the latency to struggling

behaviour [F(1,31)=4.522; p<0.05]. The latency to struggling was lower in males than in

females. No other significant behavioural differences were found in the FST between

experimental groups (table 3.3.2.2.1).

Table 3.3.2.2.1- Behaviour of male and female rats in a forced swim test after chronic cocaine exposure

during adolescence.

Male Female

saline n=9

cocaine n=9

saline n=9

cocaine n=8

Struggling

frequency 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00

duration (sec.) 47.78±14.51 27.85±8.40 37.60±7.53 38.23±8.73

latency (sec.) 0.88±0.33 1.28±0.53 2.09±0.67 5.18±2.43

Immobility

frequency 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00


latency (sec.) - - - -

Diving

frequency 0.44±0.34 1.22±1.00 0.44±0.34 0.50±0.50

duration (sec.) 0.37±0.36 1.53±1.29 0.81±0.54 0.68±0.68

latency (sec.) 253.40±30.91 253.04±31.33 252.66±31.87 269.74±30.27

Right rotation

frequency 28.67±2.48 31.22±2.52 34.44±4.60 35.34±3.08


latency (sec.) 4.85±2.48 3.36±0.71 4.12±1.67 1.57±0.62

Left rotation

frequency 36.56±2.91 32.44±3.22 33.78±4.02 28.88±3.95


latency (sec.) 2.13±0.93 3.36±1.49 4.81±1.56 3.75±1.83


same letter are significantly different. sec.- seconds.

125

Corticosterone plasma levels. At the end of FST animals were decapitated and

corticosterone levels were measured in the obtained plasma. Statistical significance

between groups was assessed using the non-parametric Mann-Whitney U Test. Statistical

analysis revealed no significant differences in the levels of corticosterone after the FST

between the experimental groups (figure 3.3.2.2.1).

0150300450600

male female

Cor

ticos

tero

ne

leve

ls (n

g/m

L)

saline cocaine

Figure 3.3.2.2.1- Effects of chronic cocaine exposure throughout adolescence on the alterations on the

plasma levels of corticosterone induced by a forced swim test, both in male and female rats. Each column

represents the mean + S.E.M for 6 animals per group.


DOPAC, HVA and 5-HIAA were measured by HPLC-EC, in the SN-VTA, dorsal raphe

nucleus, prefrontal cortex, hippocampus, amygdala and hypothalamus. The data obtained

was analyzed by the non-parametric Mann-Whitney U Test.

Within the SN-VTA, no significant differences were observed on the levels of DA

and 5-HT and their metabolites between the experimental groups (figure 3.3.2.2.6).

However, cocaine-treated males exhibited higher DOPAC/DA and (DOPAC+HVA)/DA

turnover rates as compared to control males (DOPAC/DA: [Z=-2.193 (2-tailed) p<0.05];

(DOPAC+HVA)/DA: [Z=-1.984 (2-tailed) p<0.05]) (table 3.3.2.2.6). No differences were

observed in the 5-HT turnover rate between the experimental groups.

Within the dorsal raphe nucleus, no differences were detected on the levels of DA

and 5-HT and their metabolites, and on DA and 5-HT turnover between the experimental

groups (figure 3.3.2.2.7 and table 3.3.2.2.7).

126

SN-VTA

A

0

100

200

300

400

500

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

)ng/

mg

of p

rote

in)


B

0

200

400

600

800

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the SN-VTA after a

forced swim test, determined by HPLC-EC for male and female rats. Each column represents the mean +

S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for at least 5 animals per group.

127


A

0

30

60

90

120

DA DOPAC HVANeu

rotr

ansm

itter

s co

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trat

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(ng/

mg

of p

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in)


B

0

200

400

600

800

5-HT 5-HIAANeu

rotr

ansm

itter

s co

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trat

ion

(ng/

mg

of p

rote

in)




nucleus after a forced swim test, determined by HPLC-EC for male and female rats. Each column represents


per group.

128


[5-HIAA]/[5-HT], in the SN-VTA, in cocaine- and saline-treated male and female rats, after a forced swim test.

Male Female


DOPAC/DA 0.30±0.05

(n=5)

0.46±0.05

(n=5)

0.27±0.05

(n=6)

0.39±0.07

(n=6)

HVA/DA 0.09±0.01

(n=5)

0.09±0.02

(n=5)

0.07±0.01

(n=6)

0.11±0.02

(n=6)


(n=5)

0.55±0.04

(n=5)

0.34±0.06

(n=6)

0.50±0.09

(n=6)

5-HIAA/5-HT 0.14±0.01

(n=5)

0.17±0.05

(n=5)

0.20±0.03

(n=6)

0.20±0.05

(n=6)



homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a,b p<0.05.


[5-HIAA]/[5-HT], in the dorsal raphe nucleus, in cocaine- and saline-treated male and female rats, after a

forced swim test.

Male Female


DOPAC/DA 0.73±0.11

(n=6)

0.44±0.11

(n=6)

0.54±0.12

(n=5)

0.71±0.10

(n=6)

HVA/DA 0.13±0.04

(n=6)

0.16±0.03

(n=6)

0.15±0.04

(n=5)

0.15±0.03

(n=6)


(n=6)

0.60±0.13

(n=6)

0.69±0.14

(n=5)

0.86±0.10

(n=6)

5-HIAA/5-HT 0.21±0.04

(n=6)

0.14±0.01

(n=6)

0.19±0.02

(n=5)

0.28±0.08

(n=6)



Within the prefrontal cortex, also no differences were detected on the levels of DA

and 5-HT, their metabolites and their turnover rates between the experimental groups

(figure 3.3.2.2.3 and table 3.3.2.2.3).

a a

b b

129

Prefrontal cortex

A

0

10

20

30

40

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

50

100

150

200

250

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the prefrontal cortex

after a forced swim test, determined by HPLC-EC for male and female rats. Each column represents the mean

+ S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for 6 animals per group.

130


[5-HIAA]/[5-HT], in the prefrontal cortex, in cocaine- and saline-treated male and female rats, after a forced

swim test.

Male Female


DOPAC/DA 1.88±0.50

(n=6)

1.75±0.23

(n=6)

1.66±0.44

(n=6)

1.32±0.29

(n=6)

HVA/DA 0.93±0.10

(n=6)

0.89±0.17

(n=6)

0.78±0.08

(n=6)

0.69±0.10

(n=6)


(n=6)

2.64±0.38

(n=6)

2.39±0.46

(n=6)

2.01±0.30

(n=6)

5-HIAA/5-HT 0.06±0.01

(n=6)

0.07±0.01

(n=6)

0.07±0.02

(n=6)

0.06±0.01

(n=6)



Within the hippocampus, also no differences were detected on the levels of DA

and 5-HT, their metabolites and their turnover between the experimental groups (figure

3.3.2.2.4 and table 3.3.2.2.4).

Within the amygdala, no significant differences were detected on the levels of DA

and 5-HT and their metabolites (figure 3.3.2.2.2). However, control females displayed

increased levels of 5-HT [Z =-1.922 (2-tailed) p=0.055] and decreased (DOPAC+HVA)/DA

turnover rate [Z =-1.922 (2-tailed) p=0.055] when compared with control males that almost

reached significance [Z =-1.922 (2-tailed) p=0.055]. Furthermore, it was also observed a

lower 5-HIAA/5-HT rate in control females as compared to control males [Z =-2.242 (2-

tailed) p<0.05] (table 3.3.2.2.2). These sex effects were not observed in cocaine-treated

rats. Also in this brain region, although not significantly, cocaine-treated males exhibited

lower DOPAC/DA turnover rate as compared to control males [Z =-1.922 (2-tailed)

p=0.055].

131

Hippocampus

A

0

10

20

30

40

DA DOPACNeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

50

100

150

200

250

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolite DOPAC and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hippocampus after a forced

swim test, determined by HPLC-EC for male and female rats. Each column represents the mean + S.E.M. of

monoamine levels, expressed as ng of monoamine/mg of protein, for at least 5 animals per group.

132

Amygdala

A

0

25

50

75

100

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

100

200

300

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the amygdala after a

forced swim test, determined by HPLC-EC for male and female rats. Each column represents the mean +

S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for 6 animals per group.

133


hippocampus, in cocaine- and saline-treated male and female rats, after a forced swim test.

Male Female


DOPAC/DA 2.32±0.56

(n=6)

2.47±0.21

(n=6)

1.88±0.37

(n=5)

2.03±0.41

(n=6)

5-HIAA/5-HT 0.16±0.04

(n=6)

0.13±0.02

(n=6)

0.19±0.03

(n=5)

0.23±0.05

(n=6)


dihydroxyphenylacetic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid.


[5-HIAA]/[5-HT], in the amygdala, in cocaine- and saline-treated male and female rats, after a forced swim

test.

Male Female


DOPAC/DA 0.76±0.11

(n=6)

0.57±0.06

(n=6)

0.53±0.09

(n=6)

0.55±0.08

(n=6)

HVA/DA 0.13±0.04

(n=6)

0.09±0.03

(n=6)

0.05±0.02

(n=6)

0.07±0.03

(n=6)


(n=6)

0.66±0.07

(n=6)

0.58±0.09

(n=6)

0.63±0.06

(n=6)

5-HIAA/5-HT 0.23±0.05

(n=6)

0.16±0.03

(n=6)

0.10±0.01

(n=6)

0.14±0.02

(n=6)




Within the hypothalamus, after the FST no differences were detected on the levels

of DA and 5-HT, their metabolites, and on DA and 5-HT turnover between the

experimental groups (figure 3.3.2.2.5 and table 3.3.2.2.5).

a a

134

Hypothalamus

A

0

100

200

300

400

DA DOPAC HVANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


B

0

150

300

450

600

5-HT 5-HIAANeu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)



metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hypothalamus

after a forced swim test, determined by HPLC-EC for male and female rats. Each column represents the mean

+ S.E.M. of monoamine levels, expressed as ng of monoamine/mg of protein, for 6 animals per group.

135


[5-HIAA]/[5-HT], in the hypothalamus, in cocaine- and saline-treated male and female rats, after a forced

swim test.

Male Female


DOPAC/DA 0.53±0.12

(n=6)

0.43±0.12

(n=6)

0.33±0.04

(n=6)

0.47±0.19

(n=6)

HVA/DA 0.07±0.02

(n=6)

0.04±0.01

(n=6)

0.04±0.01

(n=6)

0.06±0.01

(n=6)


(n=6)

0.48±0.13

(n=6)

0.37±0.06

(n=6)

0.53±0.10

(n=6)

5-HIAA/5-HT 0.17±0.04

(n=6)

0.14±0.03

(n=6)

0.13±0.03

(n=6)

0.23±0.06

(n=6)



136

3.4. Discussion

3.4.1. Effects of chronic cocaine treatment during adolescence on the body weight evolution

It is known that any weight reduction gain during adolescence is likely to alter the

pace of development, delaying puberty, extending the adolescence period, and perhaps

altering the normal trajectory of developmental processes (Spear, 2007). Our data

showed that male rats presented higher weight gain than did females. In fact, it is known

that sex differences in body weight appear during adolescence (Macri et al., 2002).

However, body weight evolution was not affected by the chronic cocaine administration

both in male and female adolescent rats. These findings contrast with the known anorectic

and stress-like effects of cocaine. Cocaine decreases food intake and increases metabolic

processes through the activation of the sympathetic nervous system (Spector et al., 1972;

Balopole et al., 1979). Moreover, cocaine suppresses food intake in a dose-dependent

manner, with a dose between 10 to 30 mg/kg showing to be anorectic (Cooper and van

der Hoek, 1993). On the other hand, Laviola et al (1995) have shown that chronic cocaine

(10 or 20 mg/kg/day for 4 days) reduced body weight and food consumption in adult male

rats but not in periadolescents (PND 34-39) of either sex. Giorgetti and Zhdanova (2000)

also showed that adolescent rats chronically treated with cocaine (15 mg/kg i.p., twice a

day for 9 days) did not revealed any significant alteration either in the total amount of food

consumed over a 24-h period or in the body weight. These, as well as our findings,

suggest an age effect regarding cocaine effects on body weight evolution. In addition, it

was demonstrated that adolescent rats continue to gain body weight better than adults

during both cold and restraint stress (Gomez and Dallman, 2001; Gomez et al., 2002). It

has been proposed that, facing different types of chronic stress such as cold and restrain,

adolescent rats put their resources into ponderal growth, whereas adult rats protect the

reproductive capability (Gomez and Dallman, 2001; Gomez et al., 2002). This strategy

could also underlie the absence of body weight differences between cocaine exposed and

control adolescent animals observed in this study.

3.4.2. Effects of chronic cocaine treatment during adolescence on the neuroendocrine function

The acute stimulant effects of cocaine in the HPA axis in rats and in rhesus

monkeys is well known (Rivier and Vale, 1987; Borowsky and Kuhn, 1991; Sarnyai et al.,

1992; Sarnyai et al., 1996). The stimulatory cocaine effect in human HPA axis has also

been demonstrated (Mendelson et al., 1992; Teoh et al., 1994). However, there are

137

conflicting reports in the literature as to whether chronic cocaine exhibits persistent

stimulatory effects on the HPA axis. Tolerance to the stimulatory effects of cocaine has

also been reported in rats following chronic binge pattern of administration (Zhou et al.,

1996). This tolerance has been observed as an attenuated ACTH and corticosterone

responses to cocaine (Zhou et al., 1996; Zhou et al., 2003). In the present study, male

rats displayed an activation of the HPA axis in the last day of chronic cocaine treatment.

Thirty minutes after the last administration of cocaine the ACTH levels were increased in

males. However, the increased levels of ACTH did not result in the expected subsequent

increase in the levels of corticosterone. This could be explained by an altered adrenal

sensitization to the ACTH stimulatory.

A sex difference regarding HPA axis activation following cocaine was found 30

minutes after the last administration. Contrarily to what was observed in males, in females

no differences were seen in the ACTH or corticosterone plasma levels. The sex

differences in the HPA axis function are well known. Former findings have shown greater

corticosterone and ACTH levels in female, compared with male rats, both at baseline

(Atkinson and Waddell, 1997) and following a stress response (Rivier, 1993; Handa et al.,

1994; Yoshimura et al., 2003). Moreover, it is known that males and females display a

different HPA axis response to cocaine. However, contrarily to our data, most reports

describe that female rats exhibit a greater HPA axis activation following cocaine

administration (Kuhn and Francis, 1997).

Available research indicates that sex differences in the HPA axis function emerge

over adolescence, a phenomenon reflecting the concomitant initiation of regulatory effects

of sex hormones (reviewed by McCormick and Mathews, 2007). The ACTH responses to

cocaine are absent before puberty, when levels of gonadal steroids are fairly low (Kuhn

and Francis, 1997). In addition, ovariectomy decreased the ACTH response to cocaine in

females, whereas castration did not influence the response in males (Kuhn and Francis,

1997). Moreover, estrogen treatment of ovariectomized females increases CRH

production in the hypothalamus, ACTH and corticosterone production when facing a

stressor (Lesniewska et al., 1990). Based on these data, the failure of cocaine to induce

activation of the HPA axis in adolescent females could be explained by an immature

regulation of the HPA axis activity by gonadal hormones.

In the present study data from female rats were used without regarded hormonal

fluctuations. This strategy was based on the assumption that variations in gonadal steroid

hormone levels in the physiological range through the estrous cycle would produce

smaller changes than those that exist between males and females (Kuhn and Francis,

1997). In fact, several studies using females without regard to estrous state demonstrate

138

robust gender differences in the HPA axis function (Lesniewska et al., 1990; Aloisi et al.,

1994; Chisari et al., 1995).

Interestingly, 1 day after the last administration the altered HPA axis activity was

no longer present. No differences were observed in the levels of ACTH or corticosterone

between cocaine and saline exposed male or female rats. In fact, the levels of ACTH in

cocaine exposed males did not differ between 30 minutes and 1 day after the last

administration, while the levels of ACTH in control males increased between this two

assessment time points. This may indicate that the stress caused by the binge pattern of

administration induced a decrease in plasma levels of ACTH in control males, which was

not observed in cocaine males, most likely due to the acute effect of the 3rd daily cocaine

injection on HPA axis activation. In fact, it is known that repeated stress induces

alterations in the HPA axis function. For instance, Gomez et al. (2002) reported that in

adolescent male rats, beside the habituation of ACTH responses to repeated restraint

stress across days, a clear habituation of ACTH responses to restraint occurs within three

repetitions of the stress.

Nevertheless, the absence of HPA activation, both in male and female rats, 1 day

following the last administration of cocaine was unexpected. It has been demonstrated

with a variety of paradigms that withdrawal from cocaine induces an activation of the HPA

axis. However, Mantsch et al. (2007), using adult rats, demonstrated that plasma

corticosterone and CRH mRNA levels in the paraventricular nuclei of hypothalamus were

not altered 24 hours after the final administration of chronic cocaine (30mg/kg/day, for 14

days). These findings are also consistent with reports showing that basal corticosterone

levels were unaltered after 1 day of withdrawal from chronic cocaine (Levy et al., 1994;

Zorrilla et al., 2001; Baumann et al., 2004). However, Zhou et al. (2003) reported that 1

day after chronic binge administration (14 days of 3x15mg/kg i.p.) the levels of ACTH and

corticosterone were increased in adult male rats, although the hypothalamic CRH mRNA

levels were not different from that of saline controls.

However, age-related discontinuities in the response of the HPA axis to both stress

and psychostimulants have been demonstrated (Adriani and Laviola, 2000; Laviola et al.,

2002). Upon amphetamine administration, periadolescents failed to show an increase

across the course of the session in corticosterone release, that was observed in adults

(Adriani and Laviola, 2000). Moreover, it has been demonstrated that basal corticosterone

levels are elevated in adolescents compared to adult rodents (Laviola et al., 1999). This

elevated baseline is accompanied by an attenuated percentage increase in corticosterone

levels in response to psychostimulants (Adriani and Laviola, 2000). Thus, although

139

adolescents operate at a higher baseline, their HPA axis response to psychostimulants

may be proportionately masked (Schramm-Sapyta et al., 2006).

The age-related discontinuity in the HPA axis function may be underlying the

absence of HPA axis activation on day 1 from withdrawal in adolescent rats, observed in

our study. Moreover, this observation points to a reduced HPA axis response to chronic

binge cocaine administration during adolescence, suggesting that this axis is somewhat

hyporesponsive to cocaine during adolescence.

Adrenal weight, normalized to body weight, was not affected by the cocaine

exposure during adolescence since no differences were observed between cocaine-

treated males and females and their respective controls. Nevertheless, there was a sex

effect on adrenal weight, with adrenals from male rats weighing significantly less than

those from females. This observation is concordant with data from literature showing that

in wistar rats, sex differences in adrenal weight appear at about PND 49, and from this

day onward, female relative and subsequently absolute adrenal weights are higher than in

male animals (Majchrzak and Malendowicz, 1983).

During the pubertal period, the HPG axis undergoes many dynamic changes

leading to sexual maturation. Puberty marks the final and crucial interactions between

aminergic neurotransmitters systems, gonadal steroids, and hypothalamic-pituitary

hormones (Kalra and Kalra, 1983). Without the coordinated activity of each of HPG axis

components during puberty, alterations in later reproductive functioning are highly

probable (Ojeda et al., 1992).

A number of studies have demonstrated that cocaine modulates the HPG axis. Our

data showed that male rats treated chronically with cocaine throughout adolescence

presented decreased levels of testosterone at 30 minutes after the last administration.

This result agrees with other studies that have previously referred a cocaine effect on

testosterone plasma levels in males. In male rats, acute and binge chronic cocaine

administration are reported to significantly decrease testosterone plasma levels (Berul and

Harclerode, 1989; Budziszewska et al., 1996).

However, the decreased testosterone plasma levels observed at 30 minutes after

the last cocaine administration seems to be the result of its acute effects after chronic

cocaine treatment, since 1 day after withdrawal this alteration was no longer observed. In

contrast to our results, Sarnay et al (1998) reported that in adult male rats, 22 hours after

withdrawal from both 3 and 6 weeks binge cocaine the levels of testosterone were still

decreased. Therefore, the process of maturation taking place during adolescence may

underlie these different results.

140

Chronic cocaine administration during adolescence also did not affect the testes

weight (calculated as percentage of body weight). Despite that, and although the

testosterone plasma levels were not altered in male rats 1 day after the end of chronic

cocaine administration, the repeated decrease in testosterone levels induced by the

“acute” effects of each cocaine administration throughout the exposure period, may have

induced alterations in the developmental process of the reproductive function, which can

lead to long-term impairment in the reproductive function.

The mechanisms of cocaine-induced decrease on the testosterone levels are still

unclear. One of the possible mechanisms underlying the suppression of testosterone

secretion could be the activation of the HPA axis resulting an inhibitory HPA/HPG

interaction (Rivest and Rivier, 1995; Gore et al., 2006). Alternatively, cocaine might act

directly on the testes to decrease testosterone secretion. This suggestion is supported by

morphological changes and lesions of the cells of seminiferous tubules and interstitial

cells of the testes in rats subjected to long-term cocaine administration which may

produce a decrease in testosterone secretion (Barroso-Moguel et al., 1994; George et al.,

1996).

Beside the decreased levels of testosterone on adolescent male rats treated

chronically with cocaine, our data also indicate a trend toward an increase on

progesterone levels at 30 minutes after the last administration. In fact, it has been

demonstrated that after single-, binge- and chronic-cocaine administration paradigms, in

male and female rats, the plasma levels of progesterone were increased (Quinones-Jenab

et al., 2000; Walker et al., 2001; Quinones-Jenab et al., 2008).

The findings that testosterone, but not progesterone plasma levels are reduced

following administration of cocaine suggest that this psychostimulant may interfere with

some specific metabolic processes in the synthesis of testosterone in Leydig cells (figure

3.4.1). Nevertheless, a study in female rats, has shown that cocaine stimulate

corticosterone and progesterone secretion in sham-adrenalectomized, but not in

adrenalectomized rats (Walker et al., 2001), suggesting that the adrenal gland is the

source of cocaine-stimulated progesterone. In this view, the trend to an increase in

progesterone levels may be induced by the increased ACTH levels observed in cocaine-

treated males at this assessment time point. In fact, Feder and Ruf (1969) reported that

systemic injections of ACTH increased plasma progesterone concentrations in

ovariectomized, estrogen-primed rats and guinea pigs (Feder and Ruf, 1969). In the case

of the adrenals being actually the source of progesterone increase, the decreased

testosterone levels, could be explained by the inhibitory effect of the HPA axis on the LH

release (Olster and Ferin, 1987; Petraglia et al., 1987).

141

Figure 3.4.2.1- Pathways of testosterone biosynthesis in the testes. The conversion of pregnenolone into

testosterone requires four enzymatic reactions, which are divided into two parallel pathways: one proceeding

via 17-hydroxypregnenolone, and the other proceeding via 17-hydroxyprogesterone. The relative activities of

the pathways vary among mammalian species; in rodents the pathway via 17-hydroxyprogesterone is

predominant (Norman and Litwack, 1998). 1- P450 side chain cleavage; 2,- 17α-hydroxylase/ C-17-C-20

lyase; 3- 3β-Steroid dehydrogenase; 4- 17-Ketosteroid redutase; 5- 5α-Reductase. Adapted from (Norman

and Litwack, 1998).

Preclinical studies have consistently shown that cocaine stimulates significant

increases in LH levels. Alterations in LH pulsatile release patterns and/or abnormal LH

levels could disrupt gonadotropin and ovarian steroid feedback systems. In fact, cocaine

administration disrupts estrous cyclicity and normal rates of ovulation in rats (King et al.,

1990), and menstrual cyclicity and ovulation rates in monkeys (Potter et al., 1999).

Cocores at al. reported that chronic cocaine abuse in humans was also associated with

menstrual cycle irregularities (Cocores et al., 1986). Nevertheless, the effects of cocaine

on cyclic reproductive function, including circulating LH concentrations, estrous cyclicity

and rats of ovulation in rats are dose-dependent (King et al., 1993).

Our results showed that adolescent female rats chronically exposed to binge

cocaine did not present altered levels of estradiol or progesterone when assessed 30

minutes and 1 day after the last administration. Since in our study, hormonal data from

female rats was analyzed regardless physiological hormonal fluctuations, hormonal

alterations induced by cocaine could have been masked by this approach. Although the

142

effects of cocaine on progesterone levels is known to be affected by the endocrinological

profile of female rats (Quinones-Jenab et al., 2000), several studies that also analyzed the

endocrine profile regardless of the estrous cycle have reported increased levels of

progesterone after cocaine administration (Quinones-Jenab et al., 2000; Quinones-Jenab

et al., 2008). However, cocaine modulation of progesterone plasma levels was transient,

as 3 hours post-injection progesterone plasma levels returned to control levels (Quinones-

Jenab et al., 2000). Based in this observation, it has been suggested that it is possible

that the disruptive effects of cocaine on the HPG axis are transient and/or completely

reversible. Our findings also seem to support the hypothesis that tolerance to female HPG

axis alterations may occur after continued and repetitive use of cocaine.

Moreover, data from literature also shows that cocaine-treated female rats

(20mg/kg/day) from PND 21 to 60, a period that include the pubertal development,

revealed no cocaine effects on the onset of puberty, on first ovulation age and that the

ovulation rates did not differ from the saline controls, once again suggesting that cocaine

did not induced persisting effects on HPG axis in females (Raap et al., 2000).

In what concerns estradiol, it has been reported that estradiol plasma levels are

increased by cocaine in the follicular phase but not in the midluteal phase of the rhesus

monkey cycle (Mello et al., 2000). However, no changes in estradiol levels after acute

cocaine administration have been reported in female rats (Walker et al., 2001).

Nevertheless, the stress induced by the injection protocol may also have lead to

the absence of cocaine effects in progesterone and estradiol plasma levels. It was

reported that footshock stress during 14 days had no effect on estrous cycle length,

estradiol or progesterone concentrations (Anderson et al., 1996). However, in the latter,

like in the present study, animals were housed individually, and a recent report from

Backer and Bielajew (2007) showed that single-housed rats presented greatest cycle

disruptions during post-chronic mild stress than paired housed females, this way

suggesting that housing condition, more than exposure to stress, appeared to contribute

to the disruption of estrous cycling (Baker and Bielajew, 2007). In the present study the

animals were housed individually in order to avoid variable social influences that could

play a role in cocaine behavioural, neurochemical and hormonal effects (de Jong et al.,

2007), however, this approach may have contributed to the lack of differences regarding

female gonadal hormones.

The levels of testosterone were also measured in the adolescent female rats, and

although no differences were observed between cocaine-treated and control females in

the two assessment points, in the cocaine exposed female group the levels of

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testosterone decreased from 30 minutes to 1 day after the last administration of cocaine.

As testosterone is known to play a role in the reproductive function (Drouin and Labrie,

1976; McClamrock et al., 1991; Weiss et al., 2007), this alteration may be an indication of

a certain degree of cocaine effects on the reproductive function.

During the adolescent developmental period, beside the effects of cocaine on the

HPG axis regulation, the effects of gonadal hormones on the maturating CNS represent

an important issue. Because of the profound effect of gonadal hormones on the

modulation of the CNS plasticity, cocaine-induced alterations in the HPG axis may play a

key role in the modulation of CNS neuronal functions of both males and females and thus

be an important component in the cascade of events that follow the administration of

cocaine (Quinones-Jenab et al., 2001). Thus, the altered levels of testosterone observed

in both male and female rats after chronic cocaine administration throughout adolescence

may have led to disruption of the CNS maturation process, as for instance, testosterone

seems to be implicated in cocaine-induced behavioural sensitization (Martinez-Sanchis et

al., 2002).

3.4.3. Effects of chronic cocaine treatment during adolescence on the dopaminergic and serotonergic systems function

The neural mechanisms responsible for the enhanced adolescent vulnerability for

initiating drug abuse are unclear. Drug exposure during adolescence may have unique

effects because of the dynamic ongoing neural development (Giedd et al., 1999; Spear,

2000; Cunningham et al., 2002; Chambers et al., 2003).

- Effects on the mesoaccumbens and nigrostriatal pathways

The involvement of nigrostriatal and mesolimbic DA in the mediation of

psychostimulant effects is largely supported in literature. The dopaminergic neurons of the

SN and VTA play prominent roles in novelty seeking as well as reinforcement (Le Moal

and Simon, 1991; Spanagel and Weiss, 1999). Although all of the neurochemical

mechanism for dopaminergic transmission is present soon after birth, a number of indices

of dopaminergic function change markedly during adolescence. Overexpression of D1,

D2 and D4 receptors and subsequent decreases during adolescence have been reported

in several studies (Teicher et al., 1995; Tarazi et al., 1998; Montague et al., 1999; Tarazi

et al., 1999; Andersen et al., 2002). Such pruning has also been shown in postmortem

studies of adolescent human brain for DA receptors and DAT (Meng et al., 1999). Thus, to

better understand how addictive drugs affect the DA neurotransmission as dopaminergic

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systems approach final maturation during adolescence is critical to understanding the

drug abuse vulnerability of this unique developmental period.

Our data showed that 30 minutes after the last administration, adolescent rats

treated chronically with cocaine presented no differences on the levels of DA and its

metabolites, DOPAC and HVA, in the SN-VTA and in the terminal areas, Nac and dorsal

striatum, as compared to saline controls.

Studies using microdialysis have shown that acute cocaine administration leads to

an increase in the extracellular levels of DA (Hurd and Ungerstedt, 1989; Kalivas and

Duffy, 1990). However, after chronic cocaine administration the increase in DA levels is

not so clear. Decreases in basal DA levels in dorsal striatum and Nac have been found in

rats submitted to chronic binge cocaine injection paradigm (Maisonneuve et al., 1995), in

rats injected with cocaine twice daily for 9 or 16 days (Imperato et al., 1992; Rossetti et

al., 1992) and even in rats which received a single daily injection of cocaine for 10 days

(Parsons et al., 1991) or 18 days (Robertson et al., 1991). A suggested mechanism for

this lowered basal level of DA is an increase of the DA reuptake process. For example,

the density of the DAT binding sites in the striatum of rats treated for 14 consecutive days

with cocaine were found to be significantly increased compared to controls after 3 days of

withdrawal (Claye et al., 1995). In a postmortem study of human cocaine-exposed

subjects, DAT binding was increased in the dorsal striatum and Nac (Little et al., 1993).

Thus, an enhanced clearance of synaptic DA may underlie the observed lower levels of

DA. On the other hand, super-sensitivity of D2 DA autoreceptors in the dopaminergic

terminals has been suggested as a mechanism underlying DA decreases (Dwoskin et al.,

1988). Such a change could lead to decreased DA synthesis following chronic cocaine

administration (Trulson and Ulissey, 1987; Brock et al., 1990). However, Tsukada (1996)

reported that cocaine administered to rats for 14 days in a binge pattern produced a

significant reduction in the in vivo D1 and D2 binding in striatum, due mainly to alterations

in the affinity (Tsukada et al., 1996). Nevertheless, Koeltzow and White (2003) reported

that binge cocaine-treated rats exhibited a decreased in the number of spontaneously

active VTA DA neurons. Another suggested mechanism for the lowered basal level of DA,

is that alterations in the expression of neuropeptides that modulate concentration of DA

that have found to play an important role. For example, increases in the levels of mRNA of

the striatal neuropeptide dynorphin have been found following chronic administration of

cocaine to rats (Sivam, 1989; Spangler et al., 1993; Daunais and McGinty, 1995).

Dynorphin is known to lower the striatal DA release (Reid et al., 1988). More recently, it

was reported that prolonged treatment with cocaine reduces dopaminergic terminal

density in regions receiving projections, and this reduced dopaminergic terminal density

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that follows prolonged treatment with cocaine was associated with reduced basal levels of

DA (Horne et al., 2008).

Nevertheless, and as already mentioned, our data showed no differences in levels

of DA and its metabolites in the SN-VTA, Nac and dorsal striatum, 30 minutes after the

last administration, between cocaine-treated and control male and female adolescent rats.

This absence of differences may be related to the lower basal levels of DA reported to

occur in chronically treated animals, and may not represent an effective absence of

increase in levels of DA induced by the binge cocaine administration. However, these

results contrast with previous studies that reported small increases on DA levels in the

striatum after cocaine binge during chronic exposure (Maisonneuve et al., 1995; Zhang et

al., 2003).

The immaturity of the dopaminergic neurotransmission during adolescence may

underlie the different response to the binge administration. In fact, adolescent and adult

rats use a different balance of mechanisms to attain comparable rates of DA clearance.

Relatively to adults, adolescents present a higher ratio of uptake/release, and this uptake-

dominated situation likely leads to lower extracellular DA levels during adolescence

(Walker and Kuhn, 2008). This developmental specific type of regulation could influence

drug-induced activation of the dopaminergic system (Walker and Kuhn, 2008).

