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University of Lisbon

Institute of Pharmacology and Neurosciences, Faculty of Medicine

Unit of Neurosciences, Institute of Molecular Medicine

NEUROTROPHIC FACTORS MODULATION BY ADENOSINE RECEPTORS

AND DOPAMINE RELEASE IN THE RAT STRIATUM

Catarina A. dos Reis Vale Gomes

The experimental work described in the present thesis was elaborated under supervision

of Professor Joaquim Alexandre Ribeiro.

PhD in Biomedical Sciences Specialty in Neurosciences

Lisbon 2009

ii

A impressão desta dissertação foi aprovada pela Comissão Coordenadora

do Conselho Científico da Faculdade de Medicina de Lisboa na reunião

realizada no dia 17 de Janeiro de 2009.

As opiniões expressas são da exclusiva responsabilidade do seu autor.

Este trabalho foi co-financiado pelo POCI 2010 e pelo Fundo Social

Europeu no âmbito de uma bolsa de doutoramento da Fundação para a

Ciência e a Tecnologia (SFRH/BD/13553/2003).

iii

To Lourenço and to the memory of his grandfather.

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TABLE OF CONTENTS

Table of Contents ................................................................................................. 1

Acknowledgements ............................................................................................. 7

Abbreviations list .................................................................................................. 9

Publications ........................................................................................................13

Abstract ..............................................................................................................15

Resumo ..............................................................................................................19

Figure index ........................................................................................................23

1. INTRODUCTION ............................................................................................29

1.1. The striatum: neuronal organization .......................................................30

1.2. Dopaminergic and glutamatergic neurotransmission in the dendritic

spines of the MSNs ........................................................................................34

1.2.1. Dopaminergic neurotransmission ....................................................35

1.2.2. Glutamatergic neurotransmission ....................................................37

1.3. Adenosine, a modulator of striatal physiology ........................................39

1.3.1. Overview as a modulator ................................................................39

2

1.3.2. Pathways of production, metabolism and transport ........................ 40

1.3.3. Adenosine receptors - an overview from subtypes and signalling to

distribution, heteromerization and concentration-dependent function ...... 42

1.3.4. Pathophysiological involvement and therapeutic potential of A2A

receptors in the striatum ........................................................................... 48

1.4. GDNF, another modulation system in the striatum ................................ 50

1.4.1. Brief description and overview as a trophic factor in the CNS ........ 50

1.4.2. GDNF receptor-complex - characterization, signalling and

distribution ................................................................................................. 52

1.4.3. GDNF - pathophysiological involvement and therapeutic potential in

the striatum ............................................................................................... 55

1.5. Aim ......................................................................................................... 57

2. MATERIALS AND METHODS ................................................................. 59

2.1. Animal handling and biological research preparations .......................... 61

2.1.1. Animals used .................................................................................. 61

2.1.2. Dissection and preparation of striatal synaptosomes and slices for

neurotransmitter release experiments ...................................................... 61

2.1.3. Preparation of striatal punches for western blot analysis. .............. 63

2.1.4. Preparation of striatal slices for immunochemical analysis. ........... 63

2.1.5. Preparation of total membranes and synaptosomes for synaptic

localization studies of Ret and GFRα1 ....................................................... 64

3

2.1.6. Subcellular fractionation of nerve terminals for subsynaptic

localization studies of Ret and GFRα1........................................................65

2.1.7. Preparation of striatal nerve terminals for localization and

colocalization studies .................................................................................67

2.2. Experimental procedures ........................................................................68

2.2.1. Neurotransmitter release from striatal synaptosomes and slices ....68

2.2.2. Electrode implantation and in vivo electrical cortical stimulation .....74

2.2.3. Immunocytochemical analysis of striatal slices ...............................76

2.2.4. Immunocytochemical analysis of striatal nerve terminals ...............76

2.2.5. Western blot analysis ......................................................................78

2.3. Reagents, drugs, antibodies and solutions .............................................79

3. RESULTS .......................................................................................................85

3.1. Adenosine A2A receptor modulation of GDNF effect upon the

dopaminergic input to the striatum .................................................................85

