Neuroscience 253 (2013) 29–39

ROLE OF THE BED NUCLEUS OF THE STRIA TERMINALIS INCARDIOVASCULAR CHANGES FOLLOWING CHRONIC TREATMENTWITH COCAINE AND TESTOSTERONE: A ROLE BEYOND DRUGSEEKING IN ADDICTION?

F. C. CRUZ, a,b� F. H. F. ALVES, c� R. M. LEAO, a,d�

C. S. PLANETA a,d AND C. C. CRESTANI a,d*a Laboratory of Pharmacology, Department of Natural Active

Principles and Toxicology, School of Pharmaceutical Sciences, Univ.

Estadual Paulista – UNESP, Araraquara, SP 14801-902, BrazilbBehavioral Neuroscience Branch, Intramural Research

Program, National Institute on Drug Abuse, US National Institutes

of Health, Department of Health and Human Services, Baltimore, MD,

USA

cDepartment of Pharmacology, School of Medicine of Ribeirao

Preto, University of Sao Paulo, Ribeirao Preto, SP 14090-090, Brazil

d Joint UFSCar–UNESP Graduate Program in Physiological

Sciences, Brazil

Abstract—Neural plasticity has been observed in the bed

nucleus of the stria terminalis (BNST) following exposure

to both cocaine and androgenic–anabolic steroids. Here

we investigated the involvement of the BNST on changes

in cardiovascular function and baroreflex activity following

either single or combined administration of cocaine and tes-

tosterone for 10 consecutive days in rats. Single administra-

tion of testosterone increased values of arterial pressure,

evoked rest bradycardia and reduced baroreflex-mediated

bradycardia. These effects of testosterone were not affected

by BNST inactivation caused by local bilateral microinjec-

tions of the nonselective synaptic blocker CoCl2. The single

administration of cocaine as well as the combined treatment

with testosterone and cocaine increased both bradycardiac

and tachycardiac responses of the baroreflex. Cocaine-

evoked baroreflex changes were totally reversed after BNST

inactivation. However, BNST inhibition in animals subjected

to combined treatment with cocaine and testosterone

reversed only the increase in reflex tachycardia, whereas

facilitation of reflex bradycardia was not affected by local

BNST treatment with CoCl2. In conclusion, the present study

provides the first direct evidence that the BNST play a role in

cardiovascular changes associated with drug abuse. Our

findings suggest that alterations in cardiovascular function

following subchronic exposure to cocaine are mediated by

0306-4522/13 $36.00 � 2013 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2013.08.034

*Correspondence to: C. C. Crestani, Laboratory of Pharmacology,Department of Natural Active Principles and Toxicology, School ofPharmaceutical Sciences, Sao Paulo State University – UNESP,Rodovia Araraquara-Jau Km 01, Campus Universitario, Caixa Postal502, Araraquara, SP 14801-902, Brazil. Tel: +55-16-3301-6982; fax:+55-16-3301-6980.

E-mail addresses: crestani@fcfar.unesp.br (C. C. Crestani).� These authors contributed equally to this work.

Abbreviations: AAS, androgenic-anabolic steroids; ANOVA, analysis ofvariance; BNST, bed nucleus of the stria terminalis; coc, cocaine; HR,heart rate; MAP, mean arterial pressure; T, testosterone; veh, vehicle.

29

neural plasticity in the BNST. The single treatment

with cocaine and the combined administration of

testosterone and cocaine had similar effects on

baroreflex activity, however the association with testoster-

one inhibited cocaine-induced changes in the BNST

control of reflex bradycardia. Testosterone-induced cardio-

vascular changes seem to be independent of the BNST.

� 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: addiction, steroids, cocaine, baroreflex, BNST,

extended amygdala.

INTRODUCTION

Emerging data suggest that abuse of androgenic–

anabolic steroids (AAS) is frequently followed by the use

of other psychotropic drugs (Arvary and Pope, 2000). It

has been reported that cocaine is the drug most

frequently coabused by AAS users (DuRant et al., 1993,

1995). In fact, epidemiological and clinical results

indicate that AAS users are likely to display higher

cocaine intake than non-users (McCabe et al., 2007;

Kanayama et al., 2009).

The widespread abuse of cocaine and AAS has

stimulated the interest in the study of the toxic effects of

these substances (Maraj et al., 2010; van Amsterdam

et al., 2010). Accumulating evidence suggests that the

abuse of cocaine and AAS is associated with

cardiovascular complications (Kloner et al., 1992;

Sullivan et al., 1998). Cocaine use induces both acute

and chronic cardiovascular effects (Kloner et al., 1992).

The acute effects of cocaine are well described and

include hypertension, coronary vasoconstriction and

cardiac arrhythmias (Kloner et al., 1992; Maraj et al.,

2010). Less information is available about the effects of

chronic cocaine abuse, but studies have reported

cardiomyopathies and myocarditis, arrhythmias, and

changes in baroreflex activity following long-term

cocaine exposure (Kloner et al., 1992; Engi et al., 2012;

Maraj et al., 2010). Unlike cocaine, AAS evokes minor

acute cardiovascular side effects (van Amsterdam et al.,

2010). However, chronic AAS abuse has been

associated with hypertension and cardiac pathologies

(Sullivan et al., 1998; van Amsterdam et al., 2010).