In fact, basal extracellular DA concentration measured by microdialysis are

reported to be lower in adolescent than in adult rats (Andersen and Gazzara, 1993;

Laviola et al., 2001). In contrast to lower extracellular DA in adolescents, striatal D1 and

D2 DA receptor levels are higher in adolescent than adult humans (Palacios et al., 1988;

Montague et al., 1999) and rats (Tarazi et al., 1998, 1999), and DAT density reaches

maximal expression during adolescence (Haycock et al., 2003). The combination of an

increased uptake and a decreased synthesis due to the higher D2 receptor levels, could

be the reason for the lower increase in levels of DA observes in this study after a binge

administration.

The mechanism of how a hypofunctional dopaminergic system would produced

greater vulnerability to addiction is not fully understood (Walker and Kuhn, 2008). One

possibility is that the adolescent animals have lower baseline dopaminergic activity and an

enhanced threshold for rewarding stimuli (Bjork et al., 2004).

Nevertheless, it has been hypothesized that decreases in levels of DA in the brain

are associated with the long-lasting dysphoric state and craving after cocaine withdrawal

reported in humans abstinent from cocaine (Dackis and Gold, 1985). Such a change may

play a significant role in maintaining or reinstating drug-taking to compensate for

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decreased levels of DA during withdrawal (Dackis and Gold, 1985; Weiss and Porrino,

2002).

Based on data obtained from chronic repeated cocaine-induced alterations on the

dopaminergic system, and on the age-specific characteristics of dopaminergic function of

adolescents it was expected that 1 day after withdrawal, the levels of DA in the

mesoaccumbens and nigrostriatal systems would be decreased. Our data showed that

cocaine-treated male animals did not display any difference on DA or its metabolites,

DOPAC and HVA levels in Nac as compared to control males. However, in the dorsal

striatum, DA turnover, calculated as (DOPAC+HVA)/DA was increased in cocaine-treated

males, and in the SN-VTA the levels of DA and DOPAC were increased.

Since animals were sacrificed in the room where cocaine was administrated, we

suggest that this absence of lower levels of DA could be related with the cocaine cues-

induced increases in DA. In fact, several investigators have demonstrated stimulant-like

effects when drug-free rats are exposed to environments that have been previously paired

with stimulant drugs, such as amphetamine or cocaine (Post et al., 1981; Badiani et al.,

1995). Moreover, it was shown that conditioned rewarding stimulus associated with

cocaine self-administration reverses the depression of catecholamine brain systems

following cocaine withdrawal in rats (Antkiewicz-Michaluk et al., 2006).

Dopamine, which is a neurotransmitter involved with reward and with the prediction

of reward (Wise and Rompre, 1989; Schultz et al., 1997), appears to be involved with cue-

elicited drug craving. Indeed, in laboratory animals when neutral stimuli are paired with a

rewarding drug they will, with repeated associations, acquire the ability to increase DA in

Nac and dorsal striatum, and these neurochemical responses are associated with drug-

seeking behaviour (Kiyatkin and Stein, 1996; Duvauchelle et al., 2000; Weiss et al., 2000;

Phillips et al., 2003; Di Ciano and Everitt, 2004; Vanderschuren et al., 2005). Imaging

studies in humans have also shown that in drug-addicted subjects visual exposure to drug

cues elicits DA increases in dorsal striatum and these increases are associated with the

subjective experiences of drug craving (Volkow et al., 2006; Wong et al., 2006).

Thus, during a drug-free period, on the day after cocaine administration an

increase in the levels of DA in the SN-VTA and the absence of alterations in DA and its

metabolites in Nac in rats previously treated with cocaine may be a consequence of the

exposure to stimuli associated to cocaine administration. Nevertheless, a sex difference in

DOPAC levels 30 minutes after the last administration was observed in the SN-VTA in

cocaine-treated animals.

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In fact, adolescent rats have been reported to be more sensitive to the place

conditioning effects of cocaine, showing preferences after conditioning with lower doses

than adults (Badanich et al., 2006). Clinical research suggests that adolescence is indeed

a critical period when motivational conditioning is heightened (Harrison et al., 1989).

Actually, a hypothesis was developed that defends that adolescent vulnerability to

addiction involves a developmental state that is primed to assign high salience to reward-

related cues (Brenhouse and Andersen, 2008).

The reinforcing properties of cocaine are thought to be due to the ability of cocaine

to block the uptake of DA. However, mapping of the sites of cocaine binding and neuronal

activation in DAT knockout mice suggests the involvement of serotonergic brain regions in

initiation and maintenance of cocaine self-administration (Rocha et al., 1998). Indeed,

cocaine self-administration in DAT knockout mice suggests that the serotonergic system

may provide an additional component of drug reinforcement (Rocha et al., 1998).

It is known that cocaine also inhibits the reuptake of 5-HT into serotonergic

neurons (Reith et al., 1983; Pitts and Marwah, 1987), and this effect of cocaine leads to

elevation of extracellular 5-HT levels in the brain (Bradberry et al., 1993; Parsons and

Justice, 1993a; Reith et al., 1997). Increased extracellular 5-HT levels in striatum and VTA

after acute cocaine have been reported (Bradberry et al., 1993; Reith et al., 1997; Muller

et al., 2002). After chronic treatment (20 mg/kg i.p. for 10 consecutive days) a systemic

injection of cocaine also induces higher increases of 5-HT levels in the Nac and VTA as

compared to saline control rats (Parsons and Justice, 1993a). These results contrast with

those obtained in our study. We did not observe any differences in 5-HT and 5-HIAA

levels between cocaine-treated and control animals 30 minutes after the last binge

treatment. More recently, Mills at al. (2007) reported that in rats, chronic cocaine

administration (20 mg/kg/day for 18 days) did not increase 5-HT levels in the caudate

nucleus, although produced significant increases in the Nac (Mills et al., 2007).

Some evidence supports the notion that withdrawal after repeated cocaine

administration in rats may leads to diminished synaptic 5-HT availability (Parsons et al.,

1995). Alterations in 5-HT transport may contribute to the post cocaine deficits in

serotonergic neurotransmission. Increased densities of 5-HT reuptake sites have been

demonstrated in rats following repeated cocaine exposure (Cunningham et al., 1992). In

studies using single photon emission computed tomography, acutely abstinent cocaine-

dependent patients had higher SERT binding in striatal brain regions compared to non-

drug abusing control subjects (Jacobsen et al., 2000). In monkeys, self-administration of

cocaine also induced significantly higher levels of SERT availability in the dorsal striatum

compared to control subjects (Banks et al., 2008). In fact, it has been suggested that

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chronic cocaine-produced alterations in the serotonergic neurotransmission may underlie

the dysphoria experienced by abstinent cocaine users leading to maintenance of drug

abuse (Baumann et al., 1995; Ghitza et al., 2007).

Our data also did not showed any differences in 5-HT levels between cocaine-

treated and control animals 1 day after cocaine withdrawal in Nac and dorsal striatum.

These results contrast with data from self-administration studies that reported decreased

5-HT in the Nac during withdrawal from cocaine self-administration (Parsons et al., 1996).

However, in accordance with our results, studies with passive administration of cocaine

also reported unchanged striatal levels of 5-HT, 1 day after 14 days of repeated

administration of cocaine (20 mg/kg/dose, i.p. every 12 h) (Johnson et al., 1993). The

experimental method in this study seems to approach more to our experimental

conditions, leading to similar results. Thus, the fact that cocaine has been passively

administrated may have contributed to the absence of differences between cocaine-

treated and control animals.

Nevertheless, our data also showed increased levels of 5-HT in the SN-VTA, 1 day

after the last administration in males chronically treated with cocaine.

The existence of a functional relationship between 5-HT and DA neurons in the

mesolimbic dopaminergic system has been confirmed (McMahon et al., 2001). Serotonin

receptor localization in mesolimbic circuits supports the possibility that 5-HT modulates

the flow of information through dopaminergic mesolimbic circuits, and thus these 5-HT/DA

interactions may participate in the elicitation of cocaine effects.

Neuroanatomical, electrophysiological and biochemical evidence is provided for an

interaction between serotonergic and dopaminergic systems in different brain regions,

including the SN-VTA. Dorsal raphe stimulation or local application of 5-HT agonists and

antagonists to the SN or the VTA, modulates the electrophysiological activity of

dopaminergic neurons (Kelland et al., 1990; Trent and Tepper, 1991; Pessia et al., 1994;

Prisco et al., 1994; Brodie and Bunney, 1996; Minabe et al., 1996; Cameron et al., 1997).

The 5-HT-induced release of DA has been demonstrated in vitro, in striatal (Blandina et

al., 1989) and nigral slices (Williams and Davies, 1983) and in vivo, using microdialysis in

the Nac (Parsons and Justice, 1993b; Hallbus et al., 1997), striatum (Benloucif and

Galloway, 1991), SN (Thorre et al., 1998) and in the prefrontal cortex (Iyer and Bradberry,

1996). Based on this, the observed increased levels of 5-HT in SN-VTA in male rats may

be contributing to the higher dopaminergic response to cocaine-cues observed in male

rats, this way leading to the enhanced adolescent vulnerability to addiction.

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- Effects on the amygdala

Modulation of neuronal plasticity in the mesoaccumbens dopaminergic system,

which is thought to be essential in the mediation of the behavioural effects of drugs of

abuse (Koob et al., 1998; Di Chiara et al., 2004), has been demonstrated in a number of

studies (Wolf et al., 2004; Jones and Bonci, 2005). However, psychostimulant dugs also

influence plasticity in memory-related brain regions such as the hippocampus (Thompson

et al., 2002; Thompson et al., 2005) and the amygdala (Goussakov et al., 2006).

The amygdala is an important component of prefrontal cortex networks by its

involvement in decision-making processes as it encodes the emotional value/intensity of

environmental stimuli and outcome–action associations (Winston et al., 2005; De Martino

et al., 2006). Basolateral amygdala neurons relay this information by excitatory efferents to

pyramidal neurons in the medial prefrontal cortex (Krettek and Price, 1977; Gabbott et al.,

2006; Orozco-Cabal et al., 2006). However, after chronic cocaine use, basolateral

amygdala/ medial prefrontal cortex neurotransmission may become aberrant as cocaine-

dependent individuals exhibit inappropriate utilization of emotional information to guide

behaviour (Schoenbaum et al., 2006).

Among the various neurotransmitter inputs to the amygdala, several lines of

evidence have implicated dopaminergic inputs from the VTA as having a crucial

importance (Brinley-Reed and McDonald, 1999). Amygdala also expresses both the D1

and D2 receptor (Meador-Woodruff et al., 1991; Scibilia et al., 1992). Within the

amygdala, DA stimulates D1 and D2 receptors post-synaptically on glutamatergic

projection neurons (Rosenkranz and Grace, 2002) and GABAergic interneurons (Bissiere

et al., 2003), as well as pre-synaptically on D2 DA autoreceptors (Bull et al., 1991).

In the present study no differences were observed in levels of DA or its metabolites

in amygdala 30 minutes after the last cocaine administration in both males and females as

compared with controls. Wilson et al. (1994) reported that DA and 5-HT levels, 1 h after

the last cocaine session, were significantly elevated in amygdala of rats self-administering

cocaine, but not in rats passively injected, provide additional support for the involvement

of the amygdala in the acquisition of drug seeking behaviour associated with cocaine self-

administration.

One day after the end of cocaine administration the levels of DA were not different

between cocaine-treated males and controls in the amygdala. However, cocaine-treated

adolescent females, as compared to control females, displayed increased levels of DA in

the amygdala 1 day after the last administration of cocaine.

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It was already reported that the environmental stimuli predictive of cocaine

availability elevate extracellular DA levels in the amygdala (Weiss et al., 2000).

Suggesting that increased DA transmission in this brain region may be one of the

neurobiological mechanisms underlying cocaine craving associated with exposure to

drug-related stimuli. Moreover, rats systemically administered D1 DA receptor

antagonists, reduce cocaine-seeking behaviour and Fos expression in the basolateral

amygdala in the presence of a cocaine-predictive stimulus (Ciccocioppo et al., 2001).

Additionally, intra basolateral amygdala infusion of D1 receptor antagonist abolishes the

expression of conditioned-cued reinstatement of cocaine-seeking behaviour (See et al.,

2001). While basolateral amygdala infusion of amphetamine potentiates it (Ledford et al.,

2003). Evidence from functional magnetic resonance imaging and positron-emission

tomography studies in humans also showed that cue-induced cocaine craving is

associated with activation of DA-rich forebrain regions including the amygdala (Grant et

al., 1996; Childress et al., 1999). Thus, the increased levels of DA displayed by females

may be related with the exposure, at the time of decapitation, to an environment stimulus

paired with cocaine administration. Our results seem to point to a sex-effect on the

response to cocaine-related stimuli 1 day after the last administration of cocaine, with

females showing a greater involvement of the amygdala.

In this study the levels of 5-HT in the amygdala were also measured 30 minutes

and 1 day after the last administration of cocaine. No differences were observed between

cocaine-treated and controls animals, both in male and female adolescent rats. As already

mentioned, Wilson et al. (1994) reported that when compared to the controls, in rats self-

administering cocaine, 5-HT levels 1 h after the last cocaine session, were significantly

elevated in amygdala of rats self-administering cocaine, but not of rats passively exposed

to cocaine.

- Effects on the prefrontal cortex

It is known that acute cocaine dose-dependently increase DA in the prefrontal

cortex (Pum et al., 2007). However, repeated cocaine administration has been shown to

attenuate the increase in prefrontal cortex extracellular DA levels elicited by a systemic

cocaine injection (Sorg et al., 1997; Chefer et al., 2000; Williams and Steketee, 2005b).

Our results did not show any difference in DA or its metabolites levels 30 minutes

after a binge administration in the prefrontal cortex. This could be related with the reported

attenuated increases in extracellular DA after chronic treatment. Like in our study, others

have found that the DA response to an acute cocaine injection 1 day after a repeated

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cocaine treatment (cocaine 4x15mg/kg/day i.p. for 4 days) was blunted (Williams and

Steketee, 2005b). Moreover, it has been reported that repeated cocaine produced no

statistically significant changes in baseline extracellular DA levels within the prefrontal

cortex (Sorg et al., 1997; Chefer et al., 2000; Williams and Steketee, 2005b, a). Based on

this, the lower increases in DA were probably the result of cocaine-mediated reductions in

the release of DA. It was demonstrated that this is not associated with significant changes

in DA clearance or DAT protein, but may be related with an hyporesponsivity in

mesocortical neurons mediated by a transient attenuation in depolarization-induced DA

release (Williams and Steketee, 2005a).

It was first proposed that a potential mechanism underlying the attenuation in

cocaine-induced DA overflow in the prefrontal cortex could be that repeated exposure to

cocaine would enhance DA clearance within prefrontal cortex nerve terminals. However a

recent study (although in contrast to previous findings for the Nac and dorsal striatum)

showed that repeated cocaine was not associated with significant changes of DAT protein

expression in either whole synaptosomes or the plasma membranes of the medial

prefrontal cortex (Williams and Steketee, 2005a). It appears possible that other effects of

cocaine may be taking place presynaptically in the medial prefrontal cortex at the level of

the dopaminergic nerve terminals, including cocaine-mediated changes in the DA

synthesis, vesicular storage, or autoregulation of release, which have been shown to be

altered in other mesocorticolimbic and mesostriatal brain regions after repeated

administration of psychostimulants (Trulson and Ulissey, 1987; Beitner-Johnson and

Nestler, 1991; Brown et al., 2002). Thus, the low cocaine-induced increases in DA levels

may result from a decreased DA releasability rather than an enhanced DA clearance

(Williams and Steketee, 2005a).

Within the prefrontal cortex, DA released from mesocortical neurons generally

attenuates the activity of pyramidal glutamatergic neurons, the primary excitatory efferents

of the prefrontal cortex, either by its direct effect on these cells or indirectly through

stimulation of local aminobutyric acid (GABA) release from local inhibitory interneurons

(Sesack and Bunney, 1989; Cowan et al., 1994; Grobin and Deutch, 1998). Based on the

cellular and neuroanatomical arrangement of the prefrontal cortex, attenuation in cocaine-

induce increase in dopaminergic transmission in the prefrontal cortex, after repeated

cocaine exposure, could produce a disinhibition of the prefrontal cortex pyramidal neurons

resulting in an increased excitatory output to subcortical regions. Enhanced excitatory

transmission in the medial prefrontal cortex has previously been associated with

neuroadaptive processes, within the VTA, involved in the induction of sensitization (Wolf

et al., 1995; Li et al., 1999). Furthermore, prefrontal glutamate release to the Nac has

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been shown to be critical for the expression of sensitization (Pierce et al., 1996; Pierce et

al., 1998) and the reinstatement of cocaine self-administration (McFarland et al., 2003).

In our study, 1 day after the last administration of cocaine the levels of DA in

cocaine-treated animals did not differ from controls, this is in agreement with other studies

already mentioned, that reported no significant changes in baseline extracellular DA levels

within the prefrontal cortex after repeated cocaine administration (Sorg et al., 1997;

Chefer et al., 2000; Williams and Steketee, 2005b, a).

It is also known that acute cocaine dose-dependently increases 5-HT in the

prefrontal cortex (Pum et al., 2007). However, it has been reported that chronic cocaine

does not alter the basal levels of cortical 5-HT (Baumann et al., 1993; Johnson et al.,

1993). However, it causes regionally specific alterations in 5-HT uptake binding

(Cunningham and Callahan, 1991). In the prefrontal cortex it was showed a cocaine-

induced elevation in 5-HT uptake sites (Cunningham et al., 1996). Recently, it was

reported that prolonged cocaine administration increases SERT terminal density in the

cortex and reduced 5-HT1A autoreceptors binding on serotonergic neurons projecting

from raphe, suggesting that for serotonergic neurons the autoreceptor feedback would

likely diminish following long-term treatment with cocaine (Horne et al., 2008). This

mechanism is likely to contribute towards the sprouting of serotonergic terminals. Thus,

the absence of differences between cocaine- and saline-treated animals, both 30 minutes

and 1 day after the last administration, observed in our study, may be related with

cocaine-induced adaptive processes in the serotonergic function within this area.

Prefrontal cortex serotonergic system plays a pivotal role in the locomotor

stimulant effect of cocaine (Nakamura et al., 2006). A role of the medial prefrontal cortex

in the mediation of the conditioned preference for environments associated with

psychostimulant drugs it has also been demonstrated. Excitotoxic lesions of the medial

prefrontal cortex blocked the acquisition of conditioned place preference (Tzschentke and

Schmidt, 1999). Pum et al. (2008) went further by showing that conditioned place

preference may be mediated, at least in part, by the serotonergic innervation of the medial

prefrontal cortex. Depletion of 5-HT in the medial prefrontal cortex significantly attenuated

the preference for the cocaine-associated environment and the hyperlocomotor response

to cocaine (Pum et al., 2008).

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- Effects on the hippocampus

Like observed for the prefrontal cortex, in the hippocampus no differences were

found on levels of DA or its metabolites both in males and female rats chronically exposed

to cocaine, both 30 minutes and 1 day after the last administration of cocaine. The

hippocampus is an important mediator of learning and reinforcement, but its role in

cocaine effects has received little attention.

The role of DA in the hippocampus has not been extensively studied because of

the early view that the hippocampus did not receive a significant dopaminergic innervation

(Loy et al., 1980). However, it is now clear that the hippocampus does receive such

innervation (Gasbarri et al., 1994) and there has been progress in understanding its

function. Specifically, work in the field of synaptic plasticity has provided clear evidence

that DA affects long-term potentiation (LTP), a form of synaptic plasticity thought to

encode long-term memory. The hippocampus receives dopaminergic inputs which arrive

both from the SN and the VTA (Scatton et al., 1980). Thus, cocaine-induced alterations in

the dopaminergic function may influence the hippocampus function.

It was expected that cocaine would enhances the levels of DA in hippocampus,

since Thompson et al. (2005) reported that cocaine can enhanced LTP magnitude in a

hippocampal slice assay, and that this modulation of LTP is probably a result of cocaine’s

blockade of DAT that promotes enhanced catecholamine transmitter levels, leading to the

activations of D2-like receptors. The D2 DA receptor subtype has previously been

characterized as having positive effects on LTP (Frey et al., 1990).

However, in the present study, the levels of DA were not increased 30 minutes

after the last administration of cocaine, which may be related with adaptations to the

chronic cocaine exposure already mentioned for other brain regions, such as the reduced

density of dopaminergic terminals reported by Horne at al. (2008) for the striatum (Horne

et al., 2008), or a decrease in cocaine-induced raise in DA levels in result of a decreased

DA releasability (as proposed for prefrontal cortex (Williams and Steketee, 2005a)).

Nevertheless alterations on the hippocampal function after cocaine exposure may

contribute to the maintenance of drug use. Several findings indicate that the ventral

hippocampus plays an important role in the relapse to cocaine-seeking behaviour (Rogers

and See, 2007). Selective inhibition of the ventral hippocampus attenuates relapse to

cocaine seeking following presentation of drug-paired cues or a priming injection of

cocaine itself (Rogers and See, 2007). One obvious source of regulation is the

dopaminergic input to ventral hippocampus. Lisman and Grace (2005) have recently

suggested that DA critically gates the amount of information processed by the CA1

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subregion of the hippocampus. However, the hippocampus is also involved in the

modulation of other DA-innervated regions implicated in cocaine addiction (Rogers and

See, 2007). In fact, stimulation of the ventral subiculum of the hippocampal formation

induces long-lasting DA release in the NAc (Brudzynski and Gibson, 1997; Legault et al.,

2000) and enhances the firing of mesolimbic DA neurons that originate in the VTA (Todd

and Grace, 1999; Legault et al., 2000). This Nac DA increase after ventral subiculum

stimulation depends on VTA glutamate. In fact, Nac DA increase is blocked by the

nonselective ionotropic glutamate receptor antagonist, kynurenic acid, when applied into

the VTA (Legault et al., 2000). Increased impulse flow through VTA DA neurons mediated

by excitatory glutamate inputs to this region, appears critical for ventral subiculum

stimulation to elevate Nac DA (Legault et al., 2000).

The substantial serotonergic innervation of the hippocampus (Jacobs and Azmitia,

1992), which is known to regulated hippocampal activity (Azmitia et al., 1984), raises the

possibility that cocaine also influences serotonergic transmission in the hippocampus. In

the present study, no differences were found in 5-HT as well as in 5-HIAA levels, 30

minutes after the last administration of cocaine, in both male and female adolescent rats.

In contrast, it is accepted that cocaine increases the extracellular 5-HT concentration in

the hippocampus when administrate acutely (Muller et al., 2002; Muller et al., 2004).

However, the chronic effects on 5-HT concentration are mostly unknown. Probably the

absence of differences between cocaine-treated and control animals reflects the

development of adaptation processes.

The activity of the principal cells in the hippocampus is under control of GABAergic

interneurons (Freund and Antal, 1988; Freund et al., 1990). The cocaine-induced increase

in extracellular 5-HT concentration is likely to cause a strong activation of hippocampal 5-

HT1A receptors and thus an inhibition of the GABAergic interneurons (Gulyas et al.,

1999). A reduced GABAergic inhibition of the principal cells of the hippocampus increases

activity in this region (Gulyas et al., 1999), leading to the activation of Nac neurons (Yang

and Mogenson, 1984) and DA release in the Nac (Legault et al., 2000).

Cocaine-induced alterations in the activity of hippocampus, through the interaction

with Nac and VTA, will probably contribute to the cocaine reward effects and to the

addiction state.

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- Effects on the dorsal raphe nucleus

The dorsal raphe nucleus is a major source of 5-HT in the mammalian brain

(Jacobs and Azmitia, 1992). A previous study has bound that in the raphe, under basal

conditions, 5-HT release was proportional to 5-HT recapture, leading to constant

extracellular pools of 5-HT (Adell et al., 1991). As the raphe is poor in 5-HT nerve

terminals, but sensitive to SERT blockade, it was suggested that SERT should be present

in 5-HT soma and dendrites (Adell et al., 1991). Cocaine action on blocking SERT

disrupts this equilibrium between intra- and extracellular levels of 5-HT.

In the present study, data from dorsal raphe nucleus showed that, 30 minutes after

the last administration of cocaine, males presented decreased 5-HT turnover. This may

indicate an acute effect of cocaine blocking SERT, preventing by this way the 5-HT

uptake, lowering the intracellular concentration of 5-HT and therefore reducing the levels

of 5-HT available for metabolization. In cocaine-treated females, the levels of 5-HT

decreased from 30 minutes to 1 day after the last administration, moreover at 30 minutes

these animals presented higher levels of 5-HT that almost reached significance as

compared to controls (p<0.055), this may also be explained by the acute effects of

cocaine in inhibiting the 5-HT reuptake, decreasing the 5-HT metabolization into 5-HIAA.

In fact, in cocaine exposed females the 5-HT turnover increased from 30 minutes to 1 day

after the last administration. Our data, seems to indicate that both males and females

treated with cocaine, presented extracellular increased levels of 5-HT after the last

administration of cocaine. Repeated cocaine administration has been shown to induce

increases of both 5-HT and DA in the dorsal raphe nucleus after an i.p. injection (Parsons

and Justice, 1993a). Based in this data, the absence of cocaine-induced increases in 5-

HT levels in the other analyzed brain regions seems to be mostly related with processes

occurring at terminal areas, and not mainly dependent on 5-HT synthesis area.

5-HT neurons of the dorsal raphe receive a dense dopaminergic innervation from

midbrain DA neurons (Peyron et al., 1995; Kitahama et al., 2000) and express D2-like DA

receptors (D2, D3) (Mansour et al., 1990; Suzuki et al., 1998). These anatomical features

suggest that the DA input to the dorsal raphe may play a critical role in the regulation of

the function of dorsal raphe serotonergic neurons. Thus, alterations on dopaminergic

function induced by repeated cocaine administration could interfere with serotonergic

function on dorsal raphe nucleus. Consistent with this notion, in vivo neurochemical

studies have reported that administration of DA receptor agonists increases the synthesis

and release of 5-HT in the dorsal raphe nucleus (Ferre et al., 1994; Matsumoto et al.,

1996). Direct evidence for a role of DA in the regulation of serotonergic neurons function

comes from a recent in vitro electrophysiological study showing that activation of DA

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receptors induces a membrane depolarization and thereby increases the excitability of

dorsal raphe nucleus serotonergic neurons (Haj-Dahmane, 2001). This excitatory effect is

mediated via the activation of DA D2-like receptors located on dorsal raphe nucleus 5-HT

neurons. In contrast to the midbrain DA neurons, activation of D2-like DA receptors in the

dorsal raphe nucleus strongly depolarizes and enhances the excitability of 5-HT neurons

(Haj-Dahmane, 2001). Our results did not showed any effect on the levels of DA our its

metabolites, both at 30 minutes and 1 day after the last administration of cocaine, both in

male and female adolescent rats.

- Effects on the cerebellum

Only a few studies have documented the effects of cocaine in the cerebellum.

Functional brain-imaging studies in cocaine-dependent subjects noted impairments in the

cerebellum (Gottschalk and Kosten, 2002), and an animal study has reported cocaine-

induced microscopic lesions in cerebellum (Barroso-Moguel et al., 2002). Through

development in a rat model, we have previously reported altered serotonergic turnover in

the cerebellum (Summavielle et al., 2004). Moreover, the cerebellum is activated after

exposure to cues eliciting cocaine craving (Grant et al., 1996; Wang et al., 1999).

Cerebellar dysfunction may result in neuropsychological deficits as well as impairments in

fine motor control that have been observed in cocaine abusers (Smelson et al., 1999;

Gottwald et al., 2004; Hester and Garavan, 2004).

In the present study the levels of DA and its metabolites, DOPAC and HVA, were

also analyzed in cerebellum, a brain region with low concentration of DAT (Glaser et al.,

2006). We have observed only for exposed males a decrease in DA and HVA levels

between 30 minutes and 1 day after administration, this may result for the acute effects of

cocaine of increase the extracellular levels of DA.

Although rat cerebellum is known to receive dense serotonergic projections

(Takeuchi et al., 1982; Bishop and Ho, 1985), our data did not revealed alteration on 5-HT

levels in cocaine-treated males. However, we have previously reported that neonatal

cocaine exposure affected the serotonergic cerebellar system by increasing its 5-HT

content (Summavielle et al., 2004). Repeated cocaine has been shown to increase the

expression of 5-HT3 receptor in cerebellum, which may be a consequence of an abnormal

serotonergic system (Arpin-Bott et al., 2006). Cocaine-induced increase in 5-HT in

cerebellum may be involved in counterbalancing cell damage that occurs in response to

cocaine administration.

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- Effects on the hypothalamus

Within the hypothalamus our data also showed that the 5-HT turnover rate was

decreased in cocaine-treated males 30 minutes after the last administration of cocaine,

probably due to the acute effect of cocaine, inhibiting the 5-HT reuptake, decreasing its

metabolization, and leading to increased extracellular levels of 5-HT. In fact, this animals

presented a rise in the levels of 5-HIAA from 30 minutes to 1 day after the last

administration. Moreover, 1 day after the last administration of cocaine the 5-HT turnover

rate was no longer altered. In females, this effect was not observed.

It is known that 5-HT has stimulatory effects on CRH induction in hypothalamus

(Buckingham and Hodges, 1979; Hanley and Van de Kar, 2003; Mikkelsen et al., 2004).

Thus, probably the altered HPA axis function observed 30 minutes after the last cocaine

administration in male rats, as also indicated by the increased plasma levels of ACTH,

was related with an alteration in serotonergic neurotransmission in the hypothalamus.

The HPA axis activation by either acute or chronic “binge” cocaine is apparently

mediated through the DA receptors (Borowsky and Kuhn, 1991; Spangler et al., 1997;

Zhou et al., 2001; Zhou et al., 2004). Cocaine-induced CRH mRNA increase was absent

in rats pretreated with either a selective D1-like or D2-like DA receptor antagonist,

suggesting that cocaine-induced stimulation of hypothalamic CRH gene expression is

secondary to changes in the activity of specific components of dopaminergic systems

(Zhou et al., 2004). In our study the levels of DA and its metabolites, when measured in

hypothalamus, did not reveal any significant differences between cocaine-treated and

control animals.

- Conclusion

In conclusion, the reduced number of alterations on dopaminergic function verified

30 minutes after the last administration of cocaine seems to indicate the development of

tolerance to the acute effects of cocaine. These results follow the majority studies on

chronic binge cocaine administration that report, only slight increases in extracellular

levels of DA after cocaine administration. The immaturity of the dopaminergic system in

the adolescents, and therefore increased plasticity, may explain why in this model there

are even less short-term alterations than in the adult.

To the development of tolerance, 1 day after the end of the administration another

factor must be taken into account to explain the lack of changes on dopaminergic system

in the most areas analyzed and the increase in the dopaminergic activity in SN-VTA and

in the dorsal striatum in males and in amigdala in female rats. In fact, the presentation of a

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cocaine related stimuli, may mask the expected decrease in dopaminergic

neurotransmission, justifying the absence of more pronounced differences.

Likewise, the serotonergic neurotransmission, 30 minutes after the last cocaine

administration presented only increased 5-HT in the dorsal raphe nucleus, suggesting that

the absence of cocaine-induced alterations in serotonergic neurotransmission in the other

brain regions was related with adaptive process occurring at terminal areas. Additionally,

we observed decreased 5-HT turnover in the hypothalamus, which may be related with

the increased HPA axis activity observed in cocaine-treated male rats. At 1 day after the

last administration the levels of 5-HT were increased in SN-VTA, this effect may had

contribute to the high dopaminergic activity observed in this area, at this assessment time

point.

3.4.4. Effects of chronic cocaine treatment during adolescence on the anxiogenic-like behaviour

The abrupt discontinuation of cocaine use prompts the expression of various

withdrawal symptoms, including depression, fatigue, parasomnias, anhedonia, anxiety,

and craving (Gawin and Kleber, 1986; Weddington et al., 1990; Satel et al., 1991).

Previous works carried out during adulthood have shown that withdrawal from

cocaine administration induces anxiety-like behaviour in rodents (Basso et al., 1999;

Paine et al., 2002). Our data failed to demonstrate anxiety-like behaviour after chronic

cocaine administration during adolescence, through classic EPM measured 2 days after

the last administration of cocaine. However, we have observed, in treated males,

unexpected risk-like behaviour that seems to indicate loss of risk-assessment.

The number of open arm entries and the time spent in open arms, taken as an

index of anxiety, were not affected by the cocaine treatment. Other authors have shown

that chronic cocaine administration itself failed to induce anxiety in the EPM, however

during the withdrawal period, anxiety-like behaviour was observed through decreased

time spent in open arms and decreased number of entries into the open arms were 48 h

after cocaine treatment (20 mg/ kg cocaine i.p., once a day for 14 days in rats) (Sarnyai et

al., 1995). Also, using a different paradigm, conditioned defensive burying, it was

demonstrated that severe anxiety developed within 48 h after the last cocaine injection (20

mg/kg/day, 14 days) (Harris and Aston-Jones, 1993).