3.1.1. Rationale of the study ......................................................................85

3.1.2. GDNF enhanced K+-evoked dopamine release from isolated nerve

terminals ....................................................................................................86

3.1.3. GDNF enhancement of K+-evoked dopamine release from isolated

nerve terminals is dependent on adenosine A2A receptor activation .........88

3.1.4. GDNF enhanced electrically-evoked dopamine release from striatal

slices ..........................................................................................................90

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3.1.5. GDNF effect on dopamine release from striatal slices also depends

upon A2A receptor tonic activation ............................................................. 90

3.1.6. Influence of A2A receptors and of GABA transmission blockers upon

dopamine release in striatal slices ............................................................ 93

3.1.7. Discussion ....................................................................................... 96

3.2. Adenosine A2A receptor modulation of the cortical-induced protein

phosphorylation in the striatum ..................................................................... 99

3.2.1. Rationale of the study ..................................................................... 99

3.2.2. Adenosine A2A receptor effect upon cortical stimulation-induced

protein phosphorylation ........................................................................... 100

3.2.3. Discussion ..................................................................................... 100

3.3. Adenosine A2A receptor modulation of GDNF effects on the

glutamatergic corticostriatal pathway .......................................................... 107

3.3.1. Rationale of the study ................................................................... 107

3.3.2. GDNF control of the glutamatergic input to the striatum .............. 108

3.3.3. In vivo A2A receptor modulation of the activation state of GDNF

receptor upon cortical stimulation ........................................................... 112

3.3.4. Discussion ..................................................................................... 114

3.4. GDNF receptor components distribution in the rat striatum, cellular and

subsynaptic localization and co-localization with A2A receptor.................... 116

3.4.1. Rationale of the study ................................................................... 117

3.4.2. GDNF receptor proteins are located in striatal nerve terminals.... 118

5

3.4.3. Mapping GDNF receptor components in different types of striatal

nerve terminals ........................................................................................123

3.4.4. GDNF and adenosine A2A receptor are co-located in striatal

glutamatergic and dopaminergic nerve terminals ....................................126


glutamatergic and dopaminergic nerve terminals ....................................126

4. CONCLUSION .............................................................................................135

5. REFERENCES .............................................................................................143

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ACKNOWLEDGEMENTS

To the supervisor of this thesis, Professor Joaquim Alexandre Ribeiro, I

would like to thank first the opportunity to explore such an interesting scientific

question, which led me to elect neuropharmacology as scientific field of work. I

also thank the opportunity to participate in several meetings, either in Portugal

and abroad and to collaborate with national and international research groups,

which, I believe, improves discussion, generates ideas and hence, increases

quality. Besides his well-known scientific carrer, I particularly admire his

philosophical way of thinking science and this is the great benefit I took. I hope

to be able to think about single observations in this Ribeiro-like style: broader

perspective.

All decisions about experimental design and interpretation of results

were supported by the scientific knowledge and advertisement of Professor Ana

Maria Sebastião. I would like to thank her.

I have spent increadibly pleasant moments with my colleagues. We

have also had clarifying scientific discussions. We have travelled together and

together we knew unforgetable places. Thus, Sofia, Carina, Raquel and Bruno,

Diana, Maria José, Rita, Sylvie and Vasco are now friends. In particular to

Natália, Sandra, Paula and António Francisco I wish to thank friendship and

those private meetings where we discuss literary, artistic and scientific

overtones. Finally, I would like to thank Luísa the revision of this thesis and the

wise and sensitive way of living, which she perfectly reconciles with the criticism

of the scientist.

I thank Cristina, João, Elvira and Helena for technical help and

friendship. In particular to Alexandra, I would like to thank the availability,

patience and kindness.

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I wish to thank all co-authors for their collaboration in the published

papers. In special to Doctor Sergi Ferré and to Professor Rodrigo Cunha, I

thank the wise ability to teach and share their scientific knowledge. Science can

be a hard journey and their enthusiasm and the commitement put in this project

made it easier. Both have certainly influenced my scientific and individual

growth. Paula Canas, Patrícia Simões and César Molina were more than

colleagues. Besides their technical help, they were allways kind, patient, ...

friends.