Importantly, studies in animals suggest that AAS and

cocaine are capable of mutually potentiating the

cardiovascular effects of each other (Phillis et al., 2000;

d.

30 F. C. Cruz et al. / Neuroscience 253 (2013) 29–39

Togna et al., 2003). Indeed, we have recently reported

that administration of either testosterone or cocaine for

10 consecutive days in rats produced a range of

cardiovascular effects (e.g., mild hypertension, changes

in baroreflex activity and stress-evoked cardiovascular

responses, and arrhythmias), which were more

pronounced when the substances were coadministrated

(Cruz et al., 2012; Engi et al., 2012). Although several

reports have shown the occurrence of cardiovascular

diseases following either single or combined abuse of

cocaine and AAS, the mechanisms involved in the

physiopathology of these complications are not entirely

understood.

Studies using animal models have shown that

repeated exposure to cocaine produces morphological

and functional changes throughout the brain (Belej

et al., 1996; Beveridge et al., 2004; Macey et al., 2004).

Although the overall goal of the majority of these studies

has been to evaluate neural changes in brain areas that

are considered to be components of the neurocircuitry

of addiction, significant changes have also been

reported in brain regions that regulate cardiovascular

function. Indeed, functional changes in the activity of

forebrain and brainstem autonomic regions occur even

at the initial stages of cocaine exposure and these

alterations progress with the prolonged cocaine contact

(Beveridge et al., 2004; Macey et al., 2004). Some

results have suggested that long-term AAS treatment

may also change the activity of brain regions controlling

cardiovascular functions (Rosa et al., 2005). These

results support the hypothesis that cardiovascular

complications associated with chronic exposure to

cocaine and AAS may result from neural plasticity in

central nervous system regions controlling the

autonomic activity. To our knowledge, however, no

studies have directly assessed the brain regions

involved in cardiovascular complications following

cocaine and AAS exposure.

The bed nucleus of the stria terminalis (BNST) is

localized in the rostral prosencephalon and is involved

in the control of cardiovascular functions (Gelsema

et al., 1993; Crestani et al., 2013). Previous studies

have reported that either stimulation or inhibition of the

BNST evokes changes on blood pressure, heart rate

(HR), baroreflex activity and cardiovascular

adjustments to physiological challenges (e.g., emotional

stress and physical exercise) (Ciriello and Janssen,

1993; Crestani et al., 2008, 2009; Alves et al., 2011).

Morphological and functional changes have been

observed in the BNST following repeated

administration of cocaine and AAS (Belej et al., 1996;

DeLeon et al., 2002; Beveridge et al., 2004; Macey

et al., 2004; Costine et al., 2010). These results

support data suggesting a role of the BNST in

reward-seeking, addiction, drug relapse and

behavioral changes associated with these substances

(Epping-Jordan et al., 1998; Erb and Stewart, 1999;

Aston-Jones et al., 2010; Costine et al., 2010; Koob

and Volkow, 2010). However, a possible involvement

of the BNST in cardiovascular toxicity associated with

drug abuse has never been investigated. Therefore,

the goal of the present study was to investigate the

involvement of the BNST on changes in blood

pressure, HR and baroreflex activity following either

single or combined administration of cocaine and

testosterone for 10 consecutive days in rats.

EXPERIMENTAL PROCEDURES

Animals

Twenty-six male Wistar rats weighing 200 g in the

beginning of the experiments were used. Animals were

obtained from the animal breeding facility of the Univ.

Estadual Paulista – UNESP (Botucatu, SP, Brazil) and

were housed in plastic cages in a temperature-

controlled room at 24oC in the Animal Facility of the

Laboratory of Pharmacology, School of Pharmaceutical

Sciences, Univ. Estadual Paulista – UNESP. They were

kept under a 12:12-h light–dark cycle (lights on between

6:00 am and 6:00 pm) with free access to water and

standard laboratory food. The injections of drugs and

the cardiovascular analysis were carried-out during the

light phase. Housing conditions and experimental

procedures were approved by the Ethics Committee for

Use of Animals of the School of Pharmaceutical

Science/UNESP.

Treatment

Animals were randomly divided into four groups: (i)

vehicle (almond oil, 1 ml/kg, s.c.) + vehicle (0.9% NaCl,

1 ml/kg, i.p.) (veh + veh) (n= 6); (ii) testosterone

(10 mg/kg, s.c.) + vehicle (T + veh) (n= 5); (iii)

vehicle + cocaine (20 mg/kg, i.p.) (veh + coc) (n= 6);

and (iv) testosterone + cocaine (T + coc) (n= 5).

Animals received treatments once daily for 10

consecutive days. The doses and treatment regimen

were chosen based on our previous studies (Cruz et al.,

2012; Engi et al., 2012).