Our work was conducted using adolescent rats, and several studies have shown

that cocaine induces different effects on cocaine-related behaviour across ages (Collins

and Izenwasser, 2002; Kerstetter and Kantak, 2007). In contrast to the adults,

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periadolescent rats did not show behavioural sensitization to cocaine (Collins and

Izenwasser, 2002). A similar response was observed in amphetamine exposed rats, which

showed diminished behavioural responsiveness to amphetamine during periadolescence

(PND 30-40) (Bolanos et al., 1998). Moreover, adolescent animals are known to have a

unique behavioural profile even in response to an acute administration of

psychostimulants. A profile of hyporesponsitivity to locomotor effects of an acute

administration of psycostimulants has been reported (Spear and Brake, 1983; Adriani and

Laviola, 2000). Adolescent rodents markedly differ from adults in their spontaneous

behaviour (Laviola et al., 1999; Spear, 2000), and this also could be the reason for the

lack of anxiety-like response after cocaine withdrawal observed in our results.

Additionally, aging rats prenatally exposed to either high-dose cocaine

(40mg/kg/day) or high-dose nicotine (5.0 mg/kg/day) were less timid/more impulsive,

spending most of the time in the open arms and having an high percentage of entries into

the open arms of the EPM (Sobrian et al., 2003). Moreover, in a recent work in which

adolescence mice were used, it was shown that the group-housed mice treated with a

binge pattern of cocaine were, when adults (2 weeks after the last administration), less

anxious than those treated only with saline. Interesting, such effect was not present in the

corresponding adult-treated animals (Estelles et al., 2007). All together these data

reinforces the notion that the timing of exposure strongly affects the cocaine-induced

anxiety.

Importantly, in our study, cocaine-treated male rats performed behaviours like fell-

or jump-off the apparatus and walk on the top of the closed arms during the EPM. These

unexpected behaviours were assembled in one category that we call risk-like behaviour.

This happened with an elevated frequency, six out of nine animals showed these

behaviours, and is strengthened by the fact that neither females nor male saline controls

expressed any type of risk-like behaviour. On the other hand, cocaine exposed male rats

also showed a decrease in the time spent in the central platform and a trend towards

decreased number of central platform entries. The behavioural repertoire of cocaine-

treated male rats observed during acute withdrawal is likely to be a consequence of a less

cautious behaviour while exploring the novel environment provided by the EPM

apparatus. Moreover, the risk-like behaviour was independent of any change in locomotor

activity, as there were no differences in the number of the closed arm entries, the most

reliable measure of locomotor activity on the maze (Hogg, 1996). This lack of

disturbances in motor activity during cocaine withdrawal is consistent with data obtained

from animal (Sarnyai et al., 1995) and human studies (Gawin and Kleber, 1986), and

supports the hypothesis that cocaine withdrawal during adolescence significantly impairs

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male rats risk-assessment. This is an extremely relevant result since impaired risk-

assessment may have important consequences in human adolescents.

Although elevated levels of novelty seeking (Adriani et al., 1998) and risk taking

(Macri et al., 2002), as well as reduced harm avoidance (Macri et al., 2002), are exhibited

by rodents during adolescence, our data seems to indicate that the adolescent high risk

typical behaviour was exacerbated during acute cocaine withdrawal. On the other hand,

such behavioural profile was not observed in female rats, which could be due to the fact

that, as in humans (Byrnes et al., 1999), male rats engage in more risk-taking behaviour

than females. In fact, marked sex differences were found before in mice, with males

showing significantly higher levels of novelty seeking than females (Palanza et al., 2001).

Another interesting observation is that in the cocaine exposed group, females

showed increased frequency of open arm entries, as well as an increase in the number of

closed arms entries and total arm entries. The increased frequency of closed and total

arm entries may reflect higher locomotor activity (Hogg, 1996). While increased frequency

of open arm entries is seen as an increase in anxiety levels in cocaine-treated females.

Therefore, an important sex difference was revealed by the EPM test, which may be

resultant from hormonal alterations induced by cocaine.

When looking at the possible effects of cocaine in the HPA axis it was seen that

plasma levels of corticosterone after the EPM, performed 2 days after withdrawal, were

not affected. Moreover, neuroendocrine basal data also showed no differences in basal

corticosterone plasma levels 1 day after cocaine withdrawal. These results may suggest

that the observed alterations on the EPM were not a consequence of an altered pattern of

corticosterone release.

A number of explanations can be presented for the absence of apparent

alterations of the HPA axis response during cocaine withdrawal observed in our study.

The rise in corticosterone after exposure to the novel environment provided by the EPM

apparatus could be so large that it obscures the drug treatment-induced alterations in the

increase of corticosterone (Schramm-Sapyta et al., 2006). On the other hand, age related

differences in activity of the HPA axis have been observed (Adriani and Laviola, 2000).

Adolescents have higher corticosterone levels at baseline than do adults, and such higher

baseline could contribute to a reduced percentage of increase compared to adults (Adriani

and Laviola, 2000; Schramm-Sapyta et al., 2006). Moreover, the HPA axis seems to be

somewhat hyporesponsive to external perturbations during adolescence. As shown in a

former study of response to forced novelty where acute amphetamine produce increase in

161

corticosterone release in adult mice, but only a trend toward slight elevation in adolescent

subjects (Adriani and Laviola, 2000).

Data from neurochemical analyses indicate that the DA and 5-HT metabolism were

altered in the prefrontal cortex of male rats chronically exposed to cocaine after the EPM,

as indicated by the increased DA and 5-HT turnover rates. In the amygdala there was also

an alteration on dopaminergic neurotransmission as indicated by the increased levels of

HVA levels in the same animals.

The dopaminergic system in the prefrontal cortex has been found to be involved in

the response to anxiogenic and stressfull situations in rats (Taylor et al., 1982). Decreases

in DA metabolism in the frontal cortex, but not striatum or nucleus accumbens, were

reported for up to 6 weeks following the discontinuation of chronic cocaine administration

in rats (Karoum et al., 1990), suggesting a persistent and specific down-regulation of DA

production in the frontal cortex (Goeders, 1997). Moreover, withdrawal from repeated

cocaine administration elicits some long-term neuroadaptations in the prefrontal cortex,

including reduced D2 receptor-mediated regulation of cortical excitability and reduced

responsivity of cortical cells to phasic increases in DA (Nogueira et al., 2006). The

prefrontal cortex is among the brain regions showing particularly marked ontogenetic

alterations during adolescence, with this region showing considerable synaptic culling and

a prominent loss of excitatory input (Bourgeois et al., 1994).

It is important to stress that, DA projections to the mesolimbic brain regions and

the prefrontal cortex are part of the circuitry critical for modulating risk taking, novelty

seeking, social behaviours (e.g., Le Moal and Simon, 1991) and motivational attaching to

reward (e.g., Robinson and Berridge, 2003). Because adolescents lack sufficient cortical

regulation, their behaviours tend to be more impulsive and guided by emotion impulses,

increasing risky-behaviour taking (Chambers et al., 2003). Moreover, it has been

demonstrated that prefrontocortical DA activation is necessary for coping with anxiogenic

challenges, allowing the animal to display adaptive behavioural responses (Espejo, 1997).

Therefore, the increased DA turnover rate in the prefrontal cortex we have observed, may

be involved in the impaired risk-assessment presented by cocaine exposed male rats

during the EPM.

The serotonergic system is associated with anxiety-related behaviour, with clinical

and animal studies showing increasing evidence that serotonergic neurotransmission in

the CNS modulates depression and anxiety-related behaviours (Eison, 1990; Millan et al.,

1997; van Megen et al., 1997). Results of microdialysis studies showed that aversive

conditions, such as the EPM and the conditioned fear-induced freezing behaviour, are

162

accompanied by increased 5-HT release in the prefrontal cortex (Yoshioka et al., 1995;

Inoue et al., 1996; Hashimoto et al., 1999) . Confirming this, the augmented 5-HT release

could be reduced by anxiolytic agents such as diazepam (Yoshioka et al., 1995). Rats

with lesions in the prefrontal cortex showed lower anxiety-related behaviour than their

sham-lesioned counterparts in social interaction and EPM (Gonzalez et al., 2000). Several

studies have been proposing a controlling role of 5-HT and the prefrontal cortex in

anxiety-related behaviour (Hashimoto et al., 1999; Gonzalez et al., 2000; Roberts and

Wallis, 2000), suggesting that the increase of 5-HT release in the prefrontal cortex under

aversive conditions does not mediate the “feeling” of anxiety but helps to cope with it.

Extracellular levels of 5-HT in the prefrontal cortex increases during conditioned fear-

induced freezing and this increase is followed by a resolution of the freezing (Hashimoto

et al., 1999). Therefore, the increased 5-HT turnover rate in the prefrontal cortex of

cocaine exposed male rats, which reflects a decreased availability of 5-HT, can also be

involved in the failure of these animals to cope with the challenge provided by the EPM,

suggesting, once again, a relation between cocaine-induced disruption of the prefrontal

cortex and the impaired risk-assessment.

The neurochemical data also showed an alteration in the dopaminergic system in

amygdala in chronically exposed males, as indicated by the increased levels of HVA

observed in this brain area. The amygdala has long been implicated in stimulus-reward

processes drug seeking behaviour. Exposure to a cocaine-related environment increased

the expression of c-fos in the amygdala (Neisewander et al., 2000), while amygdala

lesions disrupted acquisition of cocaine conditioned place preference (Fuchs et al., 2002).

More specifically, the amygdala dopaminergic system is involved in the cocaine self-

administration behaviour (McGregor and Roberts, 1993; Hurd et al., 1997; Hurd and

Ponten, 2000). Amygdala is also a critical structure in the neural system necessary for

implementing advantageous decisions. Amygdalar damage leads to decision-making

impairment, and this impairment is an indirect consequence of the role of the amygdala in

attaching affective attributes to stimuli (Bechara et al., 1999). It has been demonstrated

that monkeys with lesions of the amygdala have an increased tendency to approach

objects such as snakes (Zola-Morgan et al., 1991), as if the object of fear can no longer

evoke a state of fear (Bechara et al., 1999). In rats, numerous studies showed that

damaging different amygdala nuclei interferes with the processing of affective attributes of

reward stimuli (Hatfield et al., 1996). In the light of these data, the non-adaptive behaviour

observed in EPM in cocaine-treated males may be also a result of an inability to

experience sufficiently the emotional attributes of the situation provided by the EPM,

induce by an altered dopaminergic neurotransmission.

163

Adolescence is a period of increased impulsivity, which can be characterized by

decision making involving choices of high risks and low benefits (Chambers et al., 2003).

The perceived aversive consequences of risk-taking are usually reduced in adolescence

compared to young adulthood (Schramm-Sapyta et al., 2006). Recent evidence has linked

exposure to addictive drugs to an inability to use information about adverse consequences

or outcomes. For instance, addicts and drug-experienced animals fail to adapt their

behaviour to avoid adverse outcomes in gambling and reversal tasks or after changes in

the value of expected rewards (Grant et al., 2000; Bechara, 2001; Clark and Robbins,

2002).Our results do point to a cocaine-induced increase in the risk-taking behaviour in

adolescents male rats. This factor may contribute to the increased vulnerability of

adolescents to use drugs and to the maintenance of drug use, and therefore, these results

are not only biologically relevant, but also may contribute to the development of more

successful intervention strategies.

3.4.5. Effects of chronic cocaine treatment during adolescence on the depressive-like behaviour

Previous work in adult animals has shown that withdrawal from cocaine induces

depressive-like behaviour (Gawin and Kleber, 1986; Markou et al., 1992). Increased

passive behaviour responses in the FST such as immobility and decreased active

behaviours like swimming or struggling, are thought to be a clear indication of depressive-

like symptomatology (Detke et al., 1995; Lopez-Rubalcava and Lucki, 2000). However, in

our study these measures were not affected by cocaine treatment.

Following the criteria established to define immobility we were not able to observe

altered immobility behaviour in none of the studied group of animals. Former works have

shown that there was an age-dependent difference in the amount of time spent in

immobility in a forced swim test (Bilitzke and Church, 1992; Molina et al., 1994), with

juveniles showing very little evidence of immobility in contrast to adults (Hansen-Trench

and Barron, 2005). However, at least one previous study reported immobility response in

adolescent rats (Cannizzaro et al., 2007). The absence of immobility behaviour observed

could be due to methodological differences in experimental procedures, such as

apparatus dimensions, water temperature, and/or criteria for defining immobility. However,

it should be noted that there were no treatment effects in the time spent struggling in both

males and females, which allow us to propose that the behavioural strategy adopted to

cope with the stressful condition provide by the FST was not affected by the cocaine

treatment and withdrawal. The absence of significant differences between control and

164

cocaine exposed rats, may be related with the reduced behavioural and physiological

response to stressful situations in adolescent rodents (Laviola et al., 2002; Macri et al.,

2002). On the other hand, to perform hormonal measurements our rats had to be housed

in isolation and despite our efforts to minimize depressive effects through improved

handling, the isolation could interfere with the outcomes in the FST, hiding possible effects

of cocaine treatment regarding a depression-like behaviour development.

Beside the evaluation of the behavioural response to FST, alterations in

physiological responses to the stressful situation provide by the FST during withdrawal

from chronic cocaine administration were also analysed. Like in the EPM, corticosterone

levels after the FST were not significantly increased in treated animals, which supports the

already discussed possibilities: a large rise in corticosterone after exposure to the novel

environment provided by the test may obscures a possible drug treatment-induced rise in

corticosterone; the elevated basal levels of corticosterone present in adolescents or the

age-related discontinuities in the response of the HPA axis to both stress and

psychostimulants could also be reasons for the absence of alterations of the HPA axis

response to FST.

The neurochemical data indicated that the DA turnover rate in SN-VTA was higher

in cocaine-treated male rats as compared with control males. It is known that response to

stress implicates alterations in dopaminergic activity (Watanabe, 1984; Kalivas and

Abhold, 1987; Castro and Zigmond, 2001), thus an inadequate dopaminergic response to

stress may lead to a non adaptive response to a stressful stimulus. However, despite the

effect of cocaine treatment in the dopaminergic neurotransmission in the SN-VTA, no

effects of cocaine treatment were observed in the strategy adopted to cope with the

stressful situation provided by the FST.

Regarding the serotonergic system, we have determined, in the amygdala, within

the control group an increase in the 5-HT turnover rate males. The increase in

serotonergic activity has been reported in response to other stressors (Dunn, 1988; Inoue

et al., 1994). In fact, such effect has already been demonstrated in the amygdala of adult

male rats as a result of FST exposure (Connor et al., 1997). Nevertheless, data presented

in here reveals a sex effect in the serotonergic activity in control animals that was not

observed in cocaine exposed rats, which may be a consequence of cocaine treatment and

its withdrawal.

Together, these results seem to show that cocaine treatment during the

adolescence did affect the neurotransmitter systems response to the FST.

165

In conclusion, although withdrawal from chronic cocaine administration during

adolescence seems not to have a significant effect on stress-induced behavioural

alterations, neurochemical data indicate that the response to the stressful situation

provided by the FST was affected. These results are substantially different from the data

obtained in studies carried out during the adulthood, which do showed stronger influence

of cocaine in stress response (Collins and Izenwasser, 2002; O'Dell et al., 2007). These

provides further evidence supporting previous findings suggesting that adolescents

respond differently from older animals to both stress and psychostimulants (Infurna and

Spear, 1979; Adriani and Laviola, 2000; Stansfield and Kirstein, 2005). This age related

effect could be one of the reasons for the elevated prevalence of drug use and

dependence during adolescence (Schramm-Sapyta et al., 2006). Indeed, age-related

discontinuities in response to psychological stress have been indicated as a

psychobiological risk for increased vulnerability to drug abuse (Laviola et al., 1995; Adriani

et al., 1998).

3.5. Conclusion

Overall, our data lead us to conclude that the chronic exposure to cocaine

throughout adolescence produced a sex-dependent hormonal, neurochemical and

behavioural alterations.

Acute cocaine binge administration after 15 days of treatment increased the HPA

activity in male rats, as indicated by the elevated ACTH levels observed 30 minutes after

cocaine exposure. However, the HPA axis activity, evaluated 1 day after the last

administration of cocaine, did not showed to be affected in neither males nor females.

Although HPA axis activation during cocaine withdrawal has been previously

demonstrated with a variety of paradigms, our data support the idea that age-related

discontinuity in the HPA axis function may be underlying a hypoactivation of the HPA axis

during the adolescence period.

Regarding the HPG axis activity, chronic cocaine exposure throughout

adolescence also led to short-term disruption also in male rats, as indicated by the

decreased testosterone levels 30 minutes after the last administration of cocaine.

Although 1 day after withdrawal this alteration was no longer observed our long-term data

(see chapter 4) reveals important changes in the HPG axis function at later stages. In

adolescent female rats the chronic binge cocaine administration seems not to affect the

HPG axis as observed through unaltered estradiol or progesterone levels.

166

Neurochemical data revealed the development of tolerance in the dopaminergic

and serotonergic responses to the acute effects of cocaine administration.

Data obtained 1 day after the last administration, revealed an increased

dopaminergic activity, which led us to suggest that the expected decrease in

dopaminergic activity after chronic cocaine, could have been masked by the cocaine

cues-induced increases in dopaminergic function, since animals were sacrificed in the

same room where cocaine was administered. This may indicate a high susceptibility of

adolescent rats to environmental cues, with females showing a greater involvement of

amygdala in this process, while males showed a greater involvement of the nigrostriatal

pathway.

In the EPM it was observed an impaired risk-assessment in male rats. The

disrupted risk-assessment may be related with the high rates of prevalence of drug uses

during adolescence, and with increased vulnerability to relapse to drug use after cocaine

exposure throughout this developmental period. Along with impaired risk-assessment it

was observed a disrupted dopaminergic and serotonergic transmission in prefrontal cortex

and dopaminergic neurotransmission in amygdala. These neurochemical alterations may

be involved in the failure of cocaine-treated male rats to exhibit an adaptive behaviour

during the EPM.

In agreement, the 5-HT and DA response to the FST, in male rats, seems to

support the inability of animals to cope with stress.

In conclusion, the adaptive processes developed in dopaminergic and serotonergic

responses, the elevated susceptibility to the cocaine-cues, the observed impairment in

risk-assessment and the absence of depressive behaviour after cocaine treatment

throughout adolescence, are all most likely important factors contributing to the

adolescent high vulnerability to drug abuse.

These data, have increased relevance for the social-understanding of the

relationship between adolescents and drug use, and may be crucial for the design of new

strategies of drug abuse prevention in the adolescent.

167

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Chapter 4

Long-term Effects of Cocaine throughout Adolescence in the Rat

4.1. Introduction

Long-lasting effects of cocaine exposure have already been reported in an

extensive body of literature. There are numerous reports of long-lasting neurobiological

and behavioural consequences of the prenatal cocaine exposure, which last to adult life.

For example, prenatal cocaine exposure is reported to produce depression and anxiety in

aging rats (for reviews, see Sobrian et al., 2003), to produce alterations on aggressive

behaviour in adult rats (Johns et al., 1994), and to result in increased rewarding effects of

cocaine in the adult mice (Malanga et al., 2008). In adult animals, long-term

neurobiological and behavioural abnormalities induced by cocaine exposure, such as

dopaminergic neuroadaptations (Ungless et al., 2001; Kimmel et al., 2003; Nogueira et al.,

2006) and enhanced levels of anxiety behaviour (Erb et al., 2006), were also reported.

During adolescence, the developing brain may be more vulnerable to long-lasting

modifications by drug exposure (Andersen, 2003). In fact, a major concern in adolescent

psychostimulant abuse is the long-term consequence of this practice, since early drug

exposure may induce long-term adaptations, which, among other effects, may render the

organism more susceptible to drug abuse later in life (Spear, 2000). In fact, epidemiologic

data showed that drug use in adolescence is a reliable predictor of drug abuse in

adulthood (Merline et al., 2004). Studies in rodents show that early developmental

exposure to psychostimulants causes behavioural alterations in sensitivity to drugs of

abuse and natural rewards that endure into adulthood (Andersen et al., 2002; Bolanos et

al., 2003), raising the possibility that early exposure to psychostimulants can have long-

term negative consequences (Carlezon and Konradi, 2004). However, in some studies,

the adolescent brain appears resilient, showing few long-term effects of substance abuse

(Santucci et al., 2004).

The propose to determine whether adolescence represents a window of

vulnerability to the deleterious effects of cocaine or a period of increased resilience to the

effects of cocaine leading to weak long-lasting effects, represents a current and very

important issue of investigation.

The work described in this chapter was performed in order to evaluate the long-

term effects of chronic cocaine exposure during the adolescence period upon the activity

of the HPA and the HPG axes of both male and female wistar rats, by assessing the

hormonal plasma levels 5 and 10 days after cocaine withdrawal. The effects of cocaine

exposure were also evaluated upon the dopaminergic and serotonergic systems by

assessing the monoamines levels, in areas known to be involved with drug addiction, also

5 and 10 days after cocaine withdrawal.

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The discontinuation of cocaine use leads to the expression of various withdrawal

symptoms, including depression, anxiety, and craving (Gawin and Kleber, 1986;

Weddington et al., 1990; Satel et al., 1991; Coffey et al., 2000). Moreover, these

symptoms provide part of the negative reinforcement associated with cocaine

dependence and are important motivational factors for relapse and maintenance of

repetitive cycles of cocaine abuse (Gawin et al., 1989; Shaham et al., 2000; Shalev et al.,

2002). Thus, it was defined the objective of evaluate the effects of chronic cocaine

exposure during adolescence period, on behavioural strategies adopted to cope with an

environmental anxiogenic situation during withdrawal period, by submitting the animals 5

and 10 days after withdrawal to the EPM.

Psychostimulant drugs such as cocaine also have profound and long-lasting

effects on social behaviours (Wood and Spear, 1998; Estelles et al., 2005). Thus,

evaluate the long-term effects of cocaine administration during adolescence on the

aggressive behaviour, was also defined as an objective by testing male rats 10 days after

withdrawal in the resident-intruder paradigm.

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4.2. Material and Methods

4.2.1. Animals

Using the animal paradigm described in Chapter 2, we have obtained animals

chronically exposed to cocaine during adolescence that were used in the following

experiments. Briefly, on PND 30 animals were housed individually, and were randomly

assigned to the different experimental groups. Only one male and one female from each

litter were used in each experimental group. Rats were treated following a binge pattern

administration of cocaine, each day being injected 3 times intraperitonealy (i.p.) with

15mg/kg of body weight cocaine hydrochloride dissolved in 0.9% NaCl solution from PND

35 to PND 50. The daily injections were administered at hourly intervals, between 9:00

and 11:00 a.m., in a volume of 1 mL/kg. Controls were given an isovolumetric 0.9% NaCl

solution following the same experimental protocol. During the entire period of drug

administration animals were daily weighed. At least 6 animals were used in each

experimental group.

4.2.2. Behavioural tests

4.2.2.1. Elevated plus maze test

On PND 55 and on PND 60, 5 and 10 days after withdrawal, male Wistar rats were

tested in an EPM apparatus as described in Chapter 2. Spatiotemporal and behavioural

parameters were registered at the time of behaviour analyses. Spatiotemporal measures

comprised the frequencies of total, open and closed arm entries and central platform

entries, as well as the time spent in the open, closed and central part of the maze.

Behavioural measures comprised frequency and duration scores for rearing, head dipping

and grooming.

4.2.2.2. Resident-intruder paradigm

On PND 60, 10 days after cocaine withdrawal, aggressive behaviour of male rats

was evaluated using the resident-intruder paradigm, as described in Chapter 2.

Behavioural activity during test was analysed, and frequency, duration and latency data

were collected for the following behaviours divided in to three categories: (1) offensive

behaviours that included attack, bite, pinning, wrestling, chasing and aggressive

grooming; (2) defensive behaviours that included flight, avoidance of contact and

189

submissive-supine posture; (3) social investigation behaviours including sniffing,

anogenital sniffing, and approach behaviour.

4.2.3. Blood and tissue collection

Five and 10 day after the last administration of cocaine, and after the behavioural

tests, animals were sacrificed by decapitation and trunk blood was collected in lithium

heparin cover tubes and centrifuged at 1600g for 15 minutes; the obtained plasma was

kept at -70ºC until hormonal assays were conducted. Brains were also rapidly removed

and SN-VTA, Nac, dorsal striatum, prefrontal cortex, amygdala, hippocampus, dorsal

raphe nucleus, cerebellum and a hypothalamic section containing the suprachiasmatic,

pre-optic and paraventricular nuclei were dissected on ice. Pituitary glands were also

collected. Tissue samples were frozen by immersion in 2-methylbutane cooled over dry

ice and stored at -70ºC until used for further analyses. Five and 10 day after the last

administration of cocaine gonads and adrenal glands were collected and weighed, and the

data obtained was registered.

4.2.4. Hormone plasma levels determinations

The levels of progesterone, estradiol, testosterone, corticosterone, and ACTH,

were measured on plasma obtained 5 and 10 days after withdrawal. The levels of

corticosterone were also measured on the plasma obtained after the behavioural tests. All

hormones plasma levels were measured by enzyme immunoassay, using commercially

available assays.

The progesterone, estradiol, testosterone and corticosterone plasma levels were

measured using assays provided by Cayman Chemical Company (Ann Arbor, MI, USA).

For progesterone, the assay sensitivity was 0.012 ng/mL, and the intra- and inter-assay

coefficients of variation were less 8% and 12%, respectively. For estradiol, the assay

sensitivity was 0.004 ng/mL, and the intra- and inter-assay coefficients of variation were

less 10% and 12%, respectively. For testosterone, the assay sensitivity was 0.004 ng/mL,

and the intra- and inter-assay coefficients of variation were less 10% and 15%,

respectively. For corticosterone, the assay sensitivity was 0.04 ng/mL, and the intra- and

inter-assay coefficients of variation were less than 7% and 9%, respectively. The ACTH

plasma levels were measured using an assay provided by Phoenix Pharmaceuticals, Inc.

(Belmont, California, USA). The ACTH assay sensitivity was 0.18 ng/mL, and the intra-

and inter-assay coefficients of variation were less than 5 % and 14%, respectively.

190

Corticosterone plasma levels after the behavioural tests were measured using a kit

provided by Immunodiagnostic Systems Ltd (Boldon, UK). Assay sensitivity was 0.23

ng/mL. The intra- and inter-assay coefficients of variation were less than 3.1% and 7.2%,

respectively.

4.2.5. Neurochemical determinations

Concentrations of DA and 5-HT and their metabolites, 5-HIAA, DOPAC, and HVA

were quantified by HPLC-EC, in the dissected brain areas (SN-VTA, Nac, dorsal striatum,

prefrontal cortex, amygdala, hippocampus, dorsal raphe nucleus, cerebellum and

hypothalamus) collected 5 and 10 days after withdrawal, as described in Chapter 2. After

each behavioural test, levels of DA and 5-HT and their metabolites were determined in the

SN-VTA, dorsal raphe nucleus, amygdala, hippocampus, prefrontal cortex and

hypothalamus. Concentrations are expressed in ng/mg protein. The ratios of DOPAC to

DA, HVA to DA and DOPAC and HVA to DA were determined for each animal and used

as indexes of DA turnover rate, whereas the ratio of 5-HIAA to 5-HT was used as an index

of 5-HT turnover rate.

4.2.6. mRNA expression levels determination

The expression levels of mRNA transcripts for tyrosine hydroxylase (TH) were

measured in SN-VTA, Nac and dorsal striatum, and the expression levels of mRNA

transcripts for monoamine oxidase-A (MOA-A), DA transporter (DAT), DA receptors (D1,

D2, D3) and GR were measured in the SN-VTA, Nac, dorsal striatum, prefrontal cortex,

amygdala, hippocampus and hypothalamus, brain areas dissected from males sacrificed

10 days after last the administration, by qRT-PCR, as described in Chapter 2. Additionally,

the expression levels of mRNA transcripts for CRH and androgen receptor (AR) were

measured in the hypothalamus, and the expression levels of mRNA transcripts for CRH

receptor 1 (CRH-R1), GnRH receptor (GnRH-R), AR, LHβ, GR and proopiomelanocortin

(POMC) were measure in the pituitary.

The reference gene, glyceraldahyde-3-phosphate-dehydrogenase (GAPDH) was

used as internal standard for normalization.

Primer sequences and annealing temperatures (Ta) for each gene are presented

in table 4.2.2.4.1 and table 4.2.2.4.2.

191

Table 4.2.2.4.1- Primer pairs and their sequences, PCR segment size and Ta used in the qRT-PCR to study the expression of dopaminergic- related mRNAs.

Gene Primer sequence Size (bp) Tm (ºC) Ta (ºC)

GAPDH Sense: 5’-ttc aac ggc aca gtc aag g-3’

Antisense: 5’-ctc agc acc agc atc acc-3’ 114 75.9 55

TH Sense: 5’-ggc ttc tct gac cag gtg tat c-3’

Antisense: 5’-caa tct ctt ccg ctg tgt att cc-3’ 112 75.5 55

DAT Sense: 5’-gtc att gtt ctg ctc tac ttc-3’

Antisense: 5’-gtc cac act gag gta tgc-3’ 165 78.2 55

MAO-A Sense: 5’-ggc aca gag aca gca aca c-3’

Antisense: 5’-cag acc agg cac gga agg-3’ 204 77.5 59

D1 Sense: 5’-act ctg tct gtc ctt ata tcc ttc-3’

Antisense: 5’-gtt gtc atc ctc ggt gtc c-3’ 114 75.2 55

D2 Sense: 5’-caa caa tac aga cca gaa tgc gtg-3’

Antisense: 5’-cag cag agt gac gat gaa gg-3’ 100 72.8 55

D3 Sense: 5’-gtc cgc acg cct act acg-3’

Antisense: 5’-cag cac agc agc aca tac c-3’ 86 75.9 55

GR Sense: 5’-gga cag cct gac ttc ctt gg-3’

Antisense: 5’-tcc agg gct tga gta ccc at-3’ 76 74.2 55

D1- dopamine receptor 1; D2- dopamine receptor 2; D3- dopamine receptor 3; DAT- dopamine transporter; GAPDH- Glyceraldahyde-3-phosphate-dehydrogenase; GR- Glucocorticoid receptor; MAO-A- Monoamine oxidase A; TH- Tyrosine hydroxilase; Ta- anneling temperature; Tm- melting temperature.

192

Table 4.2.2.4.2- Primer pairs and their sequences, PCR segment size and Ta used in the qRT-PCR to study the the expression of HPA and HPG axes- related mRNAs.

Gene Primer sequence Size (bp) Tm (ºC) Ta (ºC)

GAPDH Sense: 5’-ttc aac ggc aca gtc aag g-3’

Antisense: 5’-ctc agc acc agc atc acc-3’ 114 75.9 55

CRH Sense: 5’-gga gaa gag aaa gga gaa gag g-3’

Antisense: 5’-aga atc ggc tga ggt tgc tg-3’ 283 83.3 62

CRH-R1 Sense: 5’-ctt ctt ctg gat gtt cgg tga g-3’

Antisense: 5’-atg agg atg cgg aca atg ttg-3’ 279 78.9 59

GnRH-R Sense: 5’-aac atc agc agt aac aga gac-3’

Antisense: 5’-aga ggc att gaa ggc agt ag-3’ 216 76.9 55

AR Sense: 5’-aag cca ttg agc cag gag tg-3’

Antisense: 5’-tta gtg aag gac cgc caa cc-3’ 235 78.5 55

POMC Sense: 5’-gaa gcg gcg ccc tgt gaa-3’

Antisense: 5’-ctc gcc ttc cag ctc cct ctt-3’ 94 78.5 59

LHβ Sense: 5’-cta ctg tcc tag cat ggt tcg-3’

Antisense: 5’-gga agg tca cag gtc att gg-3’ 231 82.7 59

GR Sense: 5’-gga cag cct gac ttc ctt gg-3’

Antisense: 5’-tcc agg gct tga gta ccc at-3’ 76 74.2 55

Ar- Androgen receptor; CRH- Corticotrophin-releasing hormone; CRH-R1- Corticotrophin-releasing hormone receptor 1; GAPDH- Glyceraldahyde-3-phosphate-dehydrogenase; GnRH-R- Gonadotropin-releasing hormone receptor; GR- Glucocorticoid receptor; LHβ- Luteinizing hormone β-subunit; POMC- Proopiomelanocortin; Ta- anneling temperature; Tm- melting temperature.

4.2.7. Statistical analysis

A 2-way ANOVA with treatment (cocaine or saline) and sex as between-subject

fixed-factors with repeated measures was used to evaluate body weight evolution during

the experimental period until long-term withdrawal. Student’s t-test was used as a post-

hoc test to analyse statistical significant differences.