The “USA experience” was particularly rich in the professional and

personal points of view. However, several months far away from home and

family also brought difficult moments. César, Carmen and Maria, which I

remember with love, were my family in USA and, once again, we have worked

together, travelled together and became friends.

Francisco has always enthusiastically encouraged my carrer in science.

He understood my fascination and this is a sensitive way of love. Without the

help of my parents this work would not be possible. By taking care of Lourenço,

they made it possible to reconcile my family and my work. Thank you family!

During the last years I have been teaching Pharmacology. I believe that

research improves teaching skills and I found that the opposite is also true. In

spite of the difficulty in reconciling two time-consuming activities, to realize how

pharmacotherapy would be with the molecules tested in the lab is a source of

motivation. I wish to thank “my” students the curiosity and enthusiasm about my

scientific work, which make me better learn and, hence, better teach.

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ABBREVIATIONS LIST

AC - adenylyl cyclise

ADA - adenosine deaminase

ADP - adenosine-5´-diphosphate

AK - adenosine kinase

AMP - adenosine 5´-monophosphate

AMPA - alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AP - anteroposterior

ATP - adenosine 5´-triphosphate

A1R - adenosine A1 receptor subtype

A2AR - adenosine A2A receptor subtype

BSA - bovine serum albumine

cAMP - cyclic adenosine 5´-monophosphate

CGP 55845 - 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-

benzyl-phosphinic acid

CGS 21680 - 2-[p-(2 carboxyethyl)phenethylamino]-5´-N-ethylcarboxamide

adenosine

CNS - central nervous system

CREB - cAMP response element-binding

CPT - 8-cyclopentyl-1,3-dimethylxanthine

DAT - dopamine transporter

D1R - dopamine D1 receptor subtype

D2R - dopamine D2 receptor subtype

Dyn - dynorphin

EDTA - ethylenediamine tetraacetic acid

EGTA - ethylene glycol tetraacetic acid

Enk - enkephalin

ERK - extracellular signal-regulated kinases

ET - equilibrative transporter

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GABA - -aminobutyric acid

GDNF - glial cell line-derived neurotrophic factor

GFL - GDNF-family ligand

GFRα1 - GDNF family receptor alpha 1

GluR1 - AMPA (glutamate) receptor subunit 1

GP - globus pallidus

GPCR - G protein-coupled receptor

GPe - external segment of the globus pallidus

GPi - internal segment of the globus pallidus

GPI - glycosylphosphatidylinositol

HEPES - N-2-hydroxyethylpiperazine-N‟-2-ethanesulfonic acid

IEG - immediate early genes

i.p. - intra-peritoneal

KHR - Krebs-HEPES-Ringer

LTD - long-term depression

LTP - long-term potentiation

MAPK - mitogen-activated protein kinase

mGluRs - metabotropic glutamate receptors

mRNA - messenger ribonucleic acid

MSNs - medium-sized spiny neurons

MSX-3 - 3,7-dihydro-8-[(1E)-2-(3-ethoxyphenyl)ethenyl]-7 methyl-3-[3-

(phosphooxy)propyl-1-(2 propynil)-1H-purine-2,6-dione

mAChR - muscarinic acetylcholine receptor

NGF - nerve growth factor

nAChR - nicotinic acetylcholine receptors

NMDA - N-methyl-D-aspartate

PBS - phosphate buffer saline

PDE - phosphodiesterase

PI3K - phosphatidylinositol 3-kinase

PKA - cAMP-dependent protein kinase

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PLC - phospholipase C

PMSF - phenylmethylsulfonylfluoride

PSD - post-synaptic density

PVP - polyvinylpyrrolidone

Ret - rearranjed during transfection

SAH - S-adenosyl homocysteine

SCH 58261 - 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4

triazolol[1,5c]pyrimidine

SDS-PAGE - sodium-dodecyl sulphate polyacrylamide gel electrophoresis

SDS - sodium-dodecyl sulphate

Ser - serine

SN - substantia nigra

SNAP - synaptosome-associated protein

SNc - substantia nigra pars compacta

SNr - substantia nigra pars reticulata

SP - substance P