Surgical preparation

On the last day of treatment, and three days before the

trial, rats were anesthetized with tribromoethanol

(250 mg/kg, i.p.). After scalp anesthesia with 2%

lidocaine the skull was exposed and stainless-steel

guide cannulas (26G) were bilaterally implanted into the

BNST at a position 1 mm above the site of injection,

using a stereotaxic apparatus (Stoelting, Wood Dale, IL,

USA). Stereotaxic coordinates for cannula implantation

into the BNST were: antero-posterior =+8.6 mm from

interaural; lateral = 4.0 mm from the medial suture,

ventral = �5.8 mm from the skull with a lateral

inclination of 23� (Paxinos and Watson, 1997).

Cannulas were fixed to the skull with dental cement and

one metal screw. After surgery, the animals received a

poly-antibiotic (Pentabiotico�, Fort Dodge, Campinas,

SP, Brazil), with streptomycins and penicillins, to

prevent infection and the non-steroidal anti-inflammatory

flunixine meglumine (Banamine�, Schering Plough,

Cotia, SP, Brazil) for post-operation analgesia.

F. C. Cruz et al. / Neuroscience 253 (2013) 29–39 31

One day before the experiment, rats were

anesthetized with tribromoethanol (250 mg/kg, i.p.) and

a catheter (a 4-cm segment of PE-10 heat-bound to a

13-cm segment of PE-50) (Clay Adams, Parsippany,

NJ, USA) was inserted into the abdominal aorta through

the femoral artery for arterial pressure and HR

recording. A second catheter was implanted into the

femoral vein for the infusion of vasoactive agents to

evoke arterial pressure changes. Both catheters were

tunneled under the skin and exteriorized on the animal’s

dorsum. After surgery, the non-steroidal anti-

inflammatory flunixine meglumine (Banamine�, Schering

Plough, Cotia, SP, Brazil) was administered for post-

operation analgesia.

Measurement of cardiovascular responses

On the day of the experiment, the arterial cannula was

connected to a pressure transducer (DPT100, Utah

Medical Products Inc., Midvale, UT, USA). Pulsatile

arterial pressure was recorded using an amplifier (Quad

Bridge Amp, ML224, ADInstruments, NSW, Australia)

and an acquisition board (PowerLab 4/30, ML866/P,

ADInstruments, NSW, Australia) connected to a

personal computer. Mean arterial pressure (MAP) and

HR values were derived from pulsatile arterial pressure

recordings.

Baroreflex assessment

The baroreflex activity was assessed through arterial

pressure changes induced by intravenous infusion of the

selective a1-adrenoceptor agonist phenylephrine (70 lg/ml at 0.4 ml/min/kg) and the nitric oxide donor sodium

nitroprusside (100 lg/ml at 0.8 ml/min/kg), using an

infusion pump (K.D. Scientific, Holliston, MA, USA)

(Head and McCarty, 1987; Crestani et al., 2010a).

Phenylephrine and sodium nitroprusside caused

incremental pressor or depressor responses,

respectively. Infusions of vasoactive drugs were

randomized and the second treatment was not realized

before cardiovascular parameters returned to control

values. The interval between infusions was

approximately 5 min. Infusions lasted for 20–30 s,

resulting in the injection of a total dose of 9–14 lg/kg of

phenylephrine and 26–40 lg/kg of sodium nitroprusside.

Method of baroreflex evaluation

Reflex bradycardia corresponding to MAP increases

evoked by phenylephrine infusion and tachycardic

responses corresponding to MAP decreases caused by

infusion of sodium nitroprusside were determined. The

HR values each 5 mmHg of MAP change until maximum

variation of 40 mmHg was calculated. Paired values of

MAP (DMAP) and HR (DHR) were plotted to generate

linear regression curves for bradycardiac and

tachycardiac responses (Crestani et al., 2010a; Karlen-

Amarante et al., 2012). The slope of the curves (gain,

bpm/mmHg) and the maximum reflex response (i.e., the

reflex HR response to 40 mmHg of MAP change) were

used to determine the baroreflex activity.

Drug microinjection into the BNST

The needles (33G, Small Parts, Miami Lakes, FL, USA)

used for microinjection into the BNST were 1 mm longer

than the guide cannulas and were connected to a 2-lLsyringe (7002-KH, Hamilton Co., Reno, NV, USA)

through PE-10 tubing (Clay Adams, Parsippany, NJ,

USA). Needles were carefully inserted into the guide

cannulas without restraining the animals and drugs were

injected in a final volume of 100 nL (Crestani et al.,

2006; Alves et al., 2009). After a 30-s period, the needle

was removed and inserted into the second guide

cannula for microinjection into the contralateral BNST.

Experimental protocol

On the trial day, animals were brought to the experimental

room in their home cages. Animals were allowed one hour

to adapt to the conditions of the experimental room, such

as sound and illumination, before starting arterial

pressure and HR recordings. The experimental room

had controlled temperature (24 �C) and was acoustically

isolated from the main laboratory.

Animals in all experimental groups were subjected to

a 30-min period of basal recording of arterial pressure

and HR. In the sequence, they received intravenous

infusion of phenylephrine and sodium nitroprusside, in a

random order. After infusions of the vasoactive agents

to determinate control parameters of the baroreflex

activity, animals received bilateral microinjection of the

nonselective synaptic blocker CoCl2 (0.1 nmol/100 nL)

into the BNST (Crestani et al., 2006). Ten min later,

phenylephrine and sodium nitroprusside were again

infused in a random order.