Adrenal and gonadal weights were converted in percentage of body weight, and

adrenal data was analyzed by a 3-way ANOVA with time point of assessment (5 and 10

days), treatment and sex as between-subject fixed-factors. Gonadal data was separated

by sex and analyzed by a 2-way ANOVA (time point of assessment (5 and 10 days) x

treatment).

For hormonal and neurochemical data obtained at 5 and 10 days after withdrawal,

obtained after the EPM and after the aggression test, statistical significance between

groups was assessed using the non-parametric Mann-Whitney U Test.

193

Student’s t-test was used to compare the expression levels of the mRNAs,

obtained by qRT-PCR, between groups.

The EPM behaviour data was analysed by a 2-way ANOVA with treatment

(cocaine or saline) and time point of assessment (5 and 10 days after withdrawal) as

between-subject fixed-factors and behavioural measurements as the dependent variable.

Student’s t-test was used as a post-hoc test to analyse statistical significant differences.

Analysis of behaviour data obtained in the aggression test was performed using

the Student’s t-test.

Differences were considered to be statistically significant when p<0.05. All data are

expressed as the mean plus standard error of the mean (S.E.M).

Statistical analyses were performed using the software SPSS 14.0 (SPSS INC. Chicago, Illinois, USA).

194

4.3. Results

4.3.1. Basal analyses

4.3.1.1. Biometric measures

Evolution of body weight. Cocaine-treated and control animals were weight

everyday from PND 35 to PND 50, and on PND 55 and PND 60. Data were analyzed with

a 2-way ANOVA (treatment x sex) with repeated measures. The body weight gain was

statistically different between male and female rats [F(1,20)= 92.787; p<0.001]), post-hoc

comparisons using Student’s t-test confirm that from PND 38 until the end of exposure

period and on PND 55 and PND 60 the weight gain was higher in males than in females

(figure 4.3.1.1.1.). The 2-way ANOVA with repeated measures also revealed that there

were no differences in body weight gain between cocaine-treated males and control males

and between cocaine-treated and control females.

0

50

100

150

200

35 37 39 41 43 45 47 49 55 60

PND

Wei

ght g

ain

(g)


Figure 4.3.1.1.1- Effects of chronic cocaine exposure on body weight gain during exposure period and

withdrawal. Values are mean ± S.E.M.. Saline male, n=7; cocaine male, n=7; saline female, n=5; cocaine

female, n=5. PND- postnatal day. &, sex effect; p<0.001.

Adrenal weight. Adrenals were collected 5 and 10 days after cocaine withdrawal

and their weights were recorded. The weight data was converted in percentage of body

weight and were analyzed with a 3-way ANOVA (time point of assessment- 5 or 10 days x

treatment x sex). The statistical analysis revealed a main effect of sex in the adrenal

weight [F(1,46)=97.731; p<0.001] (figure 4.3.1.1.2). The adrenal glands weight in females

was higher than in males. No treatment effects were found in males or females. The

adrenal weight also did not differ between 5 and 10 days after cocaine withdrawal.

&

/ /

195

Adrenal glands

0

0.02

0.04

0.06

5 days 10 days

% o

f bod

y w

eigh

t


Figure 4.3.1.1.2- Effects of chronic cocaine exposure throughout adolescence on adrenal weight of male and

female rats assessed 5 and 10 days after the withdrawal. Each column represents the mean + S.E.M. for a

least 6 animals per group.

Gonadal weight. Testes and ovaries were collected 5 and 10 days after withdrawal

of cocaine and their weight were recorded. The percentage of body weight was calculated

and data was analyzed by a 2-way ANOVA (age x treatment). In male group a 2-way

ANOVA revealed an interaction between time point of assessment (5 and 10 days) x

treatment [F(1,24)=4.306; p<0.05], a post-hoc analysis using Student’s t-test indicate that

at 10 days after withdrawal the testes weight were lower in cocaine-treated males as

compared to saline males (t(10)=-2.913; p<0.02) (figure 4.3.1.1.3-A). The statistical

analysis revealed no significant effects of cocaine treatment in the weight of female

gonads, both when assessed 5 or 10 days after withdrawal (figure 4.3.1.1.3-B). Also, no

differences were observed between time points of assessment in female gonadal weight.

196

A Male gonads B Female gonads

0

0.4

0.8

1.2

5 days 10 days

% o

f bod

y w

eigh

t


00.020.040.060.08

5 days 10 days

% o

f bod

y w

eigh

t


Figure 4.3.1.1.3- Effects of chronic cocaine exposure throughout adolescence on (A) male gonads and (B)

females gonads weights assessed 5 and 10 days after cocaine withdrawal. Each column represents the

mean + S.E.M. for at least 6 animals per group. Columns with the same letter are significantly different from

each other. a p< 0.02.

4.3.1.2. Hormone plasma levels

The levels of hormones were measured on plasma obtained 5 and 10 days after

cocaine withdrawal. The data was analyzed by the non-parametric Mann-Whitney U Test.

ACTH. The plasma levels of ACTH were not significant different between cocaine-

treated males and females, and their respective controls, in the two assessment time

points (figure 4.1.2.1-A). In saline female group, the levels of ACTH increase from 5 to 10

days after the last administration [Z=-2.000 (2-tailed) p<0.05], this effect was not observed

in saline male group. Cocaine-treated males also presented an increase in levels of ACTH

from 5 days to 10 days that almost reach significance [Z=-1.939 (2-tailed) p=0.053].

Corticosterone. No significant effects of cocaine treatment were found in both

male and female rats, at the two time point assessment (figure 4.1.2.1-B).

Testosterone. In the male group, 10 days after withdrawal cocaine-treated males

presented lower levels of testosterone [Z=-2.082 (2-tailed) p<0.05]. Saline-treated males

displayed an increase in testosterone levels from 5 to 10 days after the last administration

[Z=-2.562 (2-tailed) p<0.02], this effect was not present in cocaine-treated males. No

significant differences were observed in testosterone levels between cocaine-treated and

saline-treated females at both time points of assessment (figure 4.3.1.2.2-B).

Progesterone. In the saline-treated females the levels of progesterone decreased

from 5 to 10 days after the last administration [Z=-2.242 (2-tailed) p<0.05]. This effect was

not observed in cocaine-treated females. In fact, 10 days after withdrawal, cocaine-treated

females presented increased levels of progesterone as compared to saline females [Z=-

2.166 (2-tailed) p<0.05]. No significant differences were found in the plasma levels of

a a

197

progesterone between cocaine- and saline-treated males at the both time point

assessment (figure 4.3.1.2.2- C).

Estradiol. In the saline-treated female group, the levels of estradiol increased

from 5 to 10 days after the end of the administration period [Z=-2.571 (2-tailed) p<0.02]

(figure 4.3.1.1.2-E). This effect was not observed in cocaine-treated females.

A

0

2

4

6

8

5 days 10 days

AC

TH le

vels

(ng/

mL)


B

0

20

40

60

80

100

5 days 10 days

Cor

ticos

tero

ne le

vels

(ng/

mL)


Figure 4.3.1.2.1- Effects of chronic cocaine exposure throughout adolescence on (A) ACTH and (B)

corticosterone plasma levels assessed 5 and 10 days after withdrawal. Each column represents the mean +

S.E.M. for at least 5 animals per group. ACTH- Adrenocorticotropic hormone. Columns with the same letter

are significantly different from each other, a p<0.05.

a

a

198

A B

01234

5 days 10 daysTest

oste

rone

leve

ls

(ng/

mL)


0.00

0.05

0.10

0.15

5 days 10 daysTest

oste

rone

leve

ls

(ng/

mL)


C D

0

0.2

0.4

0.6

5 days 10 daysProg

este

rone

leve

ls

(ng/

mL)


0

50

100

150

5 days 10 daysProg

este

rone

leve

ls

(ng/

mL)


E

0

15

30

45

5 days 10 days

Estra

diol

leve

ls

(ng/

mL)


Figure 4.3.1.2.2- Effects of chronic cocaine exposure throughout adolescence on plasma levels of

testosterone in (A) males and (B) females, progesterone in (C) males and (D) females and estradiol in (E)

females, assessed 5 and 10 days after cocaine withdrawal. Each column represents the mean + S.E.M. for at

least 5 animals per group. Columns with the same letter are significantly different from each other. a,e p<0.02, b,c,d p<0.05.

4.3.1.3. Neurochemical determinations

The levels of DA, 5-HT and their metabolites, DOPAC, HVA and 5-HIAA were

measured, by HPLC-EC, 5 and 10 days after the end of the exposure period, in the

following brain regions: SN-VTA, Nac, dorsal striatum, prefrontal cortex, hippocampus,

amygdala, hypothalamus, dorsal raphe nucleus and cerebellum. These different brain

areas were selected based on their involvement in cocaine response. The data obtained

were analyzed by the non-parametric Mann-Whitney U Test.

a,b

b a

c,d

d

c

e

e

199

Within the SN-VTA, statistical analysis revealed that 10 days after withdrawal,

cocaine-treated males displayed higher levels of DOPAC as compared to saline males

and cocaine females ([Z=-2.082 (2-tailed) p<0.037] and [Z=-2.429 (2-tailed) p<0.02],

respectively) (figure 4.3.1.3.1). Cocaine-treated males also displayed an increase in 5-HT

levels from 5 to 10 days after the end of treatment [Z=-2.593 (2-tailed) p<0.02]. At 5 days

after withdrawal, no differences were detected in the levels of DA, 5-HT and their

metabolites between cocaine-treated animals and their respective controls, also no

differences were found in DA and 5-HT turnover rates between the experimental groups.

Within the Nac, no differences were detected in the levels of DA and 5-HT and

their metabolites between cocaine-treated animals and their respective controls at both

time points of analysis (figure 4.3.1.3.2). Also, no differences were found in both DA and

5-HT turnover rates between the experimental groups (table 4.3.1.3.2). However, in

cocaine-treated female group the levels of DA, 5-HT and 5-HIAA increased from 5 to 10

days after the end of treatment ([Z=-1.981 (2-tailed) p<0.05], [Z=-2.236 (2-tailed) p<0.05]

and [Z=-2.108 (2-tailed) p<0.05], respectively).

200

SN-VTA

A

0

200

400

600

800


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days

B

0

200

400

600

800


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days



determined by HPLC-EC for male and female rats, 5 and 10 days after withdrawal. Each column represents


per group. Columns with the same letter are significantly different from each other. a p<0.05, b,c p<0.02.

a a,b b

c

c

201

Nucleus accumbens

A

0

1500

3000

4500

6000


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days

B

0

200

400

600


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days


metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the Nac, determined

by HPLC-EC for male and female rats, 5 and 10 days after withdrawal. Each column represents the mean +


Columns with the same letter are significantly different from each other. a,b,c p<0.05.

a

a

b

b

c

c

202


[5-HIAA]/[5-HT], in the SN-VTA, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.16±0.01

(n=8)

0.21±0.02

(n=9)

0.16±0.01

(n=7)

0.23±0.05

(n=7)

0.24±0.06

(n=6)

0.16±0.02

(n=6)

0.16±0.02

(n=5)

0.14±0.01

(n=7)

HVA/DA 0.09±0.02

(n=8)

0.11±0.02

(n=9)

0.07±0.01

(n=7)

0.13±0.03

(n=7)

0.09±0.02

(n=6)

0.07±0.02

(n=6)

0.09±0.02

(n=5)

0.07±0.01

(n=7)


(n=8)

0.32±0.03

(n=9)

0.23±0.02

(n=7)

0.36±0.07

(n=7)

0.33±0.05

(n=6)

0.24±0.04

(n=6)

0.25±0.04

(n=5)

0.22±0.02

(n=7)

5-HIAA/5-HT 0.27±0.03

(n=8)

0.24±0.03

(n=9)

0.28±0.08

(n=7)

0.40±0.11

(n=7)

0,28±0.06

(n=6)

0.22±0.03

(n=6)

0.16±0.02

(n=5)

0.57±0.33

(n=7)



homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid.


[5-HIAA]/[5-HT], in the Nac, at 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.25±0.03

(n=8)

0.28±0.03

(n=9)

0.28±0.03

(n=7)

0.30±0.06

(n=7)

0.28±0.03

(n=6)

0.27±0.03

(n=5)

0.24±0.02

(n=6)

0.25±0.01

(n=7)

HVA/DA 0.07±0.01

(n=8)

0.10±0.01

(n=9)

0.08±0.01

(n=7)

0.11±0.03

(n=7)

0.08±0.01

(n=6)

0.07±0.01

(n=5)

0.06±0.01

(n=6)

0.08±0.01

(n=7)


(n=8)

0.38±0.04

(n=9)

0.36±0.03

(n=7)

0.41±0.09

(n=7)

0.36±0.04

(n=6)

0.33±0.03

(n=5)

0.29±0.02

(n=6)

0.33±0.02

(n=7)

5-HIAA/5-HT 0.30±0.14

(n=8)

0.37±0.12

(n=9)

0.30±0.09

(n=7)

0.33±0.12

(n=7)

0.34±0.19

(n=6)

0.59±0.27

(n=5)

0.45±0.16

(n=6)

0.34±0.12

(n=7)



Within the dorsal striatum, 5 days after withdrawal no differences were detected in

the levels of DA, 5-HT and their metabolites between cocaine-treated animals and their

respective controls. Ten days after withdrawal cocaine-treated males presented higher

HVA levels as compared to control males [Z=-2.082 (2-tailed) p<0.05] (figure 4.3.1.3.3). In

saline-treated male groups the levels of HVA decreased from 5 to 10 days after the end of

treatment [Z=-2.082 (2-tailed) p<0.02], moreover, it was also observed a decrease in

HVA/DA turnover rate [Z=-2.082 (2-tailed) p<0.05]. Ten days after the last administration,

203

saline-treated males also presented lower levels of 5-HIAA as compared to saline-treated

females [Z=-2.402 (2-tailed) p<0.02], this effects was not observed in cocaine-treated

animals.

Dorsal striatum

A

0

2000

4000

6000

8000


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days

B

0

200

400

600


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days


metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the dorsal striatum,



per group. Columns with the same letter are significantly different from each other. a p<0.05, b,c p<0.02

a,b b

c

c

a

204


[5-HIAA]/[5-HT], in the dorsal striatum, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days


saline cocaine saline cocaine saline cocaine saline Cocaine

DOPAC/DA 0.23±0.03

(n=8)

0.21±0.02

(n=8)

0.19±0.03

(n=7)

0.19±0.02

(n=7)

0.21±0.01

(n=6)

0.18±0.02

(n=6)

0.19±0.03

(n=6)

0.19±0.02

(n=7)

HVA/DA 0.11±0.02

(n=8)

0.10±0.02

(n=8)

0.08±0.01

(n=7)

0.09±0.01

(n=7)

0.05±0.01

(n=6)

0.07±0.01

(n=6)

0.06±0.01

(n=6)

0.08±0.01

(n=7)


(n=8)

0.31±0.03

(n=8)

0.27±0.03

(n=7)

0.28±0.03

(n=7)

0.26±0.02

(n=6)

0.25±0.03

(n=6)

0.25±0.03

(n=6)

0.26±0.03

(n=7)

5-HIAA/5-HT 0.36±0.17

(n=8)

0.31±0.16

(n=8)

0.25±0.17

(n=7)

0.24±0.14

(n=7)

0.38±0.18

(n=6)

0.36±0.22

(n=6)

0.35±0.19

(n=6)

0.28±0.11

(n=7)




Within the prefrontal cortex, no statistical significant differences were found on the

levels of DA, 5-HT and their metabolites or their turnover between the experimental


Within the hippocampus, the statistical analysis revealed that in cocaine-treated

female group the levels of DA and 5-HIAA increased from 5 to 10 days after cocaine

withdrawal ([Z=-2.082 (2-tailed) p<0.05] and [Z=-2.242 (2-tailed) p<0.05], respectively)

(figure 4.3.1.3.5), moreover, it was also observed a trend for an increase in 5-HT turnover

rate [Z=-1.922 (2-tailed) p=0.055] (Table 4.3.1.3.5). In saline male group there was an

increase in the 5-HT turnover rate from 5 to 10 days after the last administration [Z=-2.286

(2-tailed) p<0.05]. Ten days after withdrawal, cocaine-treated males displayed lower 5-HT

turnover rate as compared to cocaine-treated females [Z=-2.082 (2-tailed) p<0.05]. In the

saline group this difference did not reach significance.

Hippocampal levels of HVA were not detectable by HPLC-EC in a considerable

number of samples. Therefore, in this brain region, only the levels of DA metabolite

DOPAC were considered in the analysis.

a a

205

Prefrontal cortex

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5 days 10 days

B

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per group.

206

Hippocampus

A

0

5

10

15

20

DA DOPAC DA DOPAC

Neu

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5 days 10 days

B

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5 days 10 days


metabolites, DOPAC and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hippocampus determined

by HPLC-EC for male and female rats, 5 and 10 days after withdrawal. Each column represents the mean +


Columns with the same letter are significantly different from each other. a,b p<0.05.

a

a

b b

207


[5-HIAA]/[5-HT], in the prefrontal cortex, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.31±0.11

(n=7)

0.40±0.15

(n=8)

0.41±0.13

(n=8)

0.56±0.15

(n=7)

0.38±0.09

(n=5)

0.31±0.03

(n=6)

0.31±0.03

(n=6)

0.36±0.05

(n=7)

HVA/DA 0.54±0.06

(n=7)

0.54±0.07

(n=8)

0.58±0.08

(n=8)

0.57±0.12

(n=7)

0.47±0.15

(n=5)

0.49±0.09

(n=6)

0.58±0.11

(n=6)

0.43±0.07

(n=7)


(n=7)

0.95±0.17

(n=8)

0.99±0.14

(n=8)

1.13±0.22

(n=7)

0.85±0.24

(n=5)

0.80±0.09

(n=6)

0.88±0.12

(n=6)

0.79±0.09

(n=7)

5-HIAA/5-HT 0.09±0.02

(n=7)

0.12±0.03

(n=8)

0.09±0.02

(n=8)

0.14±0.08

(n=7)

0.08±0.02

(n=5)

0.11±0.03

(n=6)

0.21±0.08

(n=6)

0.22±0.08

(n=7)




hippocampus, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.73±0.38

(n=7)

0.70±0.29

(n=9)

0.84±0.23

(n=7)

0.91±0.42

(n=6)

0.24±0.05

(n=6)

0.41±0.11

(n=6)

0.54±0.16

(n=6)

0.45±0.13

(n=6)

5-HIAA/5-HT 0.18±0.05

(n=7)

0.24±0.04

(n=9)

0.21±0.02

(n=7)

0.21±0.04

(n=6)

0.41±0.07

(n=6)

0.23±0.04

(n=6)

0.24±0.04

(n=6)

0.48±0.09

(n=6)


same letter are significantly different. DA- dopamine; DOPAC- 3,4-dihydroxyphenylacetic acid; 5-HT-

serotonin; 5-HIAA- 5-hydroxyindolacetic acid. a,b p<0.05.

Within the amygdala, 10 days after withdrawal, cocaine-treated females displayed

lower levels of DOPAC as compared to control females [Z=-2.191 (2-tailed) p<0.05], and

saline males presented higher levels of DOPAC as compared to saline-treated females

[Z=-2.191 (2-tailed) p<0.05] (figure 4.3.1.3.6). In saline-treated female group the levels of

DA and DOPAC decreased from 5 to 10 days after the last administration ([Z=-2.030 (2-

tailed) p<0.05] and [Z=-2.030 (2-tailed) p<0.05], respectively). No differences were found

in the data from 5-HT system between the experimental groups.

b b a a

208

Amygdala

A


0

10

20

30

80160240320

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of p

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B

0

100

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300


Neu

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(ng/

mg

of p

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in)


5 days 10 days





per group. Columns with the same letter are significantly different from each other. a,b,c,d p<0.05.

Within the hypothalamus, no statistical significant differences were found on the

levels of DA, 5-HT and their metabolites or their turnover between the experimental


c b, c, d

d

a

a b

5 days 10 days


209

Hypothalamus

A

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200

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400


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400

600


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5 days 10 days


metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hypothalamus,



per group.

210


[5-HIAA]/[5-HT], in the amygdala, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.33±0.15

(n=7)

0.25±0.06

(n=9)

0.34±0.12

(n=7)

0.30±0.10

(n=6)

0.33±0.19

(n=6)

0.34±0.13

(n=6)

0.25±0.11

(n=5)

0.44±0.27

(n=6)

HVA/DA 0.14±0.05

(n=7)

0.17±0.08

(n=9)

0.12±0.03

(n=7)

0.18±0.14

(n=6)

0.08±0.04

(n=6)

0.08±0.02

(n=6)

0.08±0.02

(n=5)

0.07±0.02

(n=6)


(n=7)

0.42±0.10

(n=9)

0.47±0.14

(n=7)

0.49±0.16

(n=6)

0.41±0.19

(n=6)

0.42±0.13

(n=6)

0.34±0.11

(n=5)

0.51±0.28

(n=6)

5-HIAA/5-HT 0.30±0.11

(n=7)

0.39±0.16

(n=9)

0.28±0.07

(n=7)

0.29±0.08

(n=6)

0.30±0.09

(n=6)

0.47±0.15

(n=6)

0.31±0.07

(n=5)

0.36±0.10

(n=6)




[5-HIAA]/[5-HT], in the hypothalamus, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.26±0.07

(n=8)

0.27±0.07

(n=9)

0.34±0.10

(n=7)

0.37±0.06

(n=6)

0.43±0.12

(n=6)

0.41±0.09

(n=5)

0.41±0.08

(n=5)

0.48±0.13

(n=7)

HVA/DA 0.09±0.04

(n=8)

0.06±0.02

(n=9)

0.09±0.03

(n=7)

0.14±0.11

(n=6)

0.03±0.01

(n=6)

0.07±0.03

(n=5)

0.03±0.01

(n=5)

0.12±0.06

(n=7)


(n=8)

0.34±0.08

(n=9)

0.43±0.10

(n=7)

0.50±0.12

(n=6)

0.45±0.12

(n=6)

0.48±0.11

(n=5)

0.44±0.08

(n=5)

0.60±0.18

(n=7)

5-HIAA/5-HT 0.25±0.03

(n=8)

0.30±0.05

(n=9)

0.28±0.08

(n=7)

0.43±0.12

(n=6)

0.31±0.09

(n=6)

0.37±0.10

(n=5)

0.25±0.03

(n=5)

0.25±0.03

(n=7)



Within the dorsal raphe nucleus, statistical analysis revealed that 5 days after the

end of exposure period saline-treated males displayed higher levels of DA as compared to

saline-treated females [Z=-2.662 (2-tailed) p<0.01] (figure 3.3.1.3.8). No other differences

were found between the experimental groups in this brain area.

211


A

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300


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5 days 10 days

B

0

350

700

1050

1400


Neu

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itter

s co

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trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days



nucleus, determined by HPLC-EC for male and female rats, 5 and 10 days after withdrawal. Each column


6 animals per group. Columns with the same letter are significantly different from each other. a p<0.01.

a

a

212


[5-HIAA]/[5-HT], in the dorsal raphe nucleus, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.50±0.24

(n=8)

0.50±0.16

(n=9)

0.46±0.12

(n=7)

0.56±0.32

(n=7)

0.89±0.39

(n=6)

0.57±0.24

(n=6)

0.73±0.31

(n=6)

0.61±0.23

(n=7)

HVA/DA 0.18±0.07

(n=8)

0.20±0.08

(n=9)

0.16±0.12

(n=7)

0.10±0.05

(n=7)

0.10±0.06

(n=6)

0.12±0.04

(n=6)

0.11±0.03

(n=6)

0.05±0.02

(n=7)


(n=8)

0.70±0.15

(n=9)

0.63±0.15

(n=7)

0.66±0.37

(n=7)

0.99±0.41

(n=6)

0.70±0.27

(n=6)

0.84±0.32

(n=6)

0.66±0.25

(n=7)

5-HIAA/5-HT 0.32±0.06

(n=8)

0.29±0.05

(n=9)

0.46±0.21

(n=7)

0.31±0.07

(n=7)

0.67±0.37

(n=6)

0.87±0.59

(n=6)

0.28±0.05

(n=6)

0.37±0.05

(n=7)



Within the cerebellum, statistical analysis revealed that in the saline male group

the levels of HVA decreased from 5 to 10 days after the last administration [Z=-2.143 (2-

tailed) p<0.05] (figure 4.3.1.3.9). No other differences were found between the

experimental groups in the monoamine levels.

213

Cerebellum

A

0

10

20

30


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5 days 10 days

B

0

20

40

60

80

100


Neu

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ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


5 days 10 days


metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the cerebellum,



per group. Columns with the same letter are significantly different from each other. a p<0.05.

a

a

214


[5-HIAA]/[5-HT], in the cerebellum, 5 and 10 days after the end of cocaine exposure period.

5 days 10 days



DOPAC/DA 0.74±0.22

(n=7)

1.12±0.42

(n=9)

0.67±0.18

(n=7)

1.71±0.53

(n=8)

1.21±0.28

(n=6)

1.71±1.19

(n=6)

1.84±1.28

(n=6)

0.74±0.15

(n=6)

HVA/DA 1.53±0.80

(n=7)

0.92±0.12

(n=9)

0.60±0.18

(n=7)

0.55±0.13

(n=8)

1.24±0.35

(n=6)

0.68±0.21

(n=6)

0.91±0.23

(n=6)

1.34±0.62

(n=6)


(n=7)

2.03±0.37

(n=9)

1.27±0.26

(n=7)

2.26±0.44

(n=8)

2.45±0.58

(n=6)

2.39±1.37

(n=6)

2.75±1.42

(n=6)

2.08±0.75

(n=6)

5-HIAA/5-HT 0.52±0.14

(n=7)

0.25±0.03

(n=9)

0.22±0.04

(n=7)

0.36±0.10

(n=8)

0.30±0.06

(n=6)

0.22±0.06

(n=6)

0.23±0.04

(n=6)

0.34±0.08

(n=6)



Overall, our basal neurochemical and hormonal data point to much more pronounced

long-term effects of cocaine in male rats than in females. Thus, further testing, namely

determination of dopaminergic-related, HPA-related and HPG-related mRNA expression

levels and behavioural studies, such as evaluation of anxiety-like and aggressive

behaviour were only performed in male rats.

4.3.1.4. mRNA expression levels determination

4.3.1.4.1. Dopaminergic-related mRNA expression levels

The expression levels of mRNAs related with synthesis (TH), metabolization

(MAO-A), transportation (DAT) of DA, and D1, D2 and D3 receptors mRNA were

assessed by qRT-PCR in male rats at 10 days after the end of exposure protocol.

Data analysis revealed that the expression levels of TH mRNA were increased in

cocaine exposed males in the SN-VTA as compared to control males [t(4.372)=-3.089;

p<0.04] (figure 4.3.1.4.1.1.). In this brain area no differences were observed in the

expression levels of the other mRNA analysed, although a trend towards increased DAT

mRNA expression was observed in cocaine-treated animals [t(10)=-2.064; p=0.066]. In

the other brain areas studied, Nac, dorsal striatum, prefrontal cortex hippocampus,

amygdala and hypothalamus, the levels of mRNA expression of TH, MAO-A, DAT and DA

receptors D1, D2 and D3 did not differ between the cocaine-treated animals and control

animals (from figure 4.3.1.4.1.1. to figure 4.3.1.4.1.7.)

215

SN-VTA

A B

0.0000.0030.0060.0090.012

TH

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.000

0.002

0.004

0.006

DAT

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.0000.0020.0040.0060.008

MAO-A

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.00000

0.00003

0.00006

0.00009

D1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E F

0.0000

0.0002

0.0004

0.0006

D2

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.0000000.0000050.0000100.0000150.000020

D3

norm

aliz

ed m

RN

A

expr

essi

on le

vels


Figure 4.3.1.4.1.1. Effects of chronic cocaine exposure throughout adolescence on the levels of mRNA

expression of (A) TH, (B) DAT, (C) MAO-A, (D) D1, (E) D2 and (F) D3 in the SN-VTA, determined by qRT-

PCR for male rats 10 days after withdrawal. Each column represents the mean + S.E.M. of normalized mRNA

expression, for at least 5 animals per group. Columns with the same letter are significantly different from each

other. a p<0.05.

a

a

216

Nucleus accumbens

A B

0.00000

0.00002

0.00004

0.00006

TH

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.00000

0.00001

0.00002

0.00003

0.00004

DAT

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.00

0.01

0.02

0.03

0.04

MAO-A

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.0

0.3

0.6

0.9

1.2

D1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E F

0.00

0.01

0.02

0.03

D2

norm

aliz

ed m

RN

A

expr

esso

n le

vels


0.00000.00020.00040.00060.00080.0010

D3

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) TH, (B) DAT, (C) MAO-A, (D) D1, (E) D2 and (F) D3 in the Nac, determined by qRT-PCR for

male rats 10 days after withdrawal. Each column represents the mean + S.E.M. of normalized mRNA

expression, for at least 5 animals per group.

217

Dorsal striatum

A B

0.000000

0.000006

0.000012

0.000018

TH

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.000000

0.000002

0.000004

0.000006

0.000008

DAT

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.000

0.025

0.050

0.075

MAO-A

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.000

0.025

0.050

0.075

D1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E F

0.0000.0050.0100.0150.0200.025

D2

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.0000

0.0001

0.0002

0.0003

0.0004

D3

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) TH, (B) DAT, (C) MAO-A, (D) D1, (E) D2 and (F) D3 in the dorsal striatum, determined by

qRT-PCR for male rats 10 days after withdrawal. Each column represents the mean + S.E.M. of normalized

mRNA expression, for at least 5 animals per group.

218

Prefrontal cortex

A B

0.0000

0.0001

0.0002

0.0003

DAT

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.0000.0050.0100.0150.0200.025

MAO-A

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.00000.00020.00040.00060.00080.0010

D1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.00000.00030.00060.00090.00120.0015

D2

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E

0.00000

0.00006

0.00012

0.00018

D3

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) DAT, (B) MAO-A, (C) D1, (D) D2 and (E) D3 in the prefrontal cortex, determined by qRT-



219

Hippocampus

A B

0.00000

0.00001

0.00002

0.00003

DAT

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.000.020.040.060.080.10

MAO-A

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.000

0.005

0.010

0.015

0.020

D1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.0000.0010.0020.0030.004

D2

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E

0.000000.000020.000040.000060.000080.00010

D3

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) DAT, (B) MAO-A, (C) D1, (D) D2 and (E) D3 in the hippocampus, determined by qRT-PCR

for male rats 10 days after withdrawal. Each column represents the mean + S.E.M. of normalized mRNA


220

Amygdala

A B

0.0000000

0.0000002

0.0000004

0.0000006

DAT

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.000

0.003

0.006

0.009

MAO-A

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.0000

0.0002

0.0004

0.0006

D1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.00000

0.00004

0.00008

0.00012

0.00016

D2

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E

0.00000000.00000030.00000060.00000090.0000012

D3

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) DAT, (B) MAO-A, (C) D1, (D) D2 and (E) D3 in the amygdala, determined by qRT-PCR for

male rats 10 days after withdrawal. Each column represents the mean + S.E.M. of normalized mRNA


221

Hypothalamus

A B

0.00000.00020.00040.00060.00080.0010

DAT

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.00

0.06

0.12

0.18

MAO-A

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.00

0.01

0.02

0.03

0.04

D1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.000

0.001

0.002

0.003

0.004

D2

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E

0.0000

0.0001

0.0002

0.0003

D3

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) DAT, (B) MAO-A, (C) D1, (D) D2 and (E) D3 in the hypothalamus, determined by qRT-PCR

for male rats 10 days after withdrawal. Each column represents the mean + S.E.M. of normalized mRNA


4.3.1.4.2. HPA and HPG axes-related mRNA expression levels

The expression levels of mRNAs related with HPA and HPG axes were assessed

by qRT-PCR in male rats 10 days after the end of the exposure protocol.

Data analysis revealed that the expression levels of GR mRNA did not differ

between cocaine-treated males and control males in all the brain areas studied, excepted

222

for the SN-VTA, where cocaine-treated males presented lower levels of GR mRNA as

compared to control males [t(8.353)=2,329; p<0.05] (figure 4.3.1.4.2.1).

Within the hypothalamus, statistical analysis by Student’s t-test showed no

differences in the mRNA expression levels of CRH and AR in the cocaine-treated males

as compared to control males (figure 4.3.1.4.2.2).

Glucocorticoids receptor

sn-vta Nac DStriatum PFC Hippoc. Amygd. Hip. Pit.

0.000

0.002

0.004

0.006

0.04

0.08

norm

aliz

ed m

RN

A e

xpre

ssio

n le

vels


expression of GR in the SN-VTA, Nac, dorsal striatum, prefrontal cortex, hippocampus, amygdala,

hypothalamus and pituitary, determined by qRT-PCR for male rats 10 days after withdrawal. Each column

represents the mean + S.E.M. of normalized mRNA expression, for at least 5 animals per group. a p<0.05.