STn - subthalamic nucleus

TBS - Tris-buffered saline

TH - tyrosine hydroxylase

Tris - tris(hydroxymethyl-aminomethane

vAChT - vesicular acethylcholine transporter

VDCC - voltage-dependent calcium channel

vGAT - vesicular GABA transporter

vGlut1 - vesicular glutamate transporter subtype 1

vGlut2 - vesicular glutamate transporter subtype 2

VTA - ventral tegmental area

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PUBLICATIONS

The work described in this thesis was published in peer-reviewed

international scientific journals:

I. Gomes CA, Vaz SH, Ribeiro JA, Sebastião AM (2006) Glial cell line-derived

neurotrophic factor (GDNF) enhances dopamine release from striatal nerve

endings in an adenosine A2A receptor-dependent manner. Brain Res 1113:

129-136.

(Chapter 3.1)

II. Quiroz-Molina C, Gomes C, Pak AC, Ribeiro JA, Goldberg SR, Hope BT,

Ferré S (2006) Blockade of adenosine A2A receptors prevents protein

phosphorylation in the striatum induced by cortical stimulation. J Neurosci 26:

10808-10812.

(Chapter 3.2)

III. Gomes CA, Simões PF, Canas PM, Quiroz C, Sebastião AM, Ferré S,

Cunha RA, Ribeiro JA (2009) GDNF control of the glutamatergic cortico-striatal

pathway requires tonic activation of adenosine A2A receptors. J Neurochem 108:

1208-1219.

(Chapters 3.3 and 3.4)

http://www.ncbi.nlm.nih.gov/pubmed/19141075?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum


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ABSTRACT

Striatal circuits exert a crucial role in motor control, being

triggered by cortical glutamatergic inputs that are, in turn, controlled by

nigrostriatal dopaminergic innervation. Accordingly, the imbalance of

motor function characterizing some neurodegenerative diseases can

either result from dysfunction of the corticostriatal inputs, as occurs in

Huntington‟s disease, as well as from dysfunction of the nigrostriatal

system, the neurochemical hallmark of Parkinson‟s disease.

Both the neuromodulator adenosine and the neurotrophic factor

GDNF (glial cell line-derived neurotrophic factor) are tightly involved in

the control of striatal physiology and have been considered as potential

therapeutic approaches to different neurological diseases. From the four

receptor subtypes that mediate adenosine actions - A1, A2A, A2B and A3,

A2A receptors are of particular relevance in the striatum and are

abundantly expressed in this brain area. Adenosine A2A receptor

antagonists are currently a promising anti-Parkinsonian therapy and the

manipulation of the referred receptor also affords beneficial effects in

animal models of Huntington‟s disease. The administration of GDNF is

also profitable in animal models of Parkinson and Huntington‟s diseases

and, in spite of an intense debate on its clinical benefits and drawbacks,

several trials pointed out towards a therapeutic potential in Parkinson´s

disease. Considering that an important role of A2A receptors is their

ability to tightly control the action of different growth factors, it seemed of

interest to investigate if GDNF could modulate the activity of the

nigrostriatal dopaminergic and the corticostriatal glutamatergic systems

under the influence of adenosine A2A receptors.

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The final aim of this work was to evaluate the ability of GDNF to

presynaptically control neurotransmitter - dopamine and glutamate -

release from the striatum and whether adenosine A2A receptors could

modulate GDNF effect. By quantifying dopamine release from striatal

slices and isolated nerve terminals it was possible to establish that

GDNF (10 ng/ml) enhances dopamine release in a manner dependent

on A2A receptor activation. In striatal slices, blockade of GABA (-

aminobutyric acid)ergic transmission enhanced the evoked release of

dopamine, but reduced the effect of GDNF on dopamine release,

suggesting that part but not all of the excitatory effect of GDNF depends