Histological determination of the microinjection sites

At the end of experiments, animals were anesthetized

with urethane (1.25 g/kg, i.p.) and 100 nL of 1% Evan’s

blue dye was injected into the BNST as a marker of

injection sites. They were then submitted to

intracardiac perfusion with 0.9% NaCl followed by 10%

formalin. Brains were removed and post-fixed for 48 h

at 4 �C and serial 40-lm-thick sections were cut with a

cryostat (CM1900, Leica, Wetzlar, Germany). Sections

were stained with 1% neutral red for light microscopy

analysis. The actual placement of the microinjection

needles was determined by analyzing serial sections

and identified according to the rat brain atlas of

Paxinos and Watson (1997).

Drugs

Cocaine hydrochloride (Sigma, St Louis, MO, USA),

CoCl2 (Sigma), phenylephrine hydrochloride (Sigma),

sodium nitroprusside dihydrate (Sigma), urethane

(Sigma) and tribromoethanol (Sigma) were dissolved in

saline (0.9% NaCl). Testosterone (PharmaNostra, Rio

32 F. C. Cruz et al. / Neuroscience 253 (2013) 29–39

de Janeiro, RJ, Brazil) was dissolved in almond oil.

Flunixine meglumine (Banamine�, Schering Plough,

Cotia, SP, Brazil) and the poly-antibiotic preparation of

streptomycins and penicillins (Pentabiotico�, Fort

Dodge, Campinas, SP, Brazil) were used as provided.

Statistical analysis

Data are presented as mean ± S.E.M. Basal values of

MAP and HR as well as baroreflex parameters before

and after BNST pharmacological treatment in animals

subjected to single or combined treatment with cocaine

and testosterone were compared using a two-way

analysis of variance (ANOVA) for repeated

measurements (treatment vs. BNST inhibition) with

repeated measures on the second factor. When

interactions between factors were observed,

comparisons before or after BNST treatment were

performed using Bonferroni’s post hoc test, whereas the

effect of BNST inactivation in each treatment was

compared using Student’s paired t test. The change in

baroreflex curve parameters evoked by BNST inhibition

was analyzed using a one-way ANOVA followed by

Bonferroni’s post hoc test. Significance was set at

P< 0.05.

RESULTS

A representative photomicrograph of a coronal brain section

depicting bilateral microinjection sites in the BNST of one

Fig. 1. (Top) Photomicrograph of a coronal brain section from one repres

Diagrammatic representation based on the rat brain atlas of Paxinos and W

blocker CoCl2 into the BNST of animals subjected to treatment for

vehicle + cocaine (s) and testosterone + cocaine (j). ac – anterior comm

septal ventral, st – stria terminalis and f – fornix.

representative animal is presented in Fig. 1. Diagrammatic

representation showing microinjection sites of CoCl2 into

the BNST of animals subjected to single or combined

treatment with cocaine and testosterone for 10

consecutive days is also shown in Fig. 1.

Effect of BNST treatment with CoCl2 on arterialpressure and HR values in animals subjected torepeated treatment with cocaine and testosterone

A two-way ANOVA of the values of MAP and HR

indicated a significant effect of treatment (MAP:

F(3,36) = 10, P< 0.0001; HR: F(3,36) = 33, P< 0.0001),

but without influence of BNST inhibition (MAP:

F(1,36) = 0.1, P> 0.05; HR: F(1,36) = 0.9, P> 0.05).

This analysis did not detect interaction between factors

(MAP: F(3,36) = 0.5, P> 0.05; HR: F(3,36) = 2, P> 0.05).

Post hoc comparisons (Bonferroni’s post hoc test)

revealed that animals subjected to either single

treatment with testosterone or combined administration

of testosterone and cocaine for 10 consecutive days

showed increased values of MAP (T + veh group:

9 mmHg above basal, P< 0.05; T + coc group:

10 mmHg above basal, P< 0.05) and rest bradycardia

(T + veh group: 52 bpm lower basal, P< 0.001:

T + coc group: 56 bpm lower basal, P< 0.001) (Fig. 2).

However, bilateral microinjection of the nonselective

synaptic blocker CoCl2 into the BNST did not affect both

arterial pressure and HR in either control or treated

groups (Fig. 2).

entative rat showing bilateral injection sites into the BST. (Bottom)

atson (1997) indicating injection sites of the nonselective synaptic

10 days with vehicle + vehicle (h), testosterone + vehicle (d),

issure, IA – interaural coordinate, ic – internal capsule, LSV – lateral

Fig. 2. Values of mean arterial pressure (MAP) and heart rate (HR) before and after bilateral microinjection of the nonselective synaptic blocker

CoCl2 (cobalt) into the BNST of animals subjected to single or combined administration of cocaine and testosterone for 10 days. ⁄P< 0.05 vs.

vehicle + vehicle group at same condition, a two-way ANOVA followed by Bonferroni’s post hoc test.