Hypothalamus

A B

0.00

0.01

0.02

0.03

0.04

CRH

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.00

0.01

0.02

0.03

AR

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) CRH and (B) AR, in the hypothalamus, determined by qRT-PCR for male rats 10 days after

withdrawal. Each column represents the mean + S.E.M. of normalized mRNA expression, for at least 5

animals per group.


a a

223

Within the pituitary, the mRNA expression levels of CRH-R1, GnRH-R, AR, POMC

and LHβ were studied and data analysis did not reveal differences between cocaine- and

saline-treated male rats in none of mRNAs studied (figure 4.3.1.4.2.3).

Pituitary

A B

0.0000.0040.0080.0120.016

CRH-R1

norm

aliz

ed m

RN

A

expr

essi

on le

vels


0.00

0.02

0.04

0.06

GnRH-R

norm

aliz

ed m

RN

A

expr

essi

on le

vels


C D

0.0000.0050.0100.0150.020

AR

norm

aliz

ed m

RN

A

expr

essi

on le

vels


020406080

100

POMC

norm

aliz

ed m

RN

A

expr

essi

on le

vels


E

02468

LH

norm

aliz

ed m

RN

A

expr

essi

on le

vels



expression of (A) CRH-R1, (B) GnRH-R, (C) AR, (D) POMC and (E) LH in the pituitary, determined by qRT-



224

4.3.2. Behavioural studies and associated measures

4.3.2.1. Anxiety-like behaviour

Elevated plus maze test. Five and 10 days after cocaine withdrawal male rats were

tested in the EPM apparatus, in a 5 minutes session. EPM behavioural data was analysed

by a 2-way ANOVA (treatment x time point of assessment).

Statistical analysis revealed that cocaine-treated males presented no differences in

frequency of open and closed arm entries and in frequency of central platform entries,

neither 5 or 10 days after withdrawal (figure 4.3.2.1.1). Also no differences were observed

between cocaine and saline-treated male rats on the time spent in the open and closed

arms, neither 5 or 10 days after withdrawal (figure 4.3.2.1.2). ANOVA revealed a main

effect of treatment in the time spent in the central platform [F(1,32)=6.326; p<0.02].

Cocaine-treated male rats spent less time as compared to controls. Statistical analysis

also revealed that 10 days after withdrawal cocaine-treated male rats spent less time in

the central platform than controls [t(19.827)=-3.124; p<0.01] (figure 4.3.2.1.3 B).

A B

02468

5 days 10 days

Num

ber o

f ent

ries


0

20

40

60

5 days 10 days

% o

f ent

ries


C D

0

3

6

9

5 days 10 days

Num

ber o

f ent

ries


048

1216

5 days 10 days

Num

ber o

f ent

ries


Figure 4.3.2.1.1- Effects of chronic cocaine exposure throughout adolescence on the number of arm entries

during the elevated plus maze test performed 5 and 10 days after withdrawal by male rats. A- Number of open

arm entries; B- percentage of open arm entries; C- Number of closed arm entries; D- Number of total arm

entries. Each column represents the mean + S.E.M., for at least 7 animals per group.

Open arms Open arms

Closed arms Total arm entries

225

A B

0306090

120150

5 days 10 days

Dur

atio

n (s

ec.)


050

100150200250

5 days 10 days

Dur

atio

n (s

ec.)


Figure 4.3.2.1.2- Effects of chronic cocaine exposure throughout adolescence on the time spent (A) in open

arms and (B) in closed arms during the elevated plus maze test performed 5 and 10 days after withdrawal, by

male rats. Each column represents the mean + S.E.M., for at least 7 animals per group.

A B

048

1216

5 days 10 days

Num

ber o

f ent

ries


0

20

40

60

5 days 10 days

Dur

atio

n (s

ec.)


Figure 4.3.2.1.3- Effects of chronic cocaine exposure throughout adolescence (A) in the number of central

platform entries and (B) in the time spent in the central platform during the elevated plus maze test performed

5 and 10 days after cocaine withdrawal, by male rats. Each column represents the mean + S.E.M., for at least

7 animals per group. Columns with the same letter are significantly different from each other. a p<0.01.

No group differences were observed in frequency and duration of risk-like

behaviour, rearing, head-dipping and grooming during the EPM (table 4.3.2.1.1).

Open arms Closed arms

Central platform Central platform a

a

226

Table 4.3.2.1.1- Frequency and duration of behavioural categories like risk-like behaviour, rearing, head

dipping and grooming, during the 5 minutes of the elevated plus maze test, in saline- and cocaine-treated

male rats, 5 and 10 days after the last administration.

5 days 10 days

male saline

(n=7) male cocaine

(n=7) male saline

(n=11) male cocaine

(n=11)

Risk-like Behaviour frequency 0.14±0.14 0.29±0.18 0.09±0.09 0.36±0.15


Rearing frequency 15.14±1.53 16.00±2.41 16.18±1.50 13.45±1.83

duration (sec.) 32.49±4.89 39.11±6.49 42.74±4.75 31.98±4.89

Head-dipping frequency 1.86±0.67 2.71±0.36 4.73±1.10 3.36±1.05

duration (sec.) 1.30±0.58 2.74±0.71 4.75±1.68 2.68±1.07

Grooming frequency 1.71±0.36 1.71±0.42 1.27±0.19 2.09±0.65

duration (sec.) 12.34±4.21 8.57±2.47 7.90±1.87 11.30±3.70

Data expressed as the mean value ± S.E.M.; n, number of animals per group.

Corticosterone plasma levels. The animals that performed the EPM 10 days after

the end of the exposure protocol were decapitated after the behavioural test and levels of

corticosterone were measured in the obtained plasma. Statistical significance between

groups was assessed using the Student’s t-test. Analysis revealed that, 10 days after

withdrawal, cocaine-treated males had increased corticosterone plasma levels induce

after the EPM (t(16)=2.661; p<0.02) (figure 4.3.2.1.4).

0

100

200

300

Cor

ticos

tero

ne

leve

ls n

g/m

L


Figure 4.3.2.1.4- Effects of chronic cocaine exposure throughout adolescence on the alterations of the

corticosterone plasma levels induced by the elevated plus maze test 10 days after withdrawal, in male rats.

Each column represents the mean + S.E.M for at least 8 animals per group. Columns with the same letter are

significantly different from each other. a p<0.02.

a

a

227


DOPAC, HVA and 5-HIAA were measured by HPLC-EC, in the prefrontal cortex, the

hippocampus, the amygdala, the hypothalamus, the SN-VTA and the dorsal raphe

nucleus after the EPM 10 days after the end of exposure protocol. The data obtained after

the test and the basal data obtained 10 days after the end of the exposure protocol were

analyzed by the non-parametric Mann-Whitney U Test.

Within the SN-VTA, no significant differences were found on the levels of DA, 5-

HT, their metabolites or in the turnover rates between cocaine exposed and control males,

after the EPM (figure 4.3.2.1.5. and table 4.3.2.1.2). Also no significant differences were

found between basal levels and levels after the EPM.

Within the dorsal raphe nucleus, no significant differences were found on the levels

of DA, 5-HT, their metabolites or in the turnover rates between cocaine- and saline-treated

male rats after the EPM (figure 4.3.2.1.6. and table 4.3.2.1.3). After the EPM, in this brain

area, the levels of HVA were not detectable by the HPLC-EC. Statistical analysis revealed

that the levels 5-HT were higher after the EPM as compared to basal data, both in saline

and cocaine males ([Z=-2.003 (2-tailed) p<0.05] and [Z=-2.475 (2-tailed) p<0.02],

respectively) (figure 4.3.2.1.6). After the EPM, saline-treated male presented decreased 5-

HIAA/5-HT turnover rate as compared to basal conditions [Z=-2.003 (2-tailed) p<0.05]

(table 4.3.2.1.3).

228

SN-VTA

A

0

200

400

600

800


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


Basal EPM

B

0

150

300

450

600


Neu

rost

rans

mitt

ers

conc

entr

atio

n (n

g/m

g of

pro

tein

)


EPMBasal



determined 10 days after withdrawal, under basal and anxiogenic (EPM) by HPLC-EC for male rats. Each


at least 6 animals per group. Columns with the same letter are significantly different from each other. a p<0.05.

a a

229


A

0

100

200

300


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


Basal EPM

B

0

250

500

750

1000

1250

1500


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


EPMBasal



nucleus, determined 10 days after withdrawal, under basal and anxiogenic (EPM) by HPLC-EC for male rats.

Each column represents the mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of

protein, for at least 6 animals per group. nd- not detectable. Columns with the same letter are significantly

different from each other. a p<0.05, b p<0.02.

b

b

nd

a

a

230


[5-HIAA]/[5-HT], in the SN-VTA in saline- and cocaine-treated male rats, as measured 10 days after the last

administration, under basal condition and after the elevated plus maze test.

Basal EPM

saline male cocaine male saline male cocaine male

DOPAC/DA 0.24±0.06

(n=6)

0.16±0.02

(n=6)

0.21±0.01

(n=11)

0.20±0.01

(n=9)

HVA/DA 0.09±0.02

(n=6)

0.07±0.02

(n=6)

0.12±0.01

(n=11)

0.09±0.02

(n=9)


(n=6)

0.24±0.04

(n=6)

0.33±0.02

(n=11)

0.28±0.03

(n=9)

5-HIAA/5-HT 6.55±6.27

(n=6)

0.22±0.03

(n=6)

0.23±0.02

(n=11)

0.27±0.03

(n=9)




[5-HIAA]/[5-HT], in the dorsal raphe nucleus in saline- and cocaine-treated male, as measured 10 days after

the last administration, under basal condition and after the elevated plus maze test.

Basal EPM


DOPAC/DA 0.89±0.39

(n=6)

0.57±0.24

(n=6)

0.31±0.02

(n=9)

0.25±0.03

(n=9)

HVA/DA 0.10±0.06

(n=6)

0.12±0.04

(n=6) - -


(n=6)

0.70±0.27

(n=6)

- -

5-HIAA/5-HT 0.67±0.37

(n=6)

0.87±0.59

(n=6)

0.24±0.01

(n=9)

0.24±0.02

(n=9)


dihydroxyphenylacetic acid; HVA- homovanillic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. In

the same line, values with same letter are significantly different. a p<0.05.

Within the prefrontal cortex, no significant differences were found between

cocaine- and saline-treated male rats after the EPM (figure 4.3.2.1.7. and table 4.3.2.1.4).

Statistical analysis revealed that both saline- and cocaine-treated males after the EPM

presented higher HVA/DA and (DOPAC+HVA)/DA turnover rates as compared to animals

under basal conditions (saline males- HVA/DA [Z=-2.209 (2-tailed) p<0.05],

(DOPAC+HVA)/DA [Z=-2.209 (2-tailed) p<0.05]; cocaine males- HVA/DA [Z=-2.711 (2-

tailed) p<0.01], (DOPAC+HVA)/DA [Z=-2.828 (2-tailed) p<0.01]). However, cocaine-

a a

231

treated males after the EPM presented higher DOPAC/DA turnover rate as compared to

cocaine male rats under basal conditions [Z=-2.593 (2-tailed) p<0.02] and this effect was

not observed in saline-treated male rats.

Prefrontal cortex

A

0

10

20

30

40


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


Basal EPM

B

0

100

200

300

400


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


EPMBasal






232


[5-HIAA]/[5-HT], in the prefrontal cortex in saline- and cocaine-treated male, as measured 10 days after the

last administration, under basal condition and after the elevated plus maze test.

Basal EPM


DOPAC/DA 0.38±0.09

(n=5)

0.31±0.03

(n=6)

0.68±0.13

(n=11)

2.65±1.18

(n=9)

HVA/DA 0.47±0.15

(n=5)

0.49±0.09

(n=6)

0.89±0.08

(n=11)

3.58±2.59

(n=9)


(n=5)

0.80±0.09

(n=6)

1.57±0.10

(n=11)

5.83±3.24

(n=9)

5-HIAA/5-HT 0.08±0.02

(n=5)

0.11±0.03

(n=6)

0.08±0.01

(n=11)

0.11±0.01

(n=9)



the same line, values with same letter are significantly different. a p<0.02, b,d p<0.05, c,e p<0.01.

Within the hippocampus, no significant differences were found on the levels of DA,

5-HT, their metabolites and in turnover rates between cocaine- and saline-treated male

rats after the EPM (figure 4.3.2.1.8. and table 4.3.2.1.5). However, the saline-treated

males after EPM presented lower 5-HIAA/5-HT turnover rate as compared to saline males

under basal conditions [Z=-2.234 (2-tailed) p<0.05], moreover, in the cocaine-treated

animals this difference did not reach significance.

Within the amygdala, after the EPM, cocaine-treated males presented lower levels

of DOPAC and 5-HIAA as compared to saline-treated males ([Z=-3.009 (2-tailed) p<0.005]

and [Z=-1.967 (2-tailed) p<0.05], respectively) (figure 4.3.2.1.9). Saline-treated males

presented increased levels of DOPAC after EPM, as compared to data from basal

conditions [Z=-2.711 (2-tailed) p<0.01], this effects was not observed in cocaine-treated

animals.

a a

c c

e e

b b

d d

233

Hippocampus

A

0

5

10

15

20

DA DOPAC DA DOPAC

Neu

rost

rans

mitt

ers

conc

entr

atio

n (n

g/m

g of

pro

tein

)


Basal EPM

B

0

100

200

300


Neu

rotr

ansm

itter

s C

once

ntra

tion

(ng/

mg

of p

rote

in)


EPMBasal

Figure 4.3.2.1.8 - Effects of chronic cocaine exposure throughout adolescence on (A) the levels of DA, and its

metabolite DOPAC and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hippocampus, determined

10 days after withdrawal, under basal and anxiogenic (EPM) by HPLC-EC for male rats. Each column



234

Amygdala

A

0

40

80

120


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


Basal EPM

B

0

75

150

225

300


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


EPMBasal





at least 6 animals per group. Columns with the same letter are significantly different from each other. a p<0.01, b p<0.005, c p<0.05.

a,b

b

c c

a

235


hippocampus in saline- and cocaine-treated male, as measured 10 days after the last administration, under

basal condition and after the elevated plus maze test.

Basal EPM


DOPAC/DA 0.24±0.05

(n=6)

0.41±0.11

(n=6)

0.39±0.06

(n=8)

0.42±0.10

(n=6)

5-HIAA/5-HT 0.41±0.07

(n=6)

0.23±0.04

(n=6)

0.18±0.03

(n=8)

0.16±0.03

(n=6)


dihydroxyphenylacetic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. In the same line, values with

same letter are significantly different. a p<0.05.


[5-HIAA]/[5-HT], in the amygdala in saline- and cocaine-treated male, as measured 10 days after the last


Basal EPM


DOPAC/DA 0.33±0.19

(n=6)

0.34±0.13

(n=6)

0.37±0.04

(n=8)

0.36±0.07

(n=7)

HVA/DA 0.08±0.04

(n=6)

0.08±0.02

(n=6)

0.11±0.04

(n=8)

0.15±0.06

(n=7)


(n=6)

0.42±0.13

(n=6)

0.49±0.04

(n=8)

0.51±0.10

(n=7)

5-HIAA/5-HT 0.30±0.09

(n=6)

0.47±0.15

(n=6)

0.33±0.07

(n=8)

0.37±0.05

(n=7)



Within the hypothalamus, no significant differences were found on the levels of DA,

5-HT, their metabolites and in turnover rates between cocaine- and saline-treated male

rats after the EPM (figure 4.3.2.1.10. and table 4.3.2.1.7). After the EPM, in this brain area

the levels of HVA were not detectable by the HPLC-EC.

a a

236

Hypothalamus

A

0

50

100

150

200

250


Neu

rotr

ansm

itter

s co

ncen

trat

ion

(ng/

mg

of p

rote

in)


Basal EPM

B

0

200

400

600


Neu

rotr

ansm

itter

s co

ncen

trat

ions

(n

g/m

g of

pro

tein

)


EPMBasal


its metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the

hypothalamus, determined 10 days after withdrawal, under basal and anxiogenic (EPM) by HPLC-EC for male

rats. Each column represents the mean + S.E.M. of monoamine levels, expressed as ng of monoamine/mg of

protein, for at least 5 animals per group. nd- not detectable. Columns with the same letter are significantly

different from each other. a p<0.05

nd

237


[5-HIAA]/[5-HT], in the hypothalamus in saline- and cocaine-treated male, as measured 10 days after the last


Basal EPM


DOPAC/DA 0.43±0.12

(n=6)

0.41±0.09

(n=5)

0.24±0.02

(n=11)

0.21±0.04

(n=9)

HVA/DA 0.03±0.01

(n=6)

0.07±0.03

(n=5) - -


(n=6)

0.48±0.11

(n=5) - -

5-HIAA/5-HT 0.31±0.09

(n=6)

0.37±0.10

(n=5)

0.18±0.02

(n=11)

0.20±0.02

(n=9)



4.3.2.2. Aggressive behaviour

Resident-intruder paradigm. Ten days after cocaine withdrawal, aggressive

behaviour of male rats was evaluated using the resident-intruder paradigm. Behavioural

activity during test was analysed by the Student’s t-test.

Data analysis revealed that cocaine-treated males presented no differences in

frequency, duration and latency of the offensive behaviours (attack, bite, pinning,

wrestling, chasing and aggressive grooming) (figure 4.3.2.2.1). Cocaine males displayed

lower frequency of flight behaviour, a behaviour from the social investigation behaviour

category, [t(14)=-2.430; p<0.05], the latency to this behaviour was also increased in

cocaine-treated males as compared to control males [t(7.493)=2.346; p<0.05] (figure

4.3.2.2.2-A and C). Student’s t-test also revealed that the duration of the anogenital

sniffing behaviour, a behaviour from defensive behaviour category, was increased in

cocaine-treated males [t(14)=2.474; p<0.05] (figure 4.3.2.2.3-B).

238

Offensive behaviour

0

10

20

30

40

50

attack bite pinning w restling chasing aggressivegrooming

Freq

uenc

y


0

30

60

90

120

pinning w restling chasing agressive grooming

Dur

atio

n (s

ec.)


0

200

400

600

attack bite pinning w restling chasing aggressivegrooming

Late

ncy

(sec

.)


Figure 4.3.2.2.1- Effects of chronic cocaine exposure throughout adolescence on (A) frequency, (B) duration

and (C) latency of offensive behaviours exhibited by the resident animal during the resident-intruder paradigm

performed 10 days after the end of exposure protocol. Each column represents the mean + S.E.M., for at least


A

B

C

239

Defensive behaviour

0

30

60

90

flight avoidance of contact supine posture

Freq

uenc

y


0

20

40

60

80

100


Dur

atio

n (s

ec.)


0

150

300

450


Late

ncy

(sec

.)



and (C) latency of defensive behaviours performed by the resident animal during the resident-intruder

paradigm 10 days after the end of exposure protocol. Each column represents the mean + S.E.M., for at least

7 animals per group. a,b p<0.05

a a

b

b

A

B

C

240

Social investigation behaviour

0

20

40

60

80

100

sniff ing anogenital sniff ing approach behaviour

Freq

uenc

y


0

30

60

90

120


Dur

atio

n (s

ec.)


0

150

300

450


Late

ncy

(sec

.)



and (C) latency of defensive behaviours performed by the resident animal during the resident-intruder

paradigm 10 days after the end of exposure protocol. Each column represents the mean + S.E.M., for at least

7 animals per group. a p<0.05

a a

A

B

C

241

Corticosterone plasma levels. The animals that performed the resident-intruder

paradigm 10 days after the end of the exposure protocol were decapitated and

corticosterone levels were measured in the obtained plasma. Statistical significance

between groups was assessed using the Student’s t-test. Statistical analysis revealed that

10 days after the end of exposure protocol, the increase in levels of corticosterone

induced by the resident-intruder paradigm did not differ between cocaine- and saline-

treated males (figure 4.3.2.2.4). Importantly, this levels were much higher than the basal.

0

150

300

450

Cor

ticos

tero

ne le

vels

(n

g/m

L)


Figure 4.3.2.2.4- Effects of chronic cocaine exposure throughout adolescence on the alterations on the

plasma levels of corticosterone induced by the resident-intruder paradigm 10 days after withdrawal, in male

rats. Doted line represents the basal levels of corticosterone (see figure 4.3.1.2.1 B). Each column represents

the mean + S.E.M for at least 8 animals per group.


DOPAC, HVA and 5-HIAA were measured by HPLC-EC, in the prefrontal cortex,

hippocampus, amygdala, hypothalamus, SN-VTA and dorsal raphe nucleus after the

behavioural test performed 10 days after the end of exposure protocol. The data obtained

after the test and the basal data 10 days after the end of the exposure protocol were

analyzed by the non-parametric Mann-Whitney U Test.

Within the SN-VTA, after the resident-intruder paradigm cocaine-treated males had

lower 5-HIAA/5-HT turnover rate as compared to saline males [Z=-2.125 (2-tailed) p<0.05]

(table 4.3.2.2.1). Moreover, in the cocaine-treated male group the 5-HIAA/5-HT turnover

rate was decreased after the resident-intruder paradigm as compared to basal data [Z=-

2.125 (2-tailed) p<0.05]. Also in the cocaine-treated male group, the levels of DOPAC and

5-HIAA were decreased after the behavioural test, as compared to basal data ([Z=-2.357

(2-tailed) p<0.02] and [Z=-2.711 (2-tailed) p<0.01], respectively) (figure 4.3.2.2.5). These

differences were not observed in saline-treated males. After the behavioural test, in this

brain area the levels of HVA were not detectable by the HPLC-EC.

242

SN-VTA

A


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Resident-intruder paradigmBasal



determined 10 days after withdrawal, under basal conditions and after the resident-intruder paradigm, by

HPLC-EC for male rats. Each column represents the mean + S.E.M. of monoamine levels, expressed as ng of

monoamine/mg of protein, for at least 6 animals per group. nd- not detectable. Columns with the same letter

are significantly different from each other. a p<0.05, b p<0.02, c p<0.01.

nd

a

a,b

b

c c


Basal Resident-intruder paradigm

243


[5-HIAA]/[5-HT], in the SN-VTA in saline- and cocaine-treated male, as measured 10 days after the last

administration, under basal condition and after the resident-intruder paradigm.



DOPAC/DA 0.24±0.06

(n=6)

0.16±0.02

(n=6)

0.33±0.15

(n=7)

0.10±0.03

(n=9)

HVA/DA 0.09±0.02

(n=6)

0.07±0.02

(n=6) - -


(n=6)

0.24±0.04

(n=6)

- -

5-HIAA/5-HT 6.55±6.27

(n=6)

0.22±0.03

(n=6)

0.22±0.04

(n=7)

0.13±0.01

(n=9)



the same line, values with same letter are significantly different. a,b p<0.05.

Within the dorsal raphe nucleus, statistical analysis revealed no significant

differences on the levels of DA, 5-HT, their metabolites and in turnover rates between

cocaine exposed and control males after the resident-intruder paradigm (figure 4.3.2.2.6

and table 4.3.2.2.2). Nevertheless, in the saline-treated males the 5-HIAA/5-HT turnover

rate was decreased after the test as compared to basal data [Z=-2.204 (2-tailed) p<0.05].

After the test in this brain area the levels of HVA were not detectable by the HPLC-EC.

Within the prefrontal cortex, no significant differences were found on the levels of

DA, 5-HT, their metabolites and in turnover rates between cocaine exposed and control

males, after the resident-intruder paradigm (figure 4.3.2.2.7 and table 4.3.2.2.3). However,

in cocaine-treated males the levels of HVA were decreased after the behavioural test as

compared to basal data [Z=-2.121 (2-tailed) p<0.05], moreover the HVA/DA and

(DOPAC+HVA)/DA turnover rates were also decreased ([Z=-2.241 (2-tailed) p<0.05] and

[Z=-2.475 (2-tailed) p<0.02], respectively).

a a,b b

244


A


01020

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nucleus, determined 10 days after withdrawal, under basal conditions and after the resident-intruder paradigm,

by HPLC-EC for male rats. nd- not detectable. Each column represents the mean + S.E.M. of monoamine

levels, expressed as ng of monoamine/mg of protein, for at least 6 animals per group.

nd



245

Prefrontal cortex

A

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monoamine/mg of protein, for at least 5 animals per group. Columns with the same letter are significantly

different from each other. a p<0.05.

a a

246


[5-HIAA]/[5-HT], in the dorsal raphe nucleus in saline- and cocaine-treated male, as measured 10 days after

the last administration, under basal condition and after the resident-intruder paradigm.



DOPAC/DA 0.89±0.39

(n=6)

0.57±0.24

(n=6)

0.62±0.18

(n=8)

0.53±0.17

(n=9)

HVA/DA 0.10±0.06

(n=6)

0.12±0.04

(n=6) - -


(n=6)

0.70±0.27

(n=6)

- -

5-HIAA/5-HT 0.67±0.37

(n=6)

0.87±0.59

(n=6)

0.27±0.06

(n=8)

0.24±0.02

(n=9)





[5-HIAA]/[5-HT], in the prefrontal cortex in saline- and cocaine-treated male, as measured 10 days after the

last administration, under basal condition and after the resident-intruder paradigm.



DOPAC/DA 0.38±0.09

(n=5)

0.31±0.03

(n=6)

0.22±0.06

(n=7)

0.18±0.05

(n=9)

HVA/DA 0.47±0.15

(n=5)

0.49±0.09

(n=6)

0.22±0.07

(n=7)

0.20±0.06

(n=9)


(n=5)

0.80±0.09

(n=6)

0.44±0.13

(n=7)

0.38±0.11

(n=9)

5-HIAA/5-HT 0.08±0.02

(n=5)

0.11±0.03

(n=6)

0.06±0.01

(n=7)

0.11±0.03

(n=9)



the same line, values with same letter are significantly different. a p<0.05, b p<0.02.

Within the hippocampus, cocaine-treated animals present lower levels of DOPAC

after the resident-intruder paradigm, as compared to saline-treated animals [Z=-2.021 (2-

tailed) p<0.05] (figure 4.3.2.2.8). In saline-treated males the levels of DA and 5-HT were

increased after the behavioural test as compared to basal data ([Z=-2.195 (2-tailed)

p<0.05] and [Z=-2.006 (2-tailed) p<0.05], respectively), moreover the 5-HIAA/5-HT

turnover rate was decreased [Z=-2.711 (2-tailed) p<0.01] (table 4.3.2.2.4).

a a

b b

a a

247

Hippocampus

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DA DOPAC DA DOPAC

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metabolite DOPAC and on (B) the levels of 5-HT and its metabolite 5-HIAA in the hippocampus, determined

10 days after withdrawal, under basal conditions and after the resident-intruder paradigm, by HPLC-EC for

male rats. Each column represents the mean + S.E.M. of monoamine levels, expressed as ng of

monoamine/mg of protein, for at least 6 animals per group. Columns with the same letter are significantly

different from each other. a,b,c p<0.05.

a

a

c

c

b b

248


hippocampus in saline- and cocaine-treated male, as measured 10 days after the last administration, under

basal condition and after the resident-intruder paradigm.



DOPAC/DA 0.24±0.05

(n=6)

0.41±0.11

(n=6)

0.24±0.08

(n=8)

0.29±0.09

(n=9)

5-HIAA/5-HT 0.41±0.07

(n=6)

0.23±0.04

(n=6)

0.14±0.02

(n=8)

0.15±0.02

(n=9)


Dihydroxyphenylacetic acid; 5-HT- serotonin; 5-HIAA- 5-hydroxyindolacetic acid. In the same line, values with

same letter are significantly different. a p<0.01.

Within the amygdala, no significant differences were found on the levels of DA, 5-

HT, their metabolites and in turnover rates between cocaine exposed and control males,

after the resident-intruder paradigm (figure 4.3.2.2.9 and table 4.3.2.2.5). However, in

cocaine-treated males the 5-HIAA/5-HT was decreased after the behavioural test as

compared to basal data [Z=-2.326 (2-tailed) p<0.05].

Within the hypothalamus, no significant differences were found on the levels of DA,

5-HT, their metabolites and in turnover rates between cocaine exposed and control males,

after the resident-intruder paradigm (figure 4.3.2.2.10 and table 4.3.2.2.6). However, in

cocaine-treated male rats the levels of DOPAC were decreased after the test as

compared to basal data [Z=-2.333 (2-tailed) p<0.05]. Moreover, cocaine- and saline-

treated males the DOPAC/DA turnover rate was decreased after the behavioural test as

compared to basal data (cocaine-treated males [Z=-2.472 (2-tailed) p<0.02], saline-

treated males [Z=-2.326 (2-tailed) p<0.05]). The 5-HIAA/5-HT turnover rate was also

decrease after the test in cocaine-treated males as compared to cocaine-treated males

under basal conditions males [Z=-2.071 (2-tailed) p<0.05], in the saline-treated males this

difference did not reach significance.

Both in the amygdala and hypothalamus after the behavioural test the levels of

HVA were not detectable by the HPLC-EC.

a a

249

Amygdala

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monoamine/mg of protein, for at least 6 animals per group. nd-not detectable.

nd



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Hypothalamus

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its metabolites, DOPAC and HVA and on (B) the levels of 5-HT and its metabolite 5-HIAA in the

hypothalamus, determined 10 days after withdrawal, under basal conditions and after the resident-intruder

paradigm, by HPLC-EC for male rats. Each column represents the mean + S.E.M. of monoamine levels,

expressed as ng of monoamine/mg of protein, for at least 5 animals per group. nd- not detectable. Columns

with the same letter are significantly different from each other. a p<0.05.

nd

a a



251


[5-HIAA]/[5-HT], in the amygdala in saline- and cocaine-treated male, as measured 10 days after the last




DOPAC/DA 0.33±0.19

(n=6)

0.34±0.13

(n=6)

0.16±0.06

(n=7)

0.12±0.04

(n=8)

HVA/DA 0.08±0.04

(n=6)

0.08±0.02

(n=6) - -


(n=6)

0.42±0.13

(n=6) -

-

5-HIAA/5-HT 0.30±0.09

(n=6)

0.47±0.15

(n=6)

0.20±0.03

(n=7)

0.15±0.02

(n=8)





[5-HIAA]/[5-HT], in the hypothalamus in saline- and cocaine-treated male, as measured 10 days after the last




DOPAC/DA 0.43±0.12

(n=6)

0.41±0.09

(n=5)

0.14±0.03

(n=8)

0.11±0.03

(n=9)

HVA/DA 0.03±0.01

(n=6)

0.07±0.03

(n=5) - -


(n=6)

0.48±0.11

(n=5) -

-

5-HIAA/5-HT 0.31±0.09

(n=6)

0.37±0.10

(n=5)

0.15±0.02

(n=8)

0.17±0.03

(n=9)



the same line, values with same letter are significantly different. a,c p<0.05, b p<0.02.

a a b b

c c

a a

252

4.4. Discussion

4.4.1. Effects of chronic cocaine treatment during adolescence and withdrawal on the body weight evolution

The data on body weight was analyzed in terms of body weight gain, and during

the experimental period no effects of cocaine treatment were observed. These data

contradict the known anorexic effects of cocaine (Spector et al., 1972; Balopole et al.,

1979; Cooper and van der Hoek, 1993). However, other authors had already reported no

effects of cocaine administration in body weight evolution in adolescent rats (Laviola et al.,

1995; Giorgetti and Zhdanova, 2000). As already discussed in chapter 3, the absence of

cocaine effects on body weight evolution may be related with a strategy adopted during

adolescence, where the available resources are directed into ponderal growth, whereas in

the adult resources are preferentially used to maintain the reproductive function (Gomez

and Dallman, 2001; Gomez et al., 2002). Nevertheless, it was observed a sex effect in the

body weight evolution, male rats had higher weight gain than females. This effect was

expected, since sex differences in body weight appear during adolescence (Macri et al.,

2002).

4.4.2. Effects of chronic cocaine treatment during adolescence and withdrawal on the neuroendocrine function

As already discussed in chapter 3, cocaine has been reported to have effects on

the HPA axis activity. This work aimed to study the effects of chronic cocaine exposure

during adolescence on HPA axis activity throughout long-term withdrawal. Two time points

of assessment were chosen, 5 (an assessment point during adolescence period) and 10

days (beginning of adulthood) after the last cocaine administration.

It has been demonstrated that the HPA axis activity was disrupted (Sarnyai et al.,

1998; Mantsch et al., 2000) during withdrawal from chronic cocaine treatment, suggesting

that altered HPA axis function may play an important role in cocaine addiction. In fact,

acute elevation of glucocorticoids has been reported to precipitate reinstatement in rats

(Deroche et al., 1997) and craving in humans (Elman et al., 2003). Also in rats,

adrenalectomy prevents reinstatement of self-administration behaviour following extinction

(Erb et al., 1998). Elevated plasma corticosterone appears to contribute to the stress-

induced reinstatement of extinguished cocaine-seeking behaviour in animal models of

drug relapse (Deroche et al., 1997; Mantsch and Goeders, 1999). These findings indicate

that corticosterone is critical for the reinstatement of extinguished cocaine-seeking

behaviour, demonstrating an involvement of the HPA axis in the relapse to cocaine use.

253

Our data in chronic binge exposure to cocaine during adolescence indicate that

both 5 or 10 days after cocaine withdrawal the basal levels of ACTH and corticosterone in

male and female rats did not significantly differ from those of the control animals. These

results may confirm previously findings indicating that chronic cocaine administration does

not seem to have prolonged effects upon the HPA axis activity under basal conditions.

Teoh at al. (1994) found no significant differences in pulsatile release of ACTH in men

with a past history of cocaine abuse who were studied under controlled drug-free

conditions in comparison to normal controls (Teoh et al., 1994). After a brief period of

abstinence, basal and CRH-stimulated ACTH and cortisol levels in cocaine-dependent

patients were not different from healthy subjects (Vescovi et al., 1992; Baumann et al.,

1995; Jacobsen et al., 2001). In rats, Zhou et al. (2003), using a exposure paradigm

similar to the one used in the present study, showed that plasma ACTH and

corticosterone levels remained increased 4 days after withdrawal, but 10 days later were

at control levels. Rats chronically treated with cocaine (15mg/kg i.p.) for 14 days and then

challenged with an acute dose of cocaine 7 days after the last chronic cocaine treatment

displayed ACTH and corticosterone responses that did not differ from those under control

treatment conditions (Levy et al., 1992a). These findings were confirmed and extended to

periadolescent rats, after 4 days of cocaine administration (0,10 or 20 mg/kg i.p.) a

challenge dose of cocaine (10 mg/kg ip) had equivalent effects on ACTH and

corticosterone in the control and cocaine-treated groups (Laviola et al., 1995).

Nevertheless, our results showed that although no differences were observed in

corticosterone levels between groups at the same time point, saline-treated females

displayed an increase in ACTH levels from 5 to 10 days after withdrawal, with this effect

not being observed in cocaine-treated females. In males, this effects is observed in

cocaine-treated males (although no significantly) and not in control males. This may

indicate an effect of the cocaine treatment in the function of the HPA axis after withdrawal;

moreover this effect is sex-dependent.

In the present study the analysis of the effects of cocaine withdrawal on HPA axis

activity was extended besides the hormonal measures and the levels of CRH mRNA in

hypothalamus and the levels of POMC and CRH-R1 mRNAs in the pituitary were

analyzed in male rats 10 days after the end of the exposure period. The results did not

showed significant differences in the levels of expression of the analyzed mRNAs both in

hypothalamus and pituitary between cocaine-treated and control males. These measures

were only conducted for male rats, as justified in page 215.

It is known that a single injection of cocaine, or an acute binge pattern cocaine

administration elevates CRH mRNA levels in the hypothalamus in rats (Rivier and Lee,

254

1994; Zhou et al., 1996b), with no significant alterations in either CRH-R1 or POMC

mRNA levels in the anterior pituitary (Zhou et al., 1996b), indicating the importance of the

hypothalamic CRH in modulating the stimulatory effects of the HPA axis. However, Zhou

at al. (2003) showed that 1 day after a period of 14 days of chronic binge cocaine

administration did not lead to an increase in CRH mRNA levels in the hypothalamus (Zhou

et al., 2003).

CRH-R1 mRNA expression in the anterior pituitary appears to be preferentially

associated with ACTH-positive cells, and both POMC and CRH-R1 mRNA expression are

subject to negative feedback control by glucocorticoids (e.g., Zhou et al., 1996a). The

study by Zhou et al. (2003) also showed that both of the POMC and CRH-R1 mRNA

expression levels were increase 1 day after chronic binge cocaine administration but

returned to control after 4 days into withdrawal. In another study it was reported that

prolonged withdrawal from cocaine was associated with normalization of regional brain

CRH synthesis (Zhou et al., 1996b) and CRH receptor binding (Ambrosio et al., 1997) by

10 days post-withdrawal. Those data indicate that chronic cocaine binge administration

does not seem to have prolonged effects upon the HPA axis activity under basal

conditions. Our findings seems to confirm and extend this data to adolescent rats.

In the present study we also investigated the feedback inhibitory mechanisms, by

examining GR mRNA expression levels in the pituitary and brain regions putatively

involved in feedback regulation of the HPA axis. No differences were observed between

cocaine- and saline-withdrawn male rats. Although, it cannot be rule out the possibility that

GR affinity or function was altered without changes in mRNA expression. These data also

seems to indicate an absence of prolonged effects of cocaine treatment upon the HPA

axis activity under basal conditions.

Data from adrenal weight was collected 5 and 10 days after the withdrawal. The

weights were normalized to body weight and data analysis revealed that adrenal weights

were not affected by the cocaine treatment during adolescence since no differences were

observed between cocaine-treated males and females and their respective controls at

both time points of assessment. Nevertheless, and as already observed 30 minutes and 1

day after the last administration of cocaine, and discussed in chapter 3, there was a sex

effect on the adrenal weight, with adrenals of males weighing significantly less than that of

the females, which is in agreement with published literature (Majchrzak and Malendowicz,

1983).

In the present study the long-term effects of chronic cocaine exposure during

adolescence on gonadal steroid levels were examined, both in male and female rats, 5

255

and 10 days after cocaine withdrawal. As adolescence includes puberty, a period of

marked hormonal changes, external perturbation of this developmental process may lead

to long-term effects on the HPG axis function.

It has been difficult to detect changes in basal hormone levels after chronic

cocaine abuse in animal models. However, it has been infered that chronic cocaine

changes in neuroendocrine function do occur on the basis of clinical and experimental

evidence of cocaine-related disruption of the reproductive function (Siegel, 1982; Cocores

et al., 1986; Berul and Harclerode, 1989; Chen and Vandenbergh, 1994).

Our data showed that 5 days after cocaine withdrawal plasma levels of

testosterone did not differ between chronically cocaine- and saline-treated male rats.

However, saline-treated males presented an increase in levels of testosterone from 5 to

10 days; this effect was not observed in cocaine-treated males. In fact, cocaine-treated

males displayed lower levels of testosterone at 10 days after cocaine withdrawal as

compared to control males. As discussed in chapter 3, acute cocaine administration is

known to induce significantly decreases in plasma levels of testosterone (Berul and

Harclerode, 1989). The decreased testosterone plasma levels observed 10 days after

withdrawal, a time point that matches the beginning of adulthood, may be a consequence

of repeated decrease in testosterone levels after each cocaine administration throughout

the exposure period on the development of testes occurring during this period. In

agreement with this hypothesis, chronically treated males also presented decreased

relative gonadal weights at this time point, which may suggest that the observed

decreased testosterone levels may be primarily related with a reduced steroidogenic

capacity of testes, probably due to an abnormal maturation of the gonads. Other authors

have shown that morphological changes and lesions in testes are present in the rat after a

long-term exposure to cocaine. Rats long-term exposed to cocaine, starting on

peripubertal stage, presented reduced mean diameter of seminiferous tubules, thiner

germinal epithelium, and numerous degenerating cells, although in that work the levels of

testosterone were unaltered (George et al., 1996). In adult rats, Barroso-Moguel et al.

(1994), showed that in long-term exposed animals, testes were diminished in size, the

seminiferous tubules were shrunken, had small diameter and contained necrotic

spermatogenic cells and fibrosis.

In the present study no differences were found in the progesterone plasma levels

between cocaine- and saline-treated males, as accessed 5 and 10 days after cocaine

withdrawal. The fact that testosterone, but not progesterone plasma levels were reduced

at 10 days after cocaine withdrawal suggest that cocaine may interfere with some specific

metabolic processes in the synthesis of testosterone in Leydig cells. Although LH levels

256

were not measured, our data seems to strongly reflect a direct effect of cocaine on the

testes.

The study of the long-term effects of withdrawal from chronic cocaine treatment

throughout adolescence on the HPG axis activity of male rats was extended besides the

hormonal level determinations and the expression levels of GnRH-R, LHβ and AR mRNAs

in pituitary, and levels of AR mRNA in hypothalamus were analyzed at 10 days after

cocaine withdrawal. No differences were observed between cocaine- and saline-

withdrawn male rats. The absence of alterations on expression levels of AR mRNA seems

to indicate that the testosterone negative feedback mechanism at the pituitary and the

hypothalamus was not altered 10 days of withdrawal. Data on GnRH-R mRNA expression

levels seems to indicate that pituitary responsiveness to hypothalamic GnRH pulses was

not altered; moreover the LHβ mRNA levels were also unaltered as compared to control

animals. These data appears to support the hypothesis that reduced levels of testosterone

observed in cocaine-treated males result primarily from impaired testicular

steroidogenesis.

Little is known about the effects of cocaine on the endocrinological profile of

females, moreover the long-term effects have been almost unexplored. Nevertheless, the

persistent menstrual cycle abnormalities observed during cocaine withdrawal suggest that

the disruptive effects on the menstrual cycle do not only reflect an acute intoxication

outcome (Mello et al., 1997). Rather, daily cocaine exposure may induce long-lasting

disruptions of the neuroendocrine regulation of the menstrual cycle. Persistent effects of

cocaine after chronic exposure have been observed in several models (Mello and

Mendelson, 1997). In rats, estrous cycles did not return to normal after high doses of

cocaine over 5 to 6 weeks of observation (King et al., 1993b).

Our data showed that females chronically exposed to cocaine throughout

adolescence presented higher levels of progesterone at 10 days after the last cocaine

administration. These high levels of progesterone may inhibit the release of LH preventing

ovulation, leading to a disruption of the normal estrous cycle hormone regulation following

chronic cocaine administration. Moreover, saline-treated females displayed decreased

plasma levels of progesterone between 5 and 10 days after the ending of the exposure

protocol, with this effect not being observed in cocaine-treated females. In fact,

Feltenstein and See (2007) had already showed increased progesterone levels during and

after cocaine self-administration in the non-estrus phases of the cycle (Feltenstein and

See, 2007). However, Walker et al. (2001) reported that the cocaine-induced an increase

in plasma progesterone levels, which was not present in adrenalectomized animals,

257

suggesting that this effect may involve an action of cocaine on the HPA axis activity, either

through adrenally derived progesterone release or HPA axis/ovary interactions.

No differences in the plasma levels of estradiol were observed between cocaine-

and saline-treated females. Nevertheless, the levels of estradiol in saline-treated females

increased from 5 to 10 days after the last administration, with this effect not being

observed in cocaine-treated females, supporting an impaired regulation of the HPG axis

during the withdrawal period.

While suggestive, the results described here do not consider the physiological

fluctuations of ovarian hormones already present in adolescent females. Although at this

age it difficult to determine the stage of estrous cycle, this can interfere with the present

results.

Overall, our results indicate a long-lasting disruptive effect of cocaine on the HPG

axis function in male, and a trend to a long-term impaired regulation of the HPG axis in

female rats.

4.4.3. Effects of chronic cocaine treatment during adolescence on the dopaminergic and serotonergic systems function

Recently, there has been renewed interest in the neuroadaptation induced during

periods of cocaine withdrawal. Moreover, there are conceptual reasons for expecting that

drug consumption may affect adolescents differently than adult consumption. Few studies

have investigated whether the continued plasticity during this late development extends to

drug-induced changes in the CNS, although several reports indicate that cocaine (Smith

et al., 1999; Estelles et al., 2007) and alcohol (Siciliano and Smith, 2001) during

adolescence may induce some long-lasting behavioural changes. For example, Catlow

and Kirstein (2007) demonstrated that in rats pretreated with cocaine during the

adolescence there is an enhanced response of the dopaminergic system to naturally

reinforcing substances, suggesting long-term functional changes in the mesolimbic

pathway (Catlow and Kirstein, 2007). Brandon and colleagues administered 15mg/kg of

cocaine for 5 days and found that adolescent rats pretreated with cocaine showed

persistent behavioural sensitization two months after cocaine cessation (Brandon et al.,

2001). The use of cocaine during adolescence could alter the normal growth of brain

regions affected by cocaine, specifically the reward system, and impact the adult

mesolimbic system. However, there is limited literature aiming to determining whether

animals are more vulnerable to the adverse effects of drugs during adolescence (Catlow

and Kirstein, 2007). Thus, in the present study, the objective of investigate whether

258

cocaine pretreatment in adolescence altered the dopaminergic or serotonergic systems

activity at 5 or 10 (early adulthood) days after withdrawal, was also established.

- Effects on the mesoaccumbens and nigrostriatal pathways

Our data showed that, 5 days after the last administration adolescent rats treated

chronically with cocaine presented no differences on the levels of DA and its metabolites,

DOPAC and HVA, in regions expected to be affected by cocaine exposure, such as the

SN-VTA, the Nac and the dorsal striatum, as compared to saline controls. Several studies

indicate that alterations in the activity of midbrain DA neurons during withdrawal are

dependent on the type of cocaine administration protocol that is used. Whereas

intermittent cocaine injections have been shown to increase extracellular DA

concentrations in the striatum during withdrawal, continuous cocaine infusions produced

the opposite effect (King et al., 1993a). Similarly, withdrawal from continuous cocaine

administration decreased the number of spontaneously active midbrain DA neurons,

whereas withdrawal from an intermittent schedule of cocaine increased the number of

spontaneously active DA neurons (Gao et al., 1998). Considering the relatively high

dosing delivered in the binge cocaine administration experiments, it was expected that the

findings for chronic binge administration were similar to those observed in rats during

withdrawal from continuous cocaine administration, thus it was expected to find a

depression of the dopaminergic system activity.

However, the absence of differences in the activity of the dopaminergic system

between saline- and cocaine-treated animals observed in the present study may result of

increased dopaminergic activity induced by the expectancy of drug administration in

cocaine-treated animals, since animals were decapitated in the same room where the

cocaine was daily administered. As already discussed in chapter 3, several investigators

have demonstrated a stimulation of dopaminergic activity when drug-free rats were

exposed to environments that have been previously paired with stimulant drugs, such as

amphetamine or cocaine (i.e. a conditioning effect) (Post et al., 1981; Badiani et al., 1995;

Antkiewicz-Michaluk et al., 2006). In the present study, throughout the period of

withdrawal, the animals were brought daily to the room where cocaine was administered,

without receiving any injection, in order to eliminate the expectancy of drug administration,

and thus eliminate the activation of dopaminergic system induced by the cocaine cues

presentation. However, based in our data obtained at 5 days after withdrawal, we

speculated that by this time the expectancy of drug administration was still present.

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Ten days after cocaine withdrawal our data seems to point toward an increase in

dopaminergic activity in male rats, as indicated by the increased DOPAC levels in SN-

VTA and the increased levels of HVA in dorsal striatum. In fact, the mRNA expression

levels of tyrosine hydroxylase (TH) were increased in SN-VTA 10 days after withdrawal in

male rats. TH is the key enzyme for synthesizing DA in dopaminergic neurons and its

terminals. We speculate that the increase in TH in the SN-VTA may reflect a

compensatory response of dopaminergic neurons to upregulate their synthesizing

capacity. This data supports the findings from neurochemical analyses, showing that

chronic cocaine administration throughout the adolescence period has long-lasting effects

upon the dopaminergic system. Moreover, these results suggest an increase in the DA

synthesis, likely to compensate for a chronic cocaine-induced depression of the

dopaminergic system. Other authors have reported alteration in the dopaminergic system

activity at 10 days withdrawal although in the opposite direction. Antkiewicz-Michaluk and

colleagues (2006) reported that 10 days of withdrawal from self-administered cocaine

leads to an impairment of the function of the dopaminergic system, as reflected by a

significant decline in DA concentration in the Nac and striatum, but not in FC, SN and VTA

(Antkiewicz-Michaluk et al., 2006). There are two factors that vary between the latter

mentioned and the present study that could be involved in the different results obtained:

the cocaine dosing regime (i.v. self-administration versus i.p. experimenter-delivery) and

the period of exposure (adulthood versus adolescence).

It was already reported that basal levels of DA in the shell region of the Nac were

affected differentially as a function of age by repeated cocaine use (Catlow and Kirstein,

2007). Specifically, rats pretreated with cocaine in the adulthood had lower baseline levels

of DA than saline-pretreated rats. It has been postulated that a decrease in the levels of

TH within the Nac leads to decreased basal levels of DA, and this results in a postsynaptic

downregulation of the dopaminergic system (Parsons et al., 1991; Segal and Kuczenski,

1992; Maisonneuve et al., 1995). It is interesting that rats pretreated in the adolescence

with the same dose of cocaine for the same length of time did not have a reduction in

basal DA levels (Catlow and Kirstein, 2007). These results suggest that the administration

of cocaine differentially induces long-term changes in the dopaminergic transmission in

adolescent and adult rats.

Due to the increasing numbers of DATs through adolescence, higher reuptake will

decrease basal DA in the striatum (Andersen and Gazzara, 1993) and in the Nac (Philpot

and Kirstein, 1999) and upregulates cyclic-AMP (cAMP) signaling (Andersen, 2002). The

profile of the adolescent mesolimbic system, with lower basal levels of DA, increased DA

receptors and cAMP levels, could be overstimulated in the presence of cocaine.

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Persistent postsynaptic stimulation hyperpolarizes GABAergic neurons and allows an

increased release of DA into the extracellular space which could in part explain cocaine-

induced hyperactivity observed in adolescent rats (Catlow and Kirstein, 2005; Maldonado

and Kirstein, 2005b, a). During adolescence, glutamatergic projections from prefrontal

cortex to VTA and limbic areas develop (reviewed by Lewis, 1997). It is plausible that

repeated cocaine during adolescence overstimulates the mesolimbic system causing

more synaptic connections of glutamatergic afferents to the VTA. Repeated cocaine

during adolescence could result in a circuitry primed for vulnerability to addiction.

Our results seem to indicate that the nigrostriatal system of the in male rats is the

most affected dopaminergic pathway by the cocaine treatment. The striatum is thought to

have a role in motor activity, such as stereotypes (Creese and Iversen, 1974; Robbins and

Everitt, 1996), but also in rewarded “habit” learning (Everitt and Wolf, 2002), so the

present results suggest that the observed neurochemical changes may be partially

responsible for withdrawal symptoms. Moreover, the increased dopaminergic activity

observed in male rats after long-term withdrawal may render the individuals more

susceptible to the reward effects of cocaine, therefore more vulnerable to reinstatement.

In most dopaminergic cells, DAT mRNA and TH mRNA-positive neurons

completely overlap (Hoffman et al., 1998). This co-localization may be due to similar

molecular mechanisms of transcriptional regulation of these genes. The data descried in

the present study indicate that expression levels of DAT mRNA, in male rats 10 days after

withdrawal, were increased in SN-VTA in a way that almost reached significance.

Whereas acute administration of cocaine did not alter DAT mRNA levels in the VTA and

SN (Xia et al., 1992; Spangler et al., 1996), they were reported to be decreased by

chronic cocaine treatment (Xia et al., 1992; Burchett and Bannon, 1997; Letchworth et al.,

1997; Letchworth et al., 1999). Following withdrawal from cocaine, DAT mRNA expression

was unaltered in the SN at 72 hours withdrawal period (Xia et al., 1992), but decreased in

parts of the VTA at 10 days after 2 weeks i.v. cocaine treatment (Cerruti et al., 1994).

Maggos et al. (1997), using the same pattern of cocaine administration as those used in

the present study, reported unchanged DAT mRNA levels in both areas under subacute (3

days) binge, chronic (14 days) binge or 10 days withdrawal from a chronic binge pattern

cocaine. However, we did observe a trend to increased expression levels of DAT mRNA

in the SN-VTA in the cocaine-treated male rats 10 days after withdrawal. Arroyo et al.

(2000) also reported a long lasting up-regulation of the DAT mRNA levels in the VTA 10

days after animals had self-administered cocaine for 10 days. This data seems to support

that upregulation of DAT mRNA induced by cocaine administration may depend on the

period of cocaine exposure more than on the cocaine dosing regime. The upregulation of

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the gene encoding DAT could be the result of a compensatory mechanism that develops

due to the repeated DAT blockade-induced increases in extracellular DA levels following

cocaine exposure.

Nevertheless, some studies have shown no correlation between cocaine-induced

effects on DAT mRNA level and concentration and binding site densities at the synaptic

terminals, when evaluated at the same time point after non-contingent cocaine treatment

(Cerruti et al., 1994; Letchworth et al., 1997; Letchworth et al., 1999). This may reflect the

ability of cocaine to alter some of the molecular processes involved in protein synthesis

(i.e. rates of transcription, translation, mRNA degradation, axonal transport, protein

stability).

The expression levels of DA receptors D1, D2 and D3 mRNA were not altered 10

days after withdrawal in cocaine exposed male rats. There is considerable evidence that

chronic exposure to cocaine is associated with low striatal D2 receptor availability.

Moreover, imaging studies using PET have demonstrated that reduced striatal D2

receptor availability in functional subdivisions of the striatum, might be involved in the

initiation and/or maintenance of cocaine abuse. Indeed, low striatal D2 receptor availability

was observed in detoxified chronic cocaine abusers (Volkow et al., 1993), and in healthy

subjects was consider as predictive of a pleasurable experience following administration

of the psychostimulant methylphenidate (Volkow et al., 1999). A recent study by Stefanski

and colleagues (2007) reported that passively administered cocaine produced a decrease

in D2 receptor levels in the anterior and central regions of caudate-putamen, and the Nac, as measured by in vitro quantitative autoradiography. On the other hand, they reported

that self-administration of cocaine increased D2 receptor mRNA levels in the VTA,

although with no corresponding differences in D2 binding density (Stefanski et al., 2007).

This later result are consistent with previous studies demonstrating that self- administred

cocaine had no effect on D2 receptor density in the dorsal and ventral striatum of rats

(Laurier et al., 1994). Importantly, these results suggest that the reductions in striatal D2

receptor densities may be related to the direct pharmacological actions of chronic

administration of cocaine per se and not to the motivated process of reinforced

responding.

A drug-free period of 7 days after repeated intermittent treatment with cocaine for

14 days was reported to result in a functional subsensitivity of release-modulating D2

autoreceptors in the caudate (Jones et al., 1996) and in the Nac of rats (Davidson et al.,

2000). However, Svensson and Hurd (1998) reported that rats treated with cocaine (30

mg/kg, i.p. once daily) for 10 days, followed by a 10-day drug free-period displayed

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decreased expression levels of D1-receptor mRNA in the caudorostral striatum and

unaltered expression of D2-receptor mRNAs (Svensson and Hurd, 1998).

It has been reported that treatments inducing behavioural sensitization to cocaine,

all of which used experimenter administered i.p. injections, produce no change in the total

levels of D1 or D2 receptor in the VTA, Nac, striatum, or prefrontal cortex (for reviews, see

Pierce and Kalivas, 1997; Vanderschuren and Kalivas, 2000). These data agree with the

results obtained in the present study, showing no differences in the expression levels of

DA receptors mRNAs in cocaine-treated male rats 10 days after withdrawal as compared

to controls. In fact, the paradigm of drug administration used in the present study is known

to induced behavioural sensitization (Lucas et al., 1997; Caster et al., 2005).

The data obtained in cocaine-treated females, during the drug free-period point to

the absence of treatment effects in the dopaminergic neurotransmission within the SN-

VTA, Nac and dorsal striatum, again diverging from the data obtained in male rats. There

are growing evidences that females display a differential response and adaptation to

stress than do males (Dalla et al., 2008; Lin et al., 2009; also see for review Ter Horst et

al., 2009). Thus, the high levels of stress that animals were submitted into, particularly the

stress caused by the social isolation, could have masked the effects of cocaine and its

withdrawal in female rats.

Preclinical and clinical findings showed that withdrawal from chronic cocaine is

associated with impaired 5-HT neuronal function in addition to the well-accepted deficits in

DA function (Dackis and Gold, 1985). Perhaps the most compelling evidence of 5-HT

deficits in cocaine addiction is the occurrence of a psychiatric syndrome resembling major

depression that follows abstinence from binge cocaine use (Dackis and Gold, 1985;

Gawin and Kleber, 1986), coupled with the increased prevalence of suicidal ideation and

suicide attempts among cocaine addicts (Garlow et al., 2003). The established role of 5-

HT dysfunction in mediating depression and suicide (Mann, 2003) indicates that

decreased synaptic 5-HT levels could have a role in cocaine withdrawal states (Baumann

and Rothman, 1998). In addiction, increasing postsynaptic DA function does not reliably

reduce cocaine craving (Kampman et al., 1996; Handelsman et al., 1997). However,

promising results have been obtained for drugs that increase extracellular 5-HT levels

(Pollack and Rosenbaum, 1991; Batki et al., 1993; Kampman et al., 2000) or act directly

on 5-HT receptors (Buydens-Branchey et al., 1997; Buydens-Branchey et al., 1998).

In vivo microdialysis experiments reveal that rats withdrawn from i.v. cocaine self-

administration have decreased extracellular levels of 5-HT in the Nac (Parsons et al.,

1995). A feasible mechanism to explain this finding is, just like described for DA (Wilson et

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al., 1994; Mash et al., 2002), the enhanced uptake of 5-HT. Indeed, chronic cocaine

administration can increase the density of SERTs in rats (Cunningham et al., 1992) and

humans (Jacobsen et al., 2000). Increases in SERT density in the rostral ventromedial

caudate putamen, Nac shell, and olfactory tubercle were demonstrated in adult rats

pretreated with cocaine (Collins and Izenwasser, 2002). However, no significant changes

in SERT density were observed in periadolescent rats 10 days after withdrawal compared

to vehicle controls, showing that periadolescent rats have different serotonergic

adaptations to repeated cocaine administration than adult rats (Collins and Izenwasser,

2002).

In the present study neurochemical data did not showed a decrease in 5-HT levels

in cocaine-treated animals as compared to controls. However, the levels of 5-HT

increased from day 5 to day 10 after cocaine withdrawal in male rats in the SN-VTA, while

in cocaine-treated female rats the levels of 5-HT and 5-HIAA increased from 5 to 10 days

after withdrawal in the Nac. These effects were not observed in saline controls, indicating

that in fact cocaine treatment during adolescence have long-lasting effects on the

serotonergic activity, and like as for the dopaminergic activity, the increase in 5-HT levels

may represent an attempt to counteract the downregulation of the serotonergic system

activity during withdrawal.

- Effects on the prefrontal cortex

Cocaine addicts have a number of cognitive deficits that persist following

prolonged abstinence. These include impaired performance on tasks involving attention

and cognitive flexibility that are thought to be mediated by the medial and orbital prefrontal

cortex, as well as spatial, verbal, and recognition memory impairments on tasks thought to

be mediated by the hippocampus (Manschreck et al., 1990; Ardila et al., 1991; Berry et

al., 1993; Beatty et al., 1995; Hoff et al., 1996; Bolla et al., 2003).

DA is an essential contributor within the prefrontal cortex during cocaine abuse

(Kelley, 2004). DA is a critical modulator of the pyramidal neuron excitability and excitatory

synaptic transmission in the prefrontal cortex (Seamans and Yang, 2004; Floresco and

Tse, 2007). Pyramidal neurons in the medial prefrontal cortex and their afferents express

receptors for DA (Smiley et al., 1994; Krimer et al., 1997). It has been reported that after

chronic cocaine administration, dopaminergic neurotransmission in the prefrontal cortex

undergoes significant changes (Williams and Steketee, 2005), time-dependent changes

that may be caused by alteration in binding to DA receptor subtypes (Ben-Shahar et al.,

2007).

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Our study on the prefrontal cortex dopaminergic activity indicates that the levels of

DA and its metabolites DOPAC and HVA did not differ between cocaine- and saline-

treated animals, both at 5 and 10 days after withdrawal. Other studies have also revealed

that withdrawal from repeated cocaine produce no statistically significant changes in

baseline DA levels within the prefrontal cortex (Sorg et al., 1997; Chefer et al., 2000;

Williams and Steketee, 2005). However, for instance, repeated systemic administration of

cocaine produced time-dependent alterations in cocaine-induced increases in extracellular

DA levels within the medial prefrontal cortex (Williams and Steketee, 2005). In that study,

1 day after withdrawal from repeated cocaine the dopaminergic response to an acute

cocaine challenge was blunted in cocaine-pretreated animals as compared to controls,

with this attenuated response being more pronounced when examined after 7 day of

withdrawal; while after 30 days of withdrawal it was observed an augmented medial

prefrontal cortex DA response to cocaine, in cocaine-treated rats (Williams and Steketee,

2005). These results suggest that withdrawal from repeated cocaine is associated with

marked alterations in prefrontal cortex DA neurotransmission. However, in our study,

beside the absence of cocaine treatment effects revealed by the neurochemical data

obtained 5 and 10 days after withdrawal, the analysis of expression levels of D1, D2 and

D3 receptors, DAT and MAO-A mRNAs, in the prefrontal cortex of male rats, 10 days after

withdrawal, also revealed no significant differences between cocaine- and saline-treated

male rats. Nogueira et al. (2006) also reported no alterations on the effects of D1 receptor

antagonists on the current-evoked and spontaneous excitability of pyramidal cells at 2-4

weeks after a repeated cocaine treatment of 7 days, while reporting that D2 receptor-

mediated regulation of cortical excitability was reduced. After long-access cocaine self-

administration was also reported a significant decrease in D2 (but not D1) mRNA in the

medial and orbital prefrontal cortex, and D2 receptor protein in the medial prefrontal cortex

of rats (Briand et al., 2008) An extensive body of evidence describes the fundamental role

of prefrontal cortex D2 receptors in cocaine addiction. Beyer and Steketee (2002)

demonstrated that intra-medial prefrontal cortex injections of the D2 receptor agonist

quinpirole blocked cocaine-induced sensitization (Beyer and Steketee, 2002). Moreover,

in studies with human addicts, Volkow et al. (2001) reported lower levels of D2 receptor

availability in the orbitofrontal cortex, suggesting that dysregulation of this type of receptor

could underlie the loss of control and compulsive drug intake in drug addicts. The

contradictory findings between the above mentioned studies and our study could be

related with the different dosing regimen exposure and the period of exposure. In contrast

to the other studies that used adult animals, in our study, were used adolescent animals

undergoing prominent developmental transformations (Spear, 2000). Therefore, we

propose that the developmental processes may underlyie the differences observed

265

between our study and these carried out in adults. It also important to note that, although

no differences were observed in mRNA expression levels this, does not exclude the

possibility that the DA receptors concentration and binding site densities could have been

altered.

- Effects on the hippocampus

In the hippocampus, cocaine-treated males did not present alterations in levels of

DA or DOPAC both 5 and 10 days after cocaine withdrawal, with the DA turnover rate also

not being significantly different from that of the control animals. The expression levels of

D1, D2 and D3 receptors, DAT and MAO-A mRNA, determined 10 days after withdrawal

in male rats, also did not revealed any treatment effects. Together these data seems to

suggest that basal dopaminergic neurotransmission in the hippocampus was not long-

term affected by the chronic binge cocaine exposure throughout adolescence in the male

rats. However, in cocaine-treated females the levels of DA increased from 5 to 10 days

after withdrawal, while this effect was not observed in control females, suggesting a

different pattern of evolution in the dopaminergic neurotransmission between cocaine-

treated and control females. The pivotal role of the hippocampal dopaminergic system has

been demonstrated in several types of learning: intrahippocampal injections of DA

agonists enhance passive avoidance (Bernabeu et al., 1997) and win-shift positive

reinforcement learning (Packard and White, 1991). DA depletion in the hippocampus

impairs spatial navigation (Gasbarri et al., 1996). It is known that one form of synaptic

plasticity, long-term potentiation (LTP) at the CA1 Schaffer collateral synapses, is

facilitated by D1/D5 DA receptors (Frey et al., 1990; Huang and Kandel, 1995; Otmakhova

and Lisman, 1996). Thus alterations in the dopaminergic neurotransmission induced by

pre-exposure to cocaine may interfere with processes of LTP and learning/memory

formation.

Besides the observed alteration in hippocampal dopaminergic function in the

cocaine-treated female rats during withdrawal, the serotonergic neurotransmission in this

brain formation also seems to be affected by cocaine treatment. Saline-treated males

presented an increase in the 5-HIAA/5-HT turnover rate from 5 to 10 days after the last

administration, suggesting an increase in the 5-HT metabolism that was not observed in

the cocaine-treated males, and therefore, a cocaine treatment effect in the serotonergic

pattern of evolution throughout withdrawal. In cocaine-treated females, the levels of 5-

HIAA increased from 5 to 10 days after withdrawal, as well as it was observed a trend for

an enhanced 5-HIAA/5-HT turnover rate, suggesting an increase in the 5-HT metabolism

266

throughout long-term withdrawal. These data point to an altered hippocampal function

after long-term cocaine withdrawal, both in male and female rats cocaine exposed during

adolescence, Kimura et al. (2004) also reported changes in hippocampal serotonergic

modulation several days after cessation of repeated administration of methamphetamine,

even though, 10 days after withdrawal, these changes were no longer observed. Reports

on the long-term effects of cocaine on hippocampal serotonergic activity are scarce.