F. C. Cruz et al. / Neuroscience 253 (2013) 29–39 33

Effect of BNST treatment with CoCl2 on cardiacbaroreflex responses in animals subjected torepeated treatment with cocaine and testosterone

Baroreflex bradycardia. A two-way ANOVA of the gain

and maximum HR response derived from baroreflex

curves of reflex bradycardiac response indicated a

significant effect of the treatment with cocaine and/or

testosterone (gain: F(3,36) = 24, P< 0.0001; maximum

bradycardia: F(3,36) = 29, P< 0.0001) and BNST

inhibition (gain: F(1,36) = 48, P< 0.0001; maximum

bradycardia: F(1,36) = 74, P< 0.0001). An analysis of

the maximum bradycardiac response also indicated a

treatment � BNST inhibition interaction (F(3,36) = 4,

P< 0.01), whereas no interaction between factors was

detected for gain analysis (F(3,36) = 2, P> 0.05).

Post hoc analysis (Bonferroni’s post hoc test) of the

parameters derived from baroreflex curves for the reflex

bradycardiac response before BNST treatment revealed

that single treatment with testosterone for 10 days

(T + veh group) decreased the gain (P< 0.01) and

maximum bradycardia (P< 0.05), when compared with

veh + veh group (Fig. 3). Repeated treatment with

veh + coc increased reflex bradycardia gain (P< 0.05)

and maximum bradycardia (P< 0.05) (Fig. 3).

Combined treatment with testosterone and cocaine for

10 days also increased gain (P< 0.05) and maximum

response (P< 0.05) of reflex HR change caused by

arterial pressure increase (Fig. 3).

Bilateral microinjection of the nonselective synaptic

blocker CoCl2 into the BNST of control animals

(veh + veh group) increased reflex bradycardia gain

(P< 0.003) and maximum bradycardia (P< 0.0003)

(post hoc analysis, Student’s paired t test) (Fig. 3).

Baroreflex activity was not affected when microinjection

of CoCl2 reached structures surrounding the BNST,

such as the anterior commissure, fornix and internal

capsule (n= 4; data not shown). Local microinjection of

vehicle into the BNST also did not affect reflex cardiac

responses (n= 5; data not shown).

Comparison of baroreflex curves for the reflex

bradycardiac response also demonstrated that

microinjection of CoCl2 into the BNST of animals treated

with T + veh increased gain (P< 0.0002) and

maximum bradycardia (P< 0.001) (post hoc analysis,

Fig. 3. (Top) Linear regression curves correlating heart rate change (DHR) in response to mean arterial pressure increases (DMAP) before and

after BNST treatment with the nonselective synaptic blocker CoCl2 (cobalt) in animals subjected to treatment for 10 consecutive days with

vehicle + vehicle (r2 before: 0.87; r2 after: 0.74), testosterone + vehicle (r2 before: 0.62; r2 after: 0.82), vehicle + cocaine (r2 before: 0.80; r2 after:0.90) and testosterone + cocaine (r2 before: 0.70; r2 after: 0.96). (Bottom) Parameters derived from baroreflex curves generated before and after

bilateral microinjection of the nonselective synaptic blocker CoCl2 (cobalt) into the BNST of animals subjected to single or combined treatment with

cocaine and testosterone for 10 days, and changes in baroreflex parameters induced by CoCl2 microinjection into the BNST (difference between

values before and after BSNT pharmacological treatment). ⁄P< 0.05 vs. vehicle + vehicle group at same condition, two-way ANOVA followed by

Bonferroni’s post hoc test; #P < 0.05 vs. respective group before BNST pharmacological treatment; two-way ANOVA followed by Student’s t test.

34 F. C. Cruz et al. / Neuroscience 253 (2013) 29–39

Student’s paired t test) (Fig. 3). The change in baroreflex

curve parameters evoked by BNST inhibition in T + veh

group was similar to the control group (gain: P> 0.05;

maximum bradycardia: P> 0.05; one-way ANOVA

followed by Bonferroni’s post hoc test) (Fig. 3).

Inhibition of the BNST of animals treated for 10 days

with veh + coc did not affect gain (P> 0.05) and

maximum HR response (P> 0.05) caused by arterial

pressure increase (post hoc analysis, Student’s paired ttest) (Fig. 3). Conversely, the change in maximum

bradycardia evoked by BNST inhibition of cocaine-

treated animals was reduced (P< 0.01, one-way

ANOVA followed by Bonferroni’s post hoc test), when

compared with veh + veh group (Fig. 3). Change in

reflex bradycardia gain induced by microinjection of

CoCl2 into the BNST of veh + coc group was not

significantly different from control group (P> 0.05, one-

way ANOVA followed by Bonferroni’s post hoc test)

(Fig. 3).

Synaptic blockade within the BNST of animals

subjected to combined treatment with cocaine and

testosterone for 10 consecutive days increased the gain

(P< 0.003) and maximum bradycardia (P< 0.001)

(post hoc analysis, Student’s paired t test) (Fig. 3). Thechange in baroreflex curve parameters evoked by BNST

inhibition in T + coc group was similar to the control

group (gain: P> 0.05; maximum bradycardia: P> 0.05;

one-way ANOVA followed by Bonferroni’s post hoc test)

(Fig. 3).

Baroreflex tachycardia. A two-way ANOVA of the gain

and maximum HR response derived from baroreflex

curves of reflex tachycardiac response indicated a

significant effect of the treatment with cocaine and

testosterone (gain: F(3,36) = 6, P< 0.001; maximum

tachycardia: F(3,36) = 4, P< 0.02) and BNST inhibition

(gain: F(1,36) = 18, P< 0.0002; maximum tachycardia:

F(1,36) = 12, P< 0.001), as well as a treatment � BNST

inhibition interaction (gain: F(3,36) = 8, P< 0.0004;

maximum tachycardia: F(3,36) = 14, P< 0.0001).

Post hoc analysis (Bonferroni’s post hoc test) of the

parameters derived from baroreflex curves for the reflex

tachycardiac response before BNST treatment indicated

that either the single treatment with cocaine (veh + coc

group) or the combined treatment with testosterone and

cocaine increased the gain (P< 0.001) and maximum

tachycardiac response (P< 0.001), when compared

with veh + veh group (Fig. 4). Single treatment with

testosterone (T + veh group) did not affect reflex

tachycardiac response gain (P> 0.05) and maximum

Fig. 4. (Top) Linear regression curves correlating heart rate change (DHR) in response to mean arterial pressure decreases (DMAP) before and

after BNST treatment with the nonselective synaptic blocker CoCl2 (cobalt) in animals subjected to treatment for 10 consecutive days with

vehicle + vehicle (r2 before: 0.68; r2 after: 0.69), testosterone + vehicle (r2 before: 0.69; r2 after: 0.72), vehicle + cocaine (r2 before: 0.93; r2 after:0.87) and testosterone + cocaine (r2 before: 0.86; r2 after: 0.92). (Bottom) Parameters derived from baroreflex curves generated before and after

bilateral microinjection of the nonselective synaptic blocker CoCl2 (cobalt) into the BNST of animals subjected to single or combined treatment with

cocaine and testosterone for 10 days, and changes in baroreflex parameters induced by CoCl2 microinjection into the BNST (difference between

values before and after BSNT pharmacological treatment). ⁄P< 0.05 vs. vehicle + vehicle group at same condition, two-way ANOVA followed by

Bonferroni’s post hoc test; #P < 0.05 vs. respective group before BNST pharmacological treatment; two-way ANOVA followed by Student’s t test.

F. C. Cruz et al. / Neuroscience 253 (2013) 29–39 35

HR response (P> 0.05) evoked by arterial pressure

decrease (Fig. 4).

Bilateral microinjections of the nonselective synaptic

blocker CoCl2 into the BNST of both control animals

(veh + veh group) and testosterone-treated rats

(T + veh group) did not affect parameters derived from

baroreflex curves for reflex tachycardiac response

(P> 0.05; post hoc analysis, Student’s paired t test)

(Fig. 4). However, synaptic blockade within the BNST of

animals subjected to either single treatment with

cocaine (veh + coc group) or combined treatment with

cocaine and testosterone decreased the reflex

tachycardia gain (veh + coc group: P< 0.001; T + coc

group: P< 0.05) and the maximum HR response

(veh + coc group: P< 0.001; T + coc group:

P< 0.01) caused by arterial pressure decrease (post

hoc analysis, Student’s paired t test) (Fig. 4). Veh + coc

(P< 0.0001) and T + coc (P< 0.0001) treatment

increased the change in maximum tachycardiac

response following BNST inhibition (one-way ANOVA

followed by Bonferroni’s post hoc test), when compared

with the response observed in control animals. The

change in reflex tachycardia gain induced by

microinjection of CoCl2 into the BNST was not different

between the groups (P> 0.05; one-way ANOVA

followed by Bonferroni’s post hoc test) (Fig. 4). There

were not significant differences between groups in

baroreflex tachycardia parameters after BNST treatment

with CoCl2 (P> 0.05; two-way ANOVA followed by

Bonferroni’s post hoc test) (Fig. 4).

DISCUSSION

The main findings of the present study are: (1) the single

treatment with testosterone for 10 days (T + veh group)

caused a significant increase in values of arterial

pressure and evoked rest bradycardia. These effects

were not modified by either the coadministration of

cocaine (T + coc group) or bilateral microinjections of

the nonselective synaptic blocker CoCl2 into the BNST;

(2) the single treatment with testosterone also reduced

reflex bradycardia caused by blood pressure increases

without affecting reflex tachycardia caused by blood

pressure decreases. However, regardless of the effect

observed on baroreflex bradycardia before CoCl2microinjections, changes caused by BNST inhibition in

36 F. C. Cruz et al. / Neuroscience 253 (2013) 29–39

testosterone-treated animals were similar to those

observed in control animals (veh + veh group); (3) the

single treatment with cocaine (veh + coc group)

enhanced both reflex bradycardia and tachycardia, and

these effects were not identified after BSNT treatment

with CoCl2; and (4) the combined administration of

testosterone and cocaine also enhanced both

bradycardiac and tachycardiac responses of the

baroreflex. As observed in cocaine-treated animals,

BNST inhibition reversed the increase on reflex

tachycardia. However, change in baroreflex bradycardia

evoked by combined treatment with testosterone and

cocaine was still observed after BNST treatment CoCl2.