Nevertheless, for instance Przegalinski and colleagues (2003) reported that withdrawal

from chronic cocaine induces up-regulation of 5-HT1B receptors (Przegalinski et al.,

2003). Some evidence indicates that up-regulation of the 5-HT1B receptors occurs

following impairment of 5-HT neurotransmission, e.g. after 5-HT denervation with 5,7-di-

hydroxytryptamine, or after inhibition of 5-HT synthesis by chronic

parachlorophenylalanine treatment (Manrique et al., 1994; Compan et al., 1998). Thus the

5-HT1B receptor up-regulation after cocaine withdrawal may be a consequence of

impaired 5-HT neurotransmission.

- Effects on the amygdala

In the amygdala, saline-treated females presented a decrease in the levels of DA

and DOPAC from 5 to 10 days after the last administration, with this effect not being

observed in cocaine-treated females, which suggests an effect of cocaine treatment on

the evolution pattern of the dopaminergic neurotransmission in the amygdala of female

rats during long-term withdrawal. In fact, although no significant differences were

observed in DOPAC/DA turnover rate, 10 days after withdrawal cocaine-treated females

presented higher levels of DOPAC as compared to control females. Moreover, at this

time, saline-treated females presented lower levels of DOPAC as compared to saline-

treated males. This sex difference was not present in the cocaine group, and it is likely to

result from an altered DA system maturation in the amygdala of cocaine-treated female

rats. The expression levels of D1, D2 and D3 receptors, DAT and MAO-A mRNA in male

rats, determined 10 days after withdrawal also did not showed any treatment effects.

Altogether these data suggest a sex-effect regarding dopaminergic neurotransmission in

the amygdala during long-term withdrawal, with females being more sensitive than do

males to the effects of cocaine. Amygdala continues to mature during adolescence (Cao

et al., 2007), and therefore the alterations observed could be due to the cocaine effects on

the developmental process occurring during the exposure period.

Withdrawal from cocaine use is accompanied by the development of negative

emotional states characterized by intense dysphoria and anxiety (Koob and Le Moal,

267

1997; Gordon and Rosen, 1999). These observations suggest a role for the amygdala, a

component of the limbic system that serves an essential function in associative emotional

learning (Miserendino et al., 1990; Fanselow and LeDoux, 1999; LeDoux, 2000; Nader et

al., 2000; Maren, 2001). The amygdala receives dopaminergic projections from the VTA

(Koob et al., 1998; Nestler, 2001), that has been implicated, along with other brain

regions, in drug-seeking behaviour and relapse both in humans and experimental animals

(Breiter et al., 1997; Kalivas and McFarland, 2003). Because the amygdala sends

projections to key components of reward circuitry, including the Nac and prefrontal cortex

(Baxter and Murray, 2002; Wise, 2002), synaptic modifications in the amygdala resulting

from cocaine use may affect the function of these reward-associated brain structures.

Alternatively, the amygdala may contribute to brain reward function through the formation

of associations between the rewarding effects of cocaine and contextual cues (Hayes and

Gardner, 2004).

On the other hand, although serotonergic neurotransmission in the amygdala is

associated with anxiety-like behaviour (Higgins et al., 1991; Gargiulo et al., 1996;

Zangrossi et al., 1999), and anxiety has been descried as a symptom of cocaine

withdrawal (Gawin, 1991), our data seems to indicate that cocaine treatment throughout

adolescence did not have long-term effects in basal serotonergic neurotransmission in the

amygdala, since there were no alterations in the levels of 5-HT and 5-HIAA or in 5-HT

turnover rate, both 5 and 10 days after withdrawal, for both sexes.

- Effects on the hypothalamus

In the hypothalamus, cocaine treatment did not affect the levels of DA, 5-HT, their

metabolites, or turnover rates, neither in males or females as assessed 5 and 10 days

after withdrawal. Moreover, the expression levels of D1, D2 and D3 receptors, DAT and

MAO-A mRNAs were also not altered 10 days after withdrawal in cocaine-exposed males.

These data obtained in the hypothalamus seem to complement the hormonal data

indicating that chronic cocaine binge administration does not seem to have prolonged

effects upon the HPA axis activity under basal conditions, both in male and female rats.

Nevertheless, other authors have reported alterations in dopaminergic and

serotonergic neurotransmission in the hypothalamus during cocaine withdrawal. Karoun

and colleagues (2004) reported that, 1 week after 7 days of repeated cocaine

administration, the DA turnover in the hypothalamus was reduced (Karoum et al., 1994).

Differences in treatment duration, doses and period of exposure (adulthood versus

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adolescence) may be underlying the differences between our study and the results

reported by the latter study.

- Effects on the dorsal raphe nucleus

Since symptoms of depression have been associated with cocaine withdrawal, and

a dysfunction of the serotonergic neurotransmission is thought to underlie a variety of

psychiatric disorders, such as depression (Arango et al., 2002; Lira et al., 2003; Bach-

Mizrachi et al., 2006), it was expected that serotonergic neurotransmission in the dorsal

raphe nucleus would be altered during withdrawal from chronic binge cocaine. However,

in dorsal raphe nucleus, no effects of chronic binge cocaine exposure were observed on

the levels of 5-HT and 5-HIAA or 5-HT turnover rate, both in male and female rats, as

assessed 5 and 10 days after cocaine withdrawal. Nevertheless, 5 days after the last

administration, saline-treated males presented higher levels of DA as compared to saline

females, with this sex effect not being observed in the cocaine-treated group, which may

indicate some disruption of the dopaminergic neurotransmission in the dorsal raphe

nucleus. It is known that 5-HT neurons of the dorsal raphe nucleus receives a dense

innervation from midbrain dopaminergic neurons (Peyron et al., 1995; Kitahama et al.,

2000) and express D2 and D3 receptors (Mansour et al., 1990; Suzuki et al., 1998).

These anatomical evidences suggest that DA input to the dorsal raphe nucleus may play a

critical role in the regulation of the dorsal raphe nucleus 5-HT neurons activity.

Consistently with this notion, in vivo neurochemical studies have reported that

administration of DA receptor agonists increases the synthesis and release of 5-HT in the

dorsal raphe nucleus (Ferre et al., 1994; Matsumoto et al., 1996). Direct evidence for a

role of DA in the regulation of 5-HT neurons activity comes from an in vitro

electrophysiological study showing that activation of DA receptors induces a membrane

depolarization and thereby increases the excitability of dorsal raphe 5-HT neurons (Haj-

Dahmane, 2001). However, the alteration on dopaminergic neurotransmission observed 5

days after withdrawal was not accompanied by any alteration on the levels of 5-HT, 5-

HIAA or 5-HT turnover rate. Moreover the altered dopaminergic activity was no longer

present 10 days after withdrawal.

- Effects on the cerebellum

The long-term effects of exposure to psychostimulants on 5-HT neurotransmission

in the cerebellum are not well characterized. Our data indicate that the chronic binge

cocaine administration throughout adolescence did not have long-term effects on the

269

levels of 5-HT, 5-HIAA or 5-HT turnover rate. These results are consistent with those

obtained in the dorsal raphe nucleus, and altogether seem to point to an absence of long-

term effects of chronic cocaine administration throughout adolescence. Nevertheless,

saline-treated males presented decreased levels of HVA from 5 to 10 days after the last

administration, but no differences to cocaine-treated males were observed at 5 and 10

days of withdrawal, suggesting an effect of cocaine on the evolution pattern of the

dopaminergic neurotransmission, from 5 to 10 days after withdrawal.

- Interaction between the dopaminergic system and the HPA axis

As already stated in chapter 1, the interaction of glucocorticoids with the

mesocorticolimbic DA system, through the GR, may have a significant impact on the

vulnerability to self-administer psychostimulant drugs, since increased dopaminergic

transmission in these pathways is critical for the reinforcing properties of abusive drugs,

rendering the individuals more susceptible to drug reinforcement (Roberts et al., 1980;

Koob and Le Moal, 1997; Marinelli and Piazza, 2002). However, our results seem to

suggest that the increased dopaminergic activity observed 10 days after withdrawal in

cocaine-treated male rats was not associated with an augmented stimulatory effect of the

HPA axis, since no effects of cocaine were observed in basal corticosterone levels, and

the expression levels of GR mRNA was only affected in the SN-VTA of cocaine-treated

males that presented lower expression levels as compared to control animals at 10 days,

suggesting that this was not the mechanism involved in the increased dopaminergic

activity observed.

- Conclusion

In conclusion, chronic exposure to cocaine throughout the adolescence produced

long-lasting changes upon the dopaminergic system in male rats, with the data obtained

suggesting an increase in dopaminergic activity of the nigrostriatal pathway. In fact, the

data pointing to increased dopaminergic activity, is supported by an increase in the

expression levels of TH mRNA found in the area of origin of the dopaminergic system.

The increased dopaminergic activity may represent an attempt to compensate for a

chronic cocaine-induced depression of the dopaminergic system. The serotonergic activity

in male rats was also long-term affected by cocaine treatment, Increased 5-HT levels in

SN-VTA were observed, and just like as suggested for the dopaminergic activity, the

increase in the serotonergic activity may represent an attempt to counteract the

downregulation of this system’s activity during withdrawal.

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Diverging from the data obtained in male rats, in cocaine-treated females it was

observed an increase of the dopaminergic activity in the amygdala and in the

hippocampus, as well as an increase of the serotonergic activity in the hippocampus and

in the Nac throughout long-term withdrawal, these neurochemical alterations, among other

effects, could contribute to an increased vulnerability to relapse and interfere with

processes of learning and memory formation.

4.4.4. Effects of chronic cocaine treatment during adolescence on the anxiogenic-like behaviour

As already discussed in chapter 3, symptoms of anxiety and depression are

associated with withdrawal from cocaine exposure, particularly in the initial period

(Gawin, 1991). It has been postulated that anxiety persists into the later withdrawal period

and is accompanied by intense craving that may lead to relapse.

The number of entries and the time spent in the aversive open arms of the EPM

are typical measures of anxiety in this behavioural test (Pellow et al., 1985). We did not

observed both 5 and 10 days after cocaine withdrawal effects on these measures in male

rats. The locomotor activity was also not affected by the treatment, both 5 and 10 days

after withdrawal, as indicated by the absence of differences between cocaine- and saline-

treated males, in the number of closed arm entries, the most reliable measure of

locomotor activity (Hogg, 1996).

Nevertheless, data analysis indicate that male rats exposed to cocaine during

adolescence presented 10 days after withdrawal, a decrease in the time spent in the

central platform. The time spent in the center portion of the EPM has been interpreted as

a decisional component of approach/avoidance conflict task (Rodgers and Johnson, 1995;

Carobrez and Bertoglio, 2005). These results add to the alterations in risk-assessment in

young adult male rats pre-exposed to cocaine during adolescence, when exploring the

EPM apparatus.

There are numerous studies reporting that animals exhibit enhanced anxiety

during the first days after cocaine withdrawal (Sarnyai et al., 1995; Basso et al., 1999).

However, the extent to which anxiety-like responses persist over extended drug-free

periods is unclear. In some studies, cocaine pre-exposed animals exhibited anxiety-like

behaviours after drug-free periods of up to 7 days (e.g., Fontana and Commissaris, 1989;

Gordon and Rosen, 1999), whereas in other studies, cocaine pre-exposure was

ineffective in elevating anxiety beyond the first 2–3 days of withdrawal (Mutschler and

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Miczek, 1998). Erb and colleagues (2006) reported enhanced levels of anxiety in adult

rats, 10 days after cocaine withdrawal (30 mg/kg, i.p. for 7 days), but only when animals

were exposed to an environment previously paired with cocaine, suggesting that a

“reminder” of the drug experience, such as re-exposure to contextual cues previously

paired with cocaine, may be necessary to reactivate a cocaine-induced anxiety-like state

after the initial withdrawal period (Erb et al., 2006).

To our knowledge, only one study, by Estelles and colleagues have been

performed to evaluate the later impact of cocaine exposure during adolescence on anxiety

like-behaviour (Estelles et al., 2007). In mice, a binge pattern of cocaine administration

during adolescence modified the anxiety of the treated-adolescent, increasing the time

spent on the open arms of the EPM; however, housing in isolation counteracted this

cocaine effect (Estelles et al., 2007). In fact, it has been suggested that housing condition

can modulate the behavioural responses of rats to the EPM (Starkey et al., 2007). In any

animal model, the utility of the EPM is dependent on the anxiety threshold levels, low

anxiety baselines are less than optimal for detecting anti-anxiety effects and high

baselines virtually useless for detecting anxiety enhancement (Rodgers and Cole, 1993).

For example, six weeks of isolation reared rats spent less time into the open arms on

EPM compared to group housed rats (Gupta and Rana, 2007). Thus, in the present study,

the fact that animals were housed individually may have contributed to a high anxiety

baseline, which mask eventual effects of cocaine.

As already discussed in this chapter, 10 days after cocaine withdrawal, the basal

levels of corticosterone were not significantly altered in male rats, while, however after the

EPM, cocaine-treated males presented a significant increase in corticosterone, suggesting

that although chronic cocaine administration during adolescence does not seem to have

prolonged effects upon corticosterone levels under basal conditions, it does sensitize the

HPA axis activity under anxiogenic conditions.

One of the most consistently described biological abnormalities in depressive and

anxiety disorders is dysregulation of the HPA axis, which is typically normalized by

successful antidepressant or anxiolytic therapy (Nestler et al., 2002; Wichniak et al., 2004;

Mikkelsen et al., 2005). However, our data showed that the HPA axis activity was long-

termed affected, but this was not accompanied by alterations in anxiety-like behaviour as

observed in the EPM. Munoz-Abellan et al. (2008) have already reported dissociation

between HPA axis activation and the anxiety-like behaviour observed during EPM. These

authors suggested that after a very short exposure to the EPM, HPA axis activation may

reflect arousal rather than a degree of emotional reaction to the situation (Munoz-Abellan

et al., 2008).

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Corticosterone, either stress-induced or exogenously administered, is clearly

implicated in anxiety in the EPM in adult rats (e.g., Adamec et al., 2006; Matuszewich et

al., 2007). For example, corticosterone administered to the amygdala increased anxiety-

like behaviour in the EPM (Shepard et al., 2000; Myers et al., 2005) and HPA axis

response to stress (Shepard et al., 2003). Exposure to an acute stressor before testing in

the EPM increased anxiety-like behaviour, which could be attenuated by blockade of

corticosterone synthesis and reinstated by administering corticosteroid agonists (Calvo

and Volosin, 2001). Moreover, antagonists of the corticosteroid receptors attenuated the

anxiogenic effects of pre-exposure to acute stress in the EPM (Korte et al., 1995).

However, in non-stressed rats, individual differences in corticosterone concentrations

while in the EPM showed a stronger association with risk-assessment than either with

anxiety (open arm exploration) or locomotor activity (Rodgers et al., 1999; Albrechet-

Souza et al., 2007). Based in the aforementioned, the higher levels of corticosterone

displayed by cocaine exposed males after the EPM, at 10 days of withdrawal are most

likely related with the impaired risk-assessment observed in these animals, as supported

by the decreased time spent in the central platform of the maze.

The molecular mechanisms by which pretreatment with cocaine throughout

adolescence lead to sensitization of the HPA axis to an anxiogenic stimulus were not

addressed by the present study. One possible explanation to the augmented HPA axis

response to the EPM in the cocaine exposed males could be related with an impaired GR-

mediated negative feedback, which would remove inhibitory constraint from the HPA axis,

thus augmenting the glucocorticoid response to the EPM. However, and as already

mentioned in this chapter, 10 days after withdrawal, no treatment effects were found in the

expression levels of the GR mRNA in pituitary and in several brain areas implicated in

negative feedback regulation of the HPA axis. Although these findings suggest that the

augmented HPA axis response was independent of impaired GR-mediated feedback

inhibition, functional measures of feedback (e.g., sensitivity to dexamethasone

suppression) are necessary to clarify the contribution of altered negative feedback.

Further investigation is required to determine the role of adaptations within the HPA axis

that are independent of GR-mediated feedback in the intensified response.

The neurochemical results obtained after the EPM, 10 days after withdrawal,

showed that the dopaminergic and the serotonergic response in amygdala of cocaine-

treated male rats were affected under an anxiogenic situation, as indicated by the lower

levels of DOPAC and 5-HIAA, as compared to control males. In fact, control males display

an increase in DOPAC levels after the EPM that was not observed in cocaine-treated

males. As already discussed in Chapter 3, amygdala is a critical structure in the neural

273

pathways necessary for implementing advantageous decisions. The amygdala is involved

in the attachment of emotional valence to events (i.e., reward and punishment) (Bechara

et al., 1999) and is crucial for decision making and learning (e.g., Winstanley et al., 2004).

Lesions of the amygdala decrease harm avoidance (Johansson and Hansen, 2002). Thus,

the disruption in neurotransmission observed in the amygdala of cocaine-treated males

after the EPM may be related with the impaired risk assessment observed in these

animals, as suggested by the decreased time spent in the central platform of the maze.

and by this way contributing to a increased vulnerability to relapse.

These neurochemical results differ substantially from the data obtained in the

EPM 2 days after withdrawal (discussed in Chapter 3). During acute withdrawal, a

significant altered prefrontal cortex neurotransmission was observed in cocaine-treated

male rats, that was no longer found after long-term withdrawal. Besides the changes

observed in amygdala, no significant differences were observed in the neurotransmitter

profile between cocaine-treated and controls in other brain areas. However, when

comparing the profiles before and after EPM were observed important differences

between cocaine- and saline-treated male rats.

In the prefrontal cortex cocaine-treated males after EPM presented higher

DOPAC/DA turnover rate as compared to cocaine males under basal conditions and this

effect was not observed in saline-treated males, which represents a decreased availability

of DA in this region. In the hippocampus, control males presented higher 5-HT turnover

rate after EPM as compared to basal conditions, however this effect was not observed in

cocaine-treated males, which may represent an altered use of 5-HT.

In 2005, Carvalho and colleagues reported the effects of EPM exposure in the

biogenic amine content of the prefrontal cortex, amygdala, dorsal hippocampus of rats

(Carvalho et al., 2005). Their results suggest that the exposure to EPM causes a

significant decrease in the 5-HT concentration in the prefrontal cortex, amygdala and

dorsal hippocampus; with the amygdala being the only structure in which there was also a

reduction in the tissue concentration of DA and DOPAC. Moreover, their results suggest

that the observed changes in the neurotransmission of 5-HT and DA could not be

attributed to enhanced metabolism, since the turnover rates of these biogenic amines

were not different from those of control animals, not tested in the EPM. These results are

quite different from those obtained in our study. Several causes may underlie these

differences. One possible explanation may be the previous experimental manipulation that

animals in our study were submitted to, including the repeated injection protocol and long-

term social isolation, since both procedures are known to be stressful and capable of

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interfere with neurochemical processes (Suthanthirarajan and Subrahmanyam, 1983;

Persico et al., 1995; Kosten et al., 2003), this way affecting the response to the EPM.

In the SN-VTA, after the EPM, no cocaine treatment effects were detected in the

male rats. Nevertheless the higher levels of DOPAC observed in cocaine-treated males as

compared to controls under basal conditions was not found in the animals that performed

the EPM.

In the dorsal raphe nucleus, after the EPM, in both cocaine- and saline-treated

males the levels of 5-HT were higher after the EPM as compared to basal levels. The

levels of 5-HT in dorsal raphe nucleus are related with anxiety-like behaviour.

Microinfusions of drugs that increase serotonergic activity directly into the dorsal raphe

nucleus, like the benzodiazepine receptor partial inverse agonists N-methyl-beta-

carboline-3-carboxamide (FG-7142) (Lista et al., 1990), methyl 6,7-dimethoxy-4-ethyl-

beta-carboline-3-carboxylate (DMCM) (Maier et al., 1995), and the anxiety-related

neuropeptide urocortin 2 (Hammack et al., 2003; Amat et al., 2004) increase anxiety

related behavioural responses. In contrast, microinfusions of 5-HT or 5-HT1A agonists

into the dorsal raphe decrease anxiety-related behavioural responses, presumably via

activation of somatodendritic 5-HT1A autoreceptors on serotonergic neurons, resulting in

decreased 5-HT neurotransmission (Higgins et al., 1988; Higgins et al., 1992; Schreiber

and De Vry, 1993). In the present study the 5-HT turnover rate was lower in saline-treated

males after the EPM as compared to basal levels, suggesting a decrease in the 5-HT

metabolism. This effect was not observed in cocaine-treated males, which could be a sign

of increased anxiety-like behaviour, and account to explain the different strategies to

explore the EPM apparatus, as indicated by the decreased time spent in the central

platform by the cocaine-treated male rats.

Interestingly the higher levels of corticosterone were not accompanied by altered

DA or 5-HT neurotransmission in the hypothalamus. Nevertheless, a mechanism of

cocaine-induced supersensitivity of 5-HT2A receptors, in the paraventricular nucleus, have

been described after 42-h withdrawal from repeated cocaine treatment (15 mg/kg i.p.,

twice a day for 7 days) (Levy et al., 1992b). Activation of 5-HT2A receptors in the

hypothalamic paraventricular nucleus increases the secretion of ACTH, corticosterone,

oxytocin, and prolactin (Van de Kar et al., 2001). Thus, a possible alteration in the 5-HT

receptors can not be excluded.

To summarize, in the present study it was demonstrated that the EPM-induced

activation of the HPA axis is augmented in prolonged cocaine withdrawal and that despite

the absence of increased anxiogenic-like behaviour, the impaired risk-assessment

275

observed in cocaine-treated it is likely to be related with the altered HPA axis response.

Moreover, the neurotransmission disruption observed in amygdala of cocaine-treated

males in response to the EPM may also be related with changes in the behavioural

strategy adopted to cope with the anxiogenic stimulus provided by the EPM. Altogether,

these results may point to an increased susceptibility to drug use after long-term

withdrawal, since impaired risk-assessment and enhanced HPA axis response to an

environmental challenge are considered risk factors for relapse. Stress-induced HPA axis

responses can be used to predict the likelihood of drug relapse (Sinha et al., 2006), and

risk-taking behaviours are associated with increased risk for drug use onset (Rios-Bedoya

et al., 2008). Therefore, the present results may also contribute to increase knowledge on

how to structure drug abuse prevention.

4.4.5. Effects of chronic cocaine treatment during adolescence on the aggressive behaviour

In a variety of vertebrates, including primates, correlations have been established

between levels of aggressiveness and testosterone or neural testosterone metabolism

(Rose et al., 1971; Barfield et al., 1972; Albert et al., 1986). In mice, strong correlations

have been established between testosterone and the latency to attack an intruder in

resident-intruder models of offensive aggression (Van Oortmerssen et al., 1987). In rats,

castration results in a decline of aggression (Albert et al., 1986). Presumably, testosterone

would affect neural networks controlling offensive aggression, resulting in an enhanced

arousal and predisposition towards agonistic displays (Delville et al., 1996). Indeed,

testosterone is suspected to modulate offensive aggression by affecting several areas

within the limbic system (Bean and Conner, 1978; Albert et al., 1987).

As already discussed in this chapter, the present study showed that 10 days after

cocaine withdrawal male rats presented decreased levels of testosterone. Thus, due to

the role of testosterone in the aggressive behaviour, it was hypothesized that aggressive

behaviour in these animals could be altered.

There are several reports that have found an increase in aggressive and violent

behaviours in cocaine addicts (Denison et al., 1997; Chermack and Blow, 2002; Fals-

Stewart et al., 2003; Moore et al., 2008). The preclinical literature linking cocaine and

aggression is not so cohesive probably because a variety of test paradigms, animal

models, and dosing regimens have been utilized, yielding conflicting data (Miczek, 1979;

Hadfield et al., 1982; Emley and Hutchinson, 1983; Filibeck et al., 1988; Darmani et al.,

1990; Long et al., 1996). For example, Mizcek (1979) reported an inverse relationship

276

between cocaine treatment and aggression in mice treated acutely with medium- and

high-dose (8.0 and 32.0 mg/kg, respectively) cocaine, while reporting no effect on

aggression at low doses (0.5 and 2.0 mg/kg). In comparison, adult male rats treated

chronically with low doses of cocaine (1.0 mg/kg) showed elevated measures of

aggression (Long et al., 1996).

A well-established and ethologically valid model of offensive aggression used

routinely to test for aggressive behaviour is the resident–intruder paradigm (Miczek, 1979;

Koolhaas et al., 1980). In the present study, this paradigm was used to examine the long-

term effects of chronic cocaine exposure during adolescence on aggressive behaviour of

young adult male rats. The behavioural data obtained revealed no long-term effects of

chronic cocaine administration throughout adolescence on the aggressive behaviour of

the male rats, when assessed 10 days after withdrawal.

There are fewer studies evaluating the long-term effects of cocaine on social

behaviours. An increase in aggression has been found in adult rats exposed gestationally

to cocaine, using the resident-intruder paradigm (Johns et al., 1994) or toward a rival

when competing for water (Wood and Spear, 1998). Although our and the later mentioned

studies present data from long-term effects of cocaine exposure, the results can not be

compared, since the time of cocaine exposure were different (adolescence versus

gestational development). In a study using adolescent hamsters, Harrison and colleagues

(2000) also reported that chronic cocaine treatment increased the aggressive behaviour

(Harrison et al., 2000); however, this data was obtained 24 hours after the last drug

administration and in the present study aggressive behaviour was assessed 10 days after

the last drug administration. Nevertheless, in mice, Estelles and colleagues (2005)

reported that animals prenatally treated with cocaine did not exhibit an increase in

aggressive behaviours when tested as adults (Estelles et al., 2005).

In studies evaluating social behaviour, the social history of the animals employed

is very important. Isolated mice exhibit a particular behavioural profile with higher levels of

aggression than those housed in groups (White et al., 1991; Matsumoto et al., 2005). In

male rats social isolation is also reported to increase aggression (Wongwitdecha and

Marsden, 1996; Hall, 1998). Thus, the social isolation that animals in the present study

were submitted to could have increased the aggressive behaviour, by this way masking

the cocaine effects regarding this behaviour.

Of particular interest, however, was the finding that cocaine-treated male rats

displayed increased duration of the anogenital sniffing behaviour. Moreover, these

animals also exhibited lower frequency of and increased latency to flight behaviour. These

277

results appear to indicate that cocaine treatment during adolescence led to an increase in

social investigation and reduce fear at 10 days after withdrawal. This seems to contradict

the established consensus regarding the reduction in social contacts within conspecifics

that has been observed after cocaine administration in rodents (Darmani et al., 1990;

Rademacher et al., 2002). However, this assumption seems not to be so linear, as for

instance Estelles at al. (2005) reported that, isolated mice show a significant decrease in

the time dedicated to social interactions, while grouped animals prenatally treated with

cocaine exhibited an increase in the social contact and the time dedicated to each

contact, showing that some variables can influence the cocaine effects on social

investigation. In adult rats social isolation is reported to increase social investigation of an

unfamiliar rat (Niesink and van Ree, 1982; Varlinskaya et al., 1999). Therefore, rats are

routinely isolated prior to being tested in the social interaction paradigm, as this should

increase the time spent interacting with conspecifics, making alterations at this level

easier to observe (File and Seth, 2003). In our study, and contrarily to that reported by

Estelles and colleagues (2005), cocaine treatment seems to have intensified the effects of

social isolation in what concerns to the social investigation. The species in study (rat

versus mice), the time of exposure to the drug (adolescence versus gestational

development) and the withdrawal status (10 days after the last administration versus since

birth to adulthood) may have contributed to the different results between these studies.

Behavioural data suggest that chronic cocaine exposure during adolescence have

long-lasting influences in male rat reactions to social threat, providing some indications to

the capability of cocaine during adolescence to affect the ability of young adult rats to

interact properly with conspecifics. Moreover, the reduced fear presented by the cocaine-

treated animals, as indicated by the decrease in flight behaviour, suggest an altered risk-

assessment, supporting the results obtained in the EPM, also indicating an effects of

cocaine treatment in the risk-assessment.

In this study the corticosterone response to the resident-intruder paradigm was

also determined in order to evaluate the effects of cocaine treatment in the HPA axis

response to a social threat. HPA axis abnormalities in humans and rodents have often

been associated with changes in male aggression (McBurnett et al., 2000; de Kloet, 2003;

Haller et al., 2004). Paradoxically, high as well as low HPA axis activity can be associated

with excessive aggression (reviewed by Veenema and Neumann, 2007). High HPA axis

activity, often associated with a state of hyperarousal, is thought to underlie sudden

outbursts of aggression, whereas chronically low HPA axis activity, often associated with

a state of hypoarousal, might induce long-lasting changes in brain functions that promote

violence (Haller and Kruk, 2006). In clinical studies, elevated glucocorticoid responses

278

were found in individuals with high aggression levels (Gerra et al., 1997). Furthermore, an

acute activation of the HPA axis was found to promote aggressive behaviour in rodents

(Kruk et al., 2004; Mikics et al., 2004). In contrast, there are preclinical (Haller et al., 2001;

Haller et al., 2004) and clinical studies (McBurnett and Lahey, 1994; McBurnett et al.,

2000; Shoal et al., 2003) reporting an inverse relationship between pituitary-adrenocortical

(re)activity and aggressive behaviour. However, in these studies, high levels and

abnormal forms of aggression were associated with a chronic glucocorticoid deficiency.

The levels of corticosterone after the resident-intruder paradigm, although higher

than the basal, did not differ between cocaine- and saline-treated males, suggesting that

cocaine treatment did not affect the HPA axis response to the social threat provided by

the resident-intruder paradigm. As this behavioural test is known to induce a very large

adrenocortical stress response both in resident and intruder rats (Schuurman, 1980), the

increased response of the HPA axis induced by the test was the expected one. However,

it is also possible that this way mask the effects of cocaine treatment.

Classical neuroanatomical tracing and electrochemical lesion/stimulation studies

have revealed the global neural substrates of aggression (Luiten et al., 1985; Gregg and

Siegel, 2001). Several regions of cortex, amygdala, septum, hypothalamus,

periaqueductal gray and their interconnected structures are among the best documented

and characterized in this respect. In addition, more recent studies using functional

immediate-early gene expression mapping (i.e., c-FOS, p-CREB, zif-268) start to yield a

more detailed picture of the individual neurons and their neurochemical identities that

become activated within these brain regions during the expression of aggressive

behaviour (Gammie and Nelson, 2001; Halasz et al., 2002; van der Vegt et al., 2003b;

Veening et al., 2005). Despite this expansion, the 5-HT neurotransmitter system remains

the primary molecular determinant of aggression (Tuinier et al., 1995; Berman et al., 1997;

Miczek et al., 2002).

There exists a large body of evidence implicating the 5-HT as regulator of

aggressive behaviour in a number of animal models of aggression (Hadfield et al., 1982;

Vergnes et al., 1988; Higley et al., 1992; Kyes et al., 1995) and in adolescent and adult

human populations (Brown et al., 1982; Linnoila et al., 1983; Kruesi et al., 1990). In

rodents, aggressive behaviour is effectively reduced by treatment with 5-HT1A and 5-

HT1B receptor agonists (De Almeida and Lucion, 1997; Miczek et al., 1998; Simon et al.,

1998; de Boer et al., 1999; Ferris et al., 1999). In humans, excessive aggression and

impulsive violent behaviours are associated with low cerebrospinal fluid levels of the 5-HT

metabolite 5-HIAA (Berman et al., 1997). In fact, the prevailing view has been that 5-HT

exerts an inhibitory control over aggression (Linnoila and Virkkunen, 1992; Kavoussi et al.,

279

1997). However, other findings in animal research challenge this 5-HT deficiency

hypothesis of aggression, in which 5-HT is positively associated with the display of

aggression (Olivier, 2004; de Boer and Koolhaas, 2005). These opposing views might

demonstrate important differences in 5-HT functioning in abnormal or pathological forms

of aggression compared to normal and adaptive levels of aggression. Likewise, there may

be essential differences regarding the role of the 5-HT system in trait-like and state-like

aggression. Trait-like aggression can be associated or induced by chronically reduced 5-

HT activity (Miczek et al., 2002), whereas state-like aggression is characterized by

increased 5-HT activity (van der Vegt et al., 2003a; van der Vegt et al., 2003b; Summers

et al., 2005). It has also been demonstrated that 5-HT plays an important role in cocaine-

mediated aggressive behaviour (Moeller et al., 1994; DeLeon et al., 2002; Ricci et al.,

2004).

In the present study, the absence of differences in aggressive behaviour between

cocaine- and saline-treated animals was followed by the absence of differences in the

levels of 5-HT or 5-HIAA in the dorsal raphe nucleus after the resident-intruder paradigm.