The changes in arterial pressure observed in T + veh

and T + coc groups corroborate previous findings

demonstrating that either single treatment with AAS

(Grollman et al., 1940; Beutel et al., 2005; Rosa et al.,

2005) or combined administration of AAS and cocaine

evokes sustained increases in arterial pressure of rats

(Tseng et al., 1994; Engi et al., 2012). Rest bradycardia

has also been reported following long-term exposure to

AAS (Beutel et al., 2005; Cruz et al., 2012; Engi et al.,

2012). Absence of changes in arterial pressure of

cocaine-treated rats corroborates clinical reports that

cocaine abuse is not associated with hypertension

(Brecklin et al., 1998).

Several studies have demonstrated that BNST

inhibition does not affect basal parameters of both blood

pressure and HR (Crestani et al., 2006, 2010b; Nasimi

and Hatam, 2011; Granjeiro et al., 2012), suggesting

that the BNST does not have a tonic influence on

maintenance of cardiovascular function. These results

support the present findings showing that changes in

arterial pressure and HR observed in T + veh and

T + coc groups were not affected by BNST inhibition.

However, since androgen receptors are expressed in

brainstem regions tonically involved in cardiovascular

control (Gorlick and Kelley, 1986) and cocaine evokes

functional changes in a number of other forebrain and

brainstem regions associated with cardiovascular control

(Beveridge et al., 2004; Macey et al., 2004), we cannot

exclude a possible role of other neural mechanisms in

cardiovascular changes induced by long-term exposure

to testosterone and cocaine.

Although the BNST is not involved in the tonic

maintenance of cardiovascular function, several studies

have demonstrated that it plays a key role in the

integration of cardiovascular responses elicited by

peripheral stimuli (e.g., baroreflex) as well as in

autonomic adjustments during emotional stress and

physical exercise (Crestani et al., 2006, 2009; Alves

et al., 2011; Nasimi and Hatam, 2011). Indeed, the

increase of baroreflex bradycardia reported in the

present study after CoCl2 microinjection into the BNST

of control animals (veh + veh group) corroborates

previous data from our group demonstrating a tonic

inhibitory influence of the BNST on reflex bradycardiac

responses evoked by blood pressure increases

(Crestani et al., 2006; Alves et al., 2009). In addition to

its role controlling the cardiovascular function, BNST

has also been associated in the mediation of addiction

and reinstatement of drug-seeking (Smith and Aston-

Jones, 2008; Ikemoto, 2010; Koob and Volkow, 2010;

Gysling, 2012). However, to our knowledge, this is the

first study to evaluate the involvement of this brain

region in cardiovascular changes associated with drug

abuse.

Comparison of baroreflex curves before BNST

inhibition demonstrated that either single or combined

administration of cocaine and testosterone affected the

baroreflex function. These findings confirm previous

data documenting that cardiovascular responses caused

by repeated administration of these substances are

followed by changes in baroreflex control of the HR

(Beutel et al., 2005; Engi et al., 2012). Impairment of

baroreflex function has been proposed as a possible

physiopathological mechanism associated with

hypertension in humans as well as in high arterial

pressure induced in animal models of hypertension

(Grassi et al., 2006). Therefore, reduction of reflex

bradycardia could play a role in the elevation of arterial

pressure in T + veh group. However, change on

baroreflex activity does not seem to explain the rest

bradycardia following treatment with testosterone, since

the impairment of baroreflex activity is associated with

overactivity of the sympathetic tone (Grassi et al., 1995,

2006). In addition, facilitation of reflex HR responses in

animals subjected to combined treatment with cocaine

and testosterone does not support a role of baroreflex

changes altering arterial pressure in these animals.

However, exacerbated tachycardiac response may be a

risk factor for myocardial ischemia, sudden death and

cardiac failure (Dyer et al., 1980; Palatini and Julius,

1997).

Unexpectedly, the inhibition of BNST did not affect

testosterone-evoked changes in baroreflex activity.

Therefore, morphological and/or functional changes

within the BSNT following AAS exposure seem to be

more directly implicated in AAS-induced behavioral

responses (e.g., anxiety and aggression) (DeLeon et al.,

2002; Costine et al., 2010). On the other hand,

baroreflex changes induced by single treatment with

cocaine (veh + coc group) were not identified after

bilateral microinjections of CoCl2 into the BNST. The

change on maximum bradycardiac response induced by

BNST inhibition was decreased in cocaine-treated

animals, whereas BNST influence on tachycardiac

response caused by arterial pressure decrease was

enhanced. Therefore, baroreflex changes following

cocaine exposure seems to be mediated by an

enhanced influence of the BNST in baroreflex

tachycardia and a reduced control of reflex bradycardia.

As observed in cocaine-treated animals, the present

results suggest that the effect of combined

administration of testosterone and cocaine in reflex

tachycardia is mediated by a facilitatory influence of the

BNST. However, although veh + coc and T + coc

groups have shown similar alterations in baroreflex

bradycardia, the effect in T + coc group is independent

of the BNST, suggesting that testosterone reduced

cocaine-induced neural plasticity in the BNST. The

mechanisms associated with a possible interaction

F. C. Cruz et al. / Neuroscience 253 (2013) 29–39 37

between testosterone and cocaine within the BNST are

not clear. However, several pieces of evidence have

suggested that androgenic steroids decrease the

number of monoamine uptake sites (the main

pharmacological target of cocaine) as well as

monoamines content in the central nervous system

(Siddiqui and Gilmore, 1988; Borisova et al., 1996;

Vathy et al., 1997; Goel and Bale, 2010).

Significant decrease in metabolic rate was found in

the BNST after long-term cocaine exposure in primates

(Beveridge et al., 2004). Moreover, chronic cocaine self-

administration resulted in increase of noradrenaline

transporter throughout the BNST (Macey et al., 2003).

We have previously reported that the BNST, mainly by

local noradrenergic neurotransmission, exerts a tonic

inhibitory influence on baroreflex bradycardia (Crestani

et al., 2006, 2008). Therefore, a reduction in synaptic

availability of noradrenaline that in turn decreases the

BNST functional activity could explain the reduced

control of reflex bradycardia by the BNST in cocaine-

treated animals. Little information is available about the

influence of the BNST in baroreflex tachycardia

(Crestani et al., 2006; Alves et al., 2010). To our

knowledge, this is the first study to demonstrate a role

of the BNST in control of reflex cardiac responses

caused by blood pressure decreases. It is possible that

facilitatory influence of the BNST in reflex tachycardia in

cocaine-treated animals also result from changes in the

synaptic availability of monoamines. However, further

experiments are necessary to clarify local mechanisms

associated with BNST involvement in cocaine-induced

baroreflex changes.

The BNST neurons projecting to the ventral tegmental

area (mainly involved in the reward-seeking in drug

abuse) are distinct from BNST neurons projecting to

medial aspects of the hypothalamus (mainly involved in

autonomic control) (Silberman et al., 2013). Indeed, it

was recently demonstrated that selective optogenetic

stimulation of BNST-ventral tegmental area projections

increased real-time place preference without affecting

respiratory rate, whereas selective stimulation of

projections from the BNST to brainstem evoked

opposite effects (Kim et al., 2013). These results

suggest that the pathway originating in the BNST that is

involved in cardiovascular complications associated with

drug abuse may be distinct from the circuitry that

mediates drug-seeking behavior. Regarding the

cardiovascular control, numerous studies have

demonstrated that the BNST is directly connected to

medullary and supra-medullary regions controlling

sympathetic and parasympathetic activity (Holstege

et al., 1985; Gray and Magnuson, 1987; Dong and

Swanson, 2003). Baroreflex bradycardiac response is

predominantly mediated by cardiac parasympathetic

stimulation, whereas reflex tachycardia is mainly

affected by cardiac sympathetic activity (Head and

McCarty, 1987; Crestani et al., 2008, 2010c). Indeed,

we have previously demonstrated that tonic inhibitory

influence of the BNST in reflex bradycardia is mediated

by the parasympathetic nervous system (Crestani et al.,

2008). Therefore, pathways originating in the BNST

involved in mediating inhibitory influence in

parasympathetic component of the baroreflex are

possibly part of the neural substrate associated with

cocaine-induced changes in reflex bradycardia. Previous

studies have also demonstrated that the BNST controls

the sympathetic activity through mandatory synapses in

the caudal and rostral ventrolateral medulla (Ciriello and

Janssen, 1993; Giancola et al., 1993; Hatam and

Ganjkhani, 2012), thus supporting a role of the BNST in

reflex tachycardia changes following cocaine exposure.

In summary, our findings demonstrate that changes in

arterial pressure, HR and baroreflex activity induced by

repeated administration of testosterone are independent

of neural plasticity in the BNST. However, cocaine-

induced facilitation of both bradycardiac and

tachycardiac baroreflex responses is completely

reversed after BNST inhibition. Coadministration of

testosterone did not alter the effects of cocaine on

baroreflex function, but inhibited cocaine-induced

changes within the BNST associated with control of

reflex bradycardia. Importantly, the present study

provides the first direct evidence that neural plasticity

within the BNST following drug exposure are not

exclusively associated with compulsive/impulsive

behavior and reinstatement of drug-seeking in

dependence, but play a key role in cardiovascular

toxicity induced by drug exposure.

Acknowledgments—The authors wish to thank Elisabete Z.P. Le-

pera, Rosana F.P. Silva and Ivanilda Fortunato for technical

assistance. This study was supported by Sao Paulo Research

Foundation (FAPESP) grant # 2010/16192-8; National Council

for Scientific and Technological Development (CNPq) grant #

474177/2010-6; and PADC-School of Pharmaceutical Sci-

ences-Sao Paulo State University. Cleopatra S Planeta is a

CNPq research fellow.

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(Accepted 20 August 2013)(Available online 29 August 2013)