Moreover, both in control and cocaine-treated rats, the basal levels of 5-HT and 5-HIAA

also did not differ from the levels after the resident-intruder paradigm. However, in saline-

treated rats the 5-HT turnover rate decreased between basal and post-test conditions, and

this effect was not observed in cocaine-treated rats. This may point to an effect of cocaine

treatment in the 5-HT neurotransmission in the dorsal raphe nucleus of rats facing the

social conflict provided by the resident-intruder paradigm.

Moreover, in the hippocampus, the levels of 5-HT were increased in the saline-

treated rats after the resident-intruder paradigm, and again this effect was not observed in

cocaine-treated males. Adding to this in the amygdala the 5-HT turnover was decreased

after the test only in the cocaine-treated males.

The serotonergic system modulates aggressive behaviour in interaction with other

neurotransmitters, of which the corticolimbic DA continues to be of interest for its critical

role in integrating motivation and motor functions (Robbins et al., 1989).

In the present study, the levels of 5-HT after the resident-intruder paradigm in the

prefrontal cortex did not differ between cocaine- and saline-treated males, as well as no

significant differences were observed in the 5-HT neurotransmission in the prefrontal

cortex between basal and post-test conditions. Damage to or pharmacological inhibition of

the prefrontal cortex can increase aggression, and this effect is hypothesized to be

caused by loss of impulse control (Tobin and Logue, 1994). In a former microdialysis

study, decreases in 5-HT and increases in DA levels in prefrontal cortex were observed

280

using the resident-intruder paradigm (van Erp and Miczek, 2000). Differences in the

experimental procedure may be responsible for the discrepancy of data obtained in the

present study and the data reported by Van Erp and Miczek (2000), namely the housing

condition (male rats housed individually versus male rats housed with a female) since it is

known that social isolation induce an alteration of the serotonergic system in rats (Rilke et

al., 1998; Dalley et al., 2002; Bibancos et al., 2007). Nevertheless, the behavioural test

induced a decrease in the levels of HVA in cocaine male rats. In fact, after the test these

animals presented a decrease in the DA metabolism as indicated by the decreased

HVA/DA turnover rate. Moreover, the dopaminergic neurotransmission was also affected

in hippocampus, both by the test and by the cocaine treatment. In the hippocampus, the

aggressive test induced an increase in DA levels in control animals that was not observed

in the cocaine-treated animals, suggesting a cocaine effect in the dopaminergic response

to the behavioural test. Indeed, after the test the levels of DOPAC were lower in cocaine-

treated animals as compared to control animals. These data suggest a decrease in the

dopaminergic neurotransmission in cocaine-treated rat in response to the resident-intruder

paradigm as compared to control animals.

In the amygdala, the levels of HVA after the test were reduced to non detectable

levels in both cocaine- and saline-treated animals, suggesting an effect of the test on the

dopaminergic neurotransmission in the amygdala.

In the SN-VTA, the dopaminergic response to the behavioural test was affected by

the cocaine treatment, as the levels of DOPAC in the cocaine-treated rats were reduced

by the test. Also in SN-VTA the HVA levels became undetectable after the test, both in

cocaine- and saline-treated rats. In this area the serotonergic neurotransmission was also

affected by the treatment, as after the test the 5-HT metabolism was lower in cocaine-

treated rats as compared to control animals. Moreover, in cocaine-treated rats the levels

of 5-HIAA were decreased by the test and this effect was not observed in control animals.

In the present study the serotonergic response in the hypothalamus to the social

conflict provided by the resident-intruder paradigm was affected by the cocaine treatment,

as indicated by the decreased 5-HT metabolism in the cocaine-treated rats that was not

observed in the control animals. In this area it was also observed an effect of the test on

the DA neurotransmission, as the behavioural test induced a decrease in the

dopaminergic metabolism of both cocaine- and saline-treated rats, suggested by the

decreased DOPAC/DA turnover rate. Also in this area the HVA levels become

undetectable in both cocaine and control animals after the test. Nevertheless, the DOPAC

levels were decreased by the test only in the cocaine-treated rats, suggesting a potential

effect of the cocaine treatment in the dopaminergic response to the social conflict

281

provided by the resident-intruder paradigm. It seems like cocaine treatment decreased

both serotonergic and dopaminergic response of hypothalamus to the social stressful

situation provided by the resident-intruder paradigm, but these alterations were not

reflected in the HPA axis activity, since no differences were observed between cocaine-

and saline-treated animals regarding the plasma levels of corticosterone.

Taken together, these findings indicate a decrease in the dopaminergic activity in

response to the social conflict provided by the resident-intruder paradigm in the cocaine-

treated rats, 10 days after withdrawal. Cocaine treatment also seems to have influenced

the serotonergic response to the challenge provided by the test, as data obtained in this

study indicates an increased serotonergic activity in the dorsal raphe nucleus and in the

hippocampus, while decreased in the amygdala, prefrontal cortex, hypothalamus and SN-

VTA. However, these alterations in the dopoaminergic and serotonergic

neurotransmission were not reflected on alterations in the aggressive behaviour. Rather,

these data may be relevant to explain the increased social investigation and decreased

defensive behaviour observed in cocaine-treated rats. Namely, the alterations in the

amygdala and prefrontal cortex were probably related with the decreased defensive

behaviour observed in cocaine-treated male rats. The decreased defensive behaviour

appears to be related with a decrease in fear, which may result from an impaired risk

assessment, also observed in these animals when exploring the EPM, and as was already

discussed, altered function in these brain regions are associated with increased risky

behaviour (Bechara, 2001; Blanchard et al., 2005; Muller and Fendt, 2006). On the other

hand, the knowledge of the hippocampus key role in the individual recognition

(i.e.Terranova et al., 1994; Kogan et al., 2000), lead us to suggest that the increase in

social investigation observed in cocaine-treated male rats, that was probably related with

an impaired ability to learn and remember individual (see, Yamamuro, 2006), was mostly

caused by the altered function of the hippocampus.

4.5. Conclusion

The data obtained in this study showed that cocaine exposure throughout

adolescence induced sex-dependent long-lasting hormonal, neurochemical and

behavioural effects.

Chronic cocaine exposure throughout adolescence led to long-lasting disruptive

effects on the HPG function in male rats, as indicated by the decrease in testosterone

levels observed 10 days after cocaine withdrawal, with these animals also showing a

decrease in relative gonadal weight.

282

Chronic cocaine exposure did produce long-lasting changes upon the

dopaminergic system in male rats. The neurochemical data obtained in the present study

showed an increase of the dopaminergic activity in the nigrostriatal pathway, also

supported by the increased expression levels of TH mRNA in the SN-VTA. This process

may represent an attempt to compensate for a chronic cocaine-induced depression of the

dopaminergic system.

Long-lasting effects of cocaine treatment were also found in the serotonergic

activity of the male rats. Increased 5-HT levels in SN-VTA were observed in cocaine-

treated males, and as suggested for the dopaminergic activity, such increased

serotonergic activity may represent an attempt to counteract the downregulation of this

system during withdrawal.

Diverging from the data obtained in male rats, cocaine-treated females presented

an increase in the dopaminergic activity in the amygdala and hippocampus, and an

increase in serotonergic activity in the hippocampus and Nac throughout long-term

withdrawal. Among other effects, these long-term alterations could contribute to an

increased vulnerability to relapse and interfere with processes of learning and memory

formation.

After prolonged withdrawal, impaired risk-assessment was observed in cocaine-

treated male rats when exploring the EPM at 10 days after withdrawal. Simultaneously,

disrupted neurotransmission in the amygdala was observed in these animals in response

to the EPM, which may be related with changes in the behavioural strategy adopted to

cope with the anxiogenic stimulus provided by the EPM. The HPA axis response under

the anxiogenic conditions provided by the this test was also disrupted, as indicated by the

higher levels of corticosterone observed in cocaine-treated animals.

Cocaine treatment intensified social investigation and decreased defensive

behaviour during the social conflict provided by the resident-intruder paradigm, indicating

that cocaine affected the ability of male rats to properly interact with conspecifics. The

reduced defensive behaviour observed in cocaine-treated male rats, suggests an impaired

risk-assessment, supporting data obtained in the EPM. The neurochemical data showed

that cocaine decreased the dopaminergic activity in response to the social conflict

provided by the resident-intruder paradigm, with the serotonergic response also being

influenced. The neurochemical alterations may have been relevant to the increased social

investigation and reduced fear observed in cocaine-treated male rats.

283

The neurochemical, hormonal and behavioural effects of cocaine administration

observed in this study are likely contribute to an increased vulnerability to drug abuse and

relapse after withdrawal. Again, the obtained results are also relevant from the social

perspective and can be issued to better structure drug abuse prevention in the

adolescence.

284

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Chapter 5

General Conclusion

5. General Conclusion

A global analysis of the data reported in the present work allows to conclude that

the chronic exposure to cocaine throughout adolescence produced sex-dependent

hormonal, neurochemical and behavioural short and long-term alterations.

Global alterations in adolescent male rats chronically exposed to cocaine

In male rats, the HPA axis was both activated by acute cocaine administration after

chronic cocaine treatment, and affected in a long-term perspective. Although the basal

activity was not affected, a disrupted HPA axis response under anxiogenic conditions was

observed in male rats 10 days after withdrawal, as indicated by the presence of higher

corticosterone levels in response to the EPM.

Chronic cocaine exposure throughout adolescence also led to short and long-

lasting disruptive effects on the HPG axis function in male rats, as indicated by the

decreased testosterone levels observed both 30 minutes and 10 days after the last

administration of cocaine. Concurrently these animals also showed decreased relative

gonadal weights.

Within the dopaminergic and serotonergic neurotransmission systems, an overall

tolerance to the acute effects of cocaine was observed after chronic cocaine

administration. However, the neurochemical data obtained 1 day after the last

administration of cocaine, revealed increased dopaminergic activity, which led us to

suggest that the expected decrease in dopaminergic activity could have been masked by

exposure to cocaine cues-induced increases in dopaminergic activity, since the animals

were sacrificed in the same room where cocaine was daily administered. This may

indicate a high susceptibility of adolescent rats to environmental cues, with male rats

showing a greater involvement of the nigrostriatal pathway in this process.

In a long-term perspective, the dopaminergic system in male rats was undoubtedly

affected by the cocaine treatment, as demonstrated by increased dopaminergic activity in

the nigrostriatal pathway, and supported by increased expression levels of TH mRNA in

the SN-VTA. This process may represent an attempt to compensate for a chronic cocaine-

induced depression of the dopaminergic system.

Long-lasting effects of cocaine treatment were also found in the serotonergic

activity in male rats. Increased 5-HT levels in SN-VTA were observed in cocaine-treated

males, and as suggested for the dopaminergic activity, such serotonergic activity increase

303

may represent an attempt to counteract the downregulation of this system during

withdrawal.

The behavioural studies performed seem to indicate that cocaine exposure during

adolescence interfered with the adaptive responses to environmental challenges.

Impaired risk-assessment during the EPM apparatus exploration was observed in

male rats, both at short- and long-term withdrawal. Paralleling impaired risk-assessment

during short-term withdrawal, our data showed impaired dopaminergic and serotonergic

neurotransmission in the prefrontal cortex and impaired dopaminergic neurotransmission

in the amygdala. Supporting the altered risk-assessment during long-term withdrawal it

was observed impaired dopaminergic and serotonergic responses in the amygdala. These

neurochemical alterations were probably related with the failure of cocaine-treated males

to exhibit an adequate behaviour when exploring the EPM. Importantly, cocaine-induced

impaired risk-assessment presents as an important potential cause for the high rates of

drug consumption prevalence during adolescence, and with increased vulnerability to

relapse during withdrawal.

Cocaine treatment also intensified social investigation and reduced defensive

behaviour during the social conflict provided by the resident-intruder paradigm, indicating

that the cocaine treatment affected the ability of male rats to properly interact with

conspecifics. Moreover, the reduced fear presented by the cocaine-treated animals, as

indicated by the decrease in flight behaviour, suggests altered risk-assessment properties,

supporting the results obtained in the EPM. Likewise, the neurochemical data obtained

after the resident-intruder paradigm, namely the altered function observed in prefrontal

cortex, amygdala and hippocampus, supports the alterations in the social behaviour

observed in these animals.

Global alterations in adolescent female rats chronically exposed to cocaine

In female rats, the hormonal data obtained showed no alterations in HPA axis

activity and effects of cocaine treatment on the HPG axis activity were also difficult to

infer.

However, like in males, also in female rats, an overall tolerance to the acute effects

of cocaine in the dopaminergic and serotonergic neurotransmission was observed after

chronic cocaine administration. Moreover, females also presented an increased

dopaminergic activity 1 day after withdrawal, suggesting, like in males, the presence of

304

high susceptibility to environmental cues. Interestingly, females presented a greater

involvement of amygdala in this process.

Cocaine-treatment also induced long-term effects on dopaminergic and

serotonergic neurotransmission in female rats, as indicated by increased dopaminergic

activity in the amygdala and hippocampus, and increased serotonergic activity in the

hippocampus and Nac observed throughout long-term withdrawal. Among other effects,

these long-term alterations could contribute to an increased vulnerability to relapse and

interfere with processes of learning and memory formation.

Diverging from males, in female rats, no behavioural differences were observed

between cocaine-treated and controls when performing the EPM.

In conclusion, the results obtained in the present study indicate the presence of

sex-differences in the short- and long-term effects of cocaine treatment during

adolescence. Male rats were more intensively affected than females. However, certain

conditions of the experimental procedure employed in the present study may have

contributed to mask the effects of cocaine in the female rats. Namely, the social isolation

that animals were submitted to, may have contributed to this effect. Sex-differences in the

susceptibility to the effects of stress are known, and several findings indicate an increased

vulnerability of females in this field. Hence, social isolation may have masked more

prominently the cocaine exposure effects in female rats.

Although cocaine exposure effects during adulthood were not evaluated in the

present study, compared to published data, our results seem indicate less differences in

dopaminergic and serotonergic neurotransmission between saline and cocaine-treated

animals. Namely, adolescent rats treated chronically with cocaine showed few acute

effects as compared with data from studies carried out during the adulthood. This may

indicate an improved adaptive response, which may be a consequence of the increased

plasticity that characterizes the adolescent CNS maturation process. On the other hand,

our results also seem to indicate low levels of anxiety-like, depressive-like and aggression

behaviours during withdrawal from cocaine exposure during adolescence as compared

with data from literature on the effects of cocaine in adult animals. In fact, provides further

evidence in supporting previous findings suggesting that adolescents respond differently

from older animals to psychostimulants.

This age related effect may be involved in the increased vulnerability to drug use

during adolescence. Indeed, the impairment in risk assessment observed in male rats

305

exposed to cocaine during adolescence may lead to a compromise decision making

involving choices of high risks and low benefits. Moreover, this could represent an

important risk factor for the increased susceptibility to the maintenance of drug use and for

the increased risk of relapse after a drug-free period.

From a social perspective, data obtained in this study presents an increased

relevance for the understanding of the relationship between adolescents and drug use

and can be issued to better structure drug abuse prevention in the adolescence.

306

Appendix 1

Article

“Hormonal, neurochemical, and behavioral response to a Forced Swim Test in

adolescent rats throughout cocaine withdrawal”

Hormonal, Neurochemical, and BehavioralResponse to a Forced Swim Test in

Adolescent Rats throughoutCocaine Withdrawal

Cecılia Juliana Alves,a,b Ana Magalhaes,a

Teresa Summavielle,a,c Pedro Melo,a Liliana De Sousa,a,b

Maria Amelia Tavares,a,d and Pedro R.R. Monteiroa,c

aIBMC–Instituto de Biologia Molecular e Celular, Universidade do Porto,Porto, Portugal

bInstitute of Biomedical Sciences Abel Salazar, University of Porto, Porto, PortugalcEscola Superior de Tecnologia da Saude do Porto do IPP, Porto, Portugal

dInstitute of Anatomy, Medical School of the University of Porto, Porto, Portugal

The use of cocaine in adults has been linked to depression and/or anxiety. Severalstudies have shown an association between cocaine-primed craving and depressivesymptoms. In animal models, the forced swim test (FST) is frequently used for screen-ing depressive-like behavior. This study aimed to verify the presence of depression-likesymptoms in adolescent rats after chronic cocaine exposure by analyzing behavior ina FST. The subsequent alterations in neurotransmitters and hypothalamus-pituitary-adrenal axis activity induced by this test were also analyzed. Both male and femaleadolescent Wistar rats were submitted to a chronic “binge” pattern of administrationof cocaine hydrochloride, and subjects were tested in a forced swim test 2 days aftercocaine’s last administration. At the end of the behavioral test, trunk blood was col-lected for quantification of corticosterone plasma levels, and hypothalamus, prefrontalcortex, amygdala, and hippocampus were dissected for neurochemical determinations.No significant differences were found in the behavior on the FST of both males andfemales after withdrawal from chronic cocaine administration. Nevertheless, plasmalevels of corticosterone were increased in cocaine-treated males, although not signifi-cantly (P = 0.065). In females cocaine failed to affect corticosterone levels. Of interest,neurochemical analyses showed that dopamine turnover was decreased in amygdalain cocaine-treated males (not significantly, P = 0.055). No significant differences werefound on neurotransmitter levels in the other brain regions analyzed. Withdrawal fromchronic cocaine administration during adolescence did not have a significant effecton stress-induced behavioral alterations, although the neurochemical response to thestressful situation provided by FTS seemed to be affected.

Key words: cocaine; chronic exposure; withdrawal; adolescence; rat; forced swim test;behavior; monoamines; corticosterone

Address for correspondence: Cecılia Juliana Alves, Unidade de Neuro-comportamento, IBMC–Instituto de Biologia Molecular e Celular, Ruado Campo Alegres, 823, 4150-180 Porto, Portugal. Voice: +351-226-074-900; fax: +351-226-099-157. [email protected]

Introduction

Several studies have shown that cocaineabuse and withdrawal has been linkedto the development of depressive and/oranxiety-like symptoms.1,2 In humans and rats,

Ann. N.Y. Acad. Sci. 1139: 366–373 (2008). C© 2008 New York Academy of Sciences.doi: 10.1196/annals.1432.047 366

Alves et al.: Cocaine Withdrawal and Stress Response 367

antidepressants can attenuate symptoms of co-caine withdrawal,2,3 confirming that depres-sion may be involved.4 Moreover, severe anxi-ety and depression provide part of the negativereinforcement associated with cocaine depen-dence and are an important motivational factorfor relapse and maintenance of repetitive cyclesof cocaine abuse.2

The forced swim test (FST) was proposed byPorsolt et al.5 as a model of depression and itis still used to screen antidepressants in rats.6,7

The FST is also often employed as a measure offear or emotionality in response to a stressor.8,9

In fact, this test is a potent psychophysiologi-cal stressor that is accompanied by a variety ofneurochemical, endocrine, and immunologicchanges.10,11 Alterations in the serotoninergicsystem induced by exposure to the FST, namely,an increase in the serotonin (5-HT) turnover inseveral brain areas has been reported.12 Thetest is also recognized to increase the activityof the hypothalamus-pituitary-adrenal (HPA)axis.13

Cocaine also seems to alter the behavioralresponse to stressful situations. Repeated co-caine decreases the avoidance response to anovel stimulus in rats,14 and in the FST post-natal exposure to cocaine increases immobilityin juvenile rats.15 On the other hand, stress isknown to contribute to reinstatement of drugabuse.16,17

Although adolescents represent a risk groupregarding drug abuse and addiction, experi-mental research evaluating the effects of psy-chostimulants has been mostly focused on theassessment of adult animals. In fact, previousstudies have shown that most illicit drug usebegins in adolescence,18,19 and that adolescentsare more likely to progress from use to depen-dence than adults.20

Adolescence is an ontogenetic transitionfrom the dependence of youth to the indepen-dence of the adulthood, and during this phasethe brain and the hormonal systems are still un-dergoing many complex changes.21 Disruptionof the development of the central nervous sys-tem at this stage may increase the probability

of developing psychopathologies such as drugdependence (reviewed by Spear21). Therefore,there is a growing interest in assessing the influ-ence that drug use can have on the developingadolescent.

The present study aims to verify the presenceof depressive symptoms in male and femaleadolescent rats after withdrawal from chroniccocaine administration, assessing the behaviorin a FST. The effects of cocaine treatment onthe subsequent alterations on brain neurotrans-mitters and in hypothalamus-pituitary-adrenalaxis activity induced by the FST were alsoanalyzed.

Material and Methods

Animal Model

Wistar rats born from nulliparous femalespurchased from Charles River LaboratoriesEspana S.A. (Barcelona, Spain) were used inthis experiment. Animals were maintained un-der a 12-h light/dark cycle in a temperature-and humidity-controlled room and given ad li-

bitum access to food and water. Institutionalguidelines regarding animal experimentationwere followed. All procedures used were ap-proved by the Portuguese Agency for An-imal Welfare (General Board of VeterinaryMedicine in compliance with the InstitutionalGuidelines and the European Convention).

On the day after birth, postnatal day 1 (PND1), litters were standardized, whenever possible,to 10 sex-balanced pups. Animals were weanedon PND 21 and housed individually on PND30 to avoid hormonal co-regulation. In orderto minimize the effects of social isolation, bothcontrol and treated animals were housed indi-vidually handled daily and were given cylindri-cal plastic tubes and soft paper for nest con-struction. One male and one female from eightlitters received intraperitoneal (i.p.) injections ofcocaine (3 × 15 mg/kg) from PND 35 to PND50, following a “binge” pattern regime: threetimes daily at hourly intervals, between 9 AM

368 Annals of the New York Academy of Sciences

and 11 AM. Controls were given isovolumetricsaline (0.9%) following the same experimentalprotocol. Cocaine was obtained from SigmaChemical Company (St. Louis, MO, USA).

Forced Swim Test

The FST procedure used was similar to theone described by Porsolt et al.5 Two days aftercocaine’s last administration, rats were placedinto a glass cylinder (19 cm in diameter and40 cm deep) of 22 ± 1 ◦C water filled to a depthof 30 cm for 15 min of pretest. Twenty-fourhours later, rats were again placed into the wa-ter for 5 min. Activity during the second swimtest was video-recorded for subsequent behav-ioral analysis. Videotapes were scored by anobserver using the Noldus Observer software(Observer 4.1 Noldus Information Technology,Wageningen, the Netherlands) and the follow-ing behavior categories were analyzed: immo-bility, struggling, diving, as well as right andleft rotation. The data were collected for fre-quency, duration, and latency. Immobility wasdefined as lack of movement of three paws andonly minimal movement of the fourth, or lackof movement of all four paws.22 Struggling wasdefined as vigorous movement of all four paws,with the rat in a relatively upright position.23

Blood and Tissue Collection

At the end of the behavioral session animalswere decapitated and the brain was rapidlyremoved. Amygdala, hippocampus, prefrontalcortex, and suprachiasmatic, pre-optic and par-aventricular nuclei of hypothalamus were dis-sected on ice (according to the atlas by Paxi-nos and Watson24), frozen in 2-methylbutanecooled over dry ice, and stored at −70 ◦C un-til used for neurotransmitter determinations.Trunk blood was collected in lithium heparin-coated tubes and centrifuged at 1600 × g for15 min, and the plasma obtained was kept at−70 ◦C until corticosterone (CORT) hormoneassay.

Hormone Assays

CORT plasma levels were measured byenzyme immunoassay, using a commerciallyavailable kit provided by IDS, Ltd. (Boldon,UK). The intra- and interassay coefficientsof variation were less than 6.6% and 8.6%,respectively.

Neurotransmitter Determinations

Concentrations of dopamine (DA) andserotonin (5-HT) and their metabolites, 5-hydroxyindole-3-acetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), andhomovanillic acid (HVA) from the brain ar-eas collected were quantified by a modifiedmethod25 of high-performance liquid chro-matography with electrochemical detection(HPLC/EC) in a Gilson Medical Electronicssystem (Gilson, Middleton, WI, USA). The an-alytic column was a Supelco, Inc. (Bellefonte,PA, USA) Supelcosil LC-18 of 3 μM (7.5 cm ×4.6 mm). Concentrations of neurotransmitterswere calculated using standard curves gener-ated using standard amine purchased from theSigma Chemical Co. Neurotransmitter con-centrations were expressed in ng/mg protein.Protein content was determined by the Bio-Rad protein assay (Munchen, Germany), usingbovine serum albumin as a standard. The ratioof DOPAC and HVA to DA was determined foreach animal and used as an index of dopamineturnover rate; likewise, the ratio of 5-HIAA to5-HT was also determined and used as an in-dex of serotonin turnover rate.

Statistical Analyses

FST behavior during chronic cocaine with-drawal was analyzed by two-way ANOVAwith treatment (cocaine or saline) and sex asbetween-subject fixed-factors and behavioralmeasurements as the dependent variable. Thet-test was used as a post hoc test to analyze sta-tistically significant differences. For hormonaland neurochemical data, statistical significance


between groups was assessed using the non-parametric Mann–Whitney U Test. The sta-tistical level of significance was considered atP < 0.05. All data are expressed as mean ±SEM. Statistical analyses were performed us-ing SPSS 14.0 software (SPSS INC. Chicago,Illinois, USA).

Results

Behavioral Data

Statistic analyses revealed an interaction be-tween sex and treatment for the latency to strug-gling behavior [F(1.28) = 4.969; P < 0.05]. Post

hoc comparisons using the t-test indicate that inthe control group the latency to struggling waslower in males [t(14) =−2.293 (two-tailed), P <

0.05]. No significant differences were found onthe behavior in the FST of both males and fe-males after withdrawal from chronic cocaineadministration (Table 1).

Hormonal Data

Analysis of hormonal data revealed thatwithin the male group, plasma levels of corti-costerone were increased after FST in cocaine-treated animals, although not significantly [Z =−1.848 (two-tailed), P = 0.065] (Fig. 1). Infemales the treatment did not affect CORTlevels.

Neurochemical Data

In the amygdala, within the control group, fe-males displayed increased levels of 5-HT, whencompared with those of control males, that al-most reached significance [Z = −1.922 (two-tailed) P = 0.055] (Fig. 2A) and also a lowerratio 5-HIAA/5-HT [Z = −2.242 (two-tailed)P < 0.05] (Fig. 2B). Also in this brain re-gion, cocaine decreased (although not signif-icantly) the ratio DOPAC/DA in male [Z =−1.922 (two-tailed), P = 0.055], but not in fe-male rats (Fig. 2C). Analysis of neurochemicaldata from prefrontal cortex, hippocampus, and TA

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Figure 1. Effects of withdrawal from cocaine ex-posure during adolescence on plasma levels of corti-costerone induced by forced swim test, both in maleand female rats. Each column represents the mean ±S.E.M for 6 animals per group. CORT, corticosterone.

hypothalamus after FST showed no differencesin levels of DA or 5-HT and their metabolitesor in turnover rates.

Discussion

Previous work in adult animals hasshown that withdrawal from cocaine inducesdepressive-like behavior.2,3 However, our re-sults did not show any significant effect ofcocaine withdrawal during adolescence on thebehavior of male and female rats on the FST.Increased passive behavioral responses in theFST such as immobility and decreased ac-tive behaviors like swimming or struggling arethought to be a clear indication of depressive-like symptoms,6,26 but in our study these mea-sures were not affected by cocaine treatment.

Following the criteria established to defineimmobility, we were not able to observe alteredimmobility behavior in any studied group ofanimals. Some studies have shown that therewas an age-dependent difference in the amountof time spent in immobility in a forced swimtest,8,27 with juveniles showing very little ev-idence of immobility in contrast to adults.23

However, at least one previous study showedthat the immobility response was present inadolescent rats.28 This absence of immobilitybehavior observed in our study could be due tomethodologic differences in experimental pro-cedures, such as apparatus dimensions, watertemperature, and/or criteria for defining im-

Figure 2. Effects of withdrawal from cocaine ex-posure during adolescence on the levels (ng/mgprotein) of 5-HT (A), on 5-HIAA/5-HT (B), and onDOPAC/DA ratio (C) in amygdala after a forcedswim test both in male and female rats. Each columnrepresents the mean ± SEM for 6 animals per group.DA, dopamine; DOPAC, 3,4-dihydroxyphenylaceticacid; 5-HT, serotonin; 5-HIAA, 5-hydroxyindolaceticacid. Columns with the same letter are significantlydifferent: a = P < 0.05.

mobility.29 However, it should be noted thatthere were no treatment effects in the timespent struggling either in males and females,which allow us to propose that the behavioralstrategy adopted to cope with the stressful con-dition provide by the FST was not affected bythe cocaine treatment and withdrawal. This ab-sence of significant differences between controland cocaine-exposed rats could be related tothe reduced behavioral and physiological re-sponse to stressful situations in rodents dur-ing the adolescent period indicated by somestudies.30,31 Moreover, cocaine was reported todisplay lower behavioral effects in adolescence


than in adult rats.32 In addition, our experi-mental design requires animals to be housedindividually, in order to avoid variable socialinfluences that could normalize hormonal ef-fects, masking the possible effects of cocaineexposure.33 However, housing isolation itself,despite our efforts to minimize it through im-proved handling, may contribute to the devel-opment of depressive behavior,34 which couldinterfere with the possible effects of cocainetreatment regarding depressive-like behavioraldevelopment.

Beside the evaluation of the behavioral re-sponse to FST, alterations in physiological re-sponses to the stressful situation provided by theFST during withdrawal from chronic cocaineadministration were also analyzed. Althoughplasma levels of CORT were increased afterFST in cocaine-treated males, this increase wasnot significantly different. Thus, the hormonaldata from both male and female adolescentrats seems to indicate that the plasma levelsof CORT after performing the FST were notinfluenced by the withdrawal from chronic co-caine administration. Other authors have alsoshown a lack of cocaine effects on HPA re-sponse to stress. Seven days of exposure to15 mg/kg of cocaine failed to alter the CORTresponse to 20 min of immobilization 42 h af-ter the final injection,35 and no changes weredemonstrated in the CORT response to 60 minof immobilization 21 h after the final injectionof a 3- or 6-week “binge” cocaine regimen.36

However, Mantsch et al. reported a CORT re-sponse to a stressor, 30 min of restraint, duringacute (24-h) withdrawal from chronic cocaineadministration.37 In our study, the absence ofdifferences in CORT response to FST could berelated with the elevated basal levels of CORTpresent in adolescents. The fact that adoles-cents usually display higher CORT levels atbaseline than those of adults could contributeto a reduced percentage of increase when com-pared to adults.38,39 Moreover, age-related dis-continuities in the response of the HPA toboth stress and psychostimulants have been

demonstrated.30 The HPA axis seems to be hy-poresponsive to external perturbations duringadolescence.39 Since both saline and cocaine-treated rats showed a similar CORT responseto the FST, it seems that chronic cocaine ad-ministration did not result in the developmentof tolerance or sensitization of the HPA axisresponse to a novel stressor.

The neurochemical data presented indicatethat within the control group there was an in-crease in the 5-HT turnover in the amygdalain the adolescent males. This increase is con-sistent with previous reports that examined theeffects of other stressors on the 5-HT turnoverin the brain.40,41 Besides, such an effect hasalready been demonstrated in the amygdalaof adult male rats as a result of FST expo-sure.12 Data presented here reveal a sex ef-fect in the serotonergic activity of control ani-mals that was not observed in cocaine-exposedrats, which may be a consequence of cocainewithdrawal. Moreover, although not signifi-cantly, withdrawal from chronic cocaine de-creases the ratio DOPAC/DA in male rats. To-gether, these results seem to show that cocainetreatment during the adolescence did affectthe neurotransmitter system’s response to theFST.

In conclusion, although withdrawal fromchronic cocaine administration during adoles-cence seems not to have had a significant effecton stress-induced behavioral alterations, neu-rochemical data indicate that the response tothe stressful situation created by the FTS wasaffected. These results are substantially differ-ent from the data obtained in studies carriedout in adult rats, which do show stronger ef-fects.38,42 Thus, the data provided by this workmay confirm previous findings suggesting thatadolescents respond differently from older ani-mals to both stress and psychostimulants.39,43,44

This age-related effect could be one of thereasons for the elevated prevalence of druguse and dependence during adolescence.38 In-deed, age-related discontinuities in response topsychological stress have been indicated as a


psychobiological risk for increased vulnerabil-ity to drug abuse.45,46

Acknowledgments

This work was supported by Fundacaopara a Ciencia e Tecnologia (FCT) ProjectPOCTI/ESP/43630/1999 and Programade Financiamento Plurianual (IBMC).Cecılia Juliana Alves, Ana Magalhaes,and Teresa Summavielle were supportedby FCT Grants SFRH/BD/17195/2004,SFRH/BPD/19200/2004, and SFRH/BPD/20997/2004, respectively.

Conflicts of Interest

The authors declare no conflicts of interest.

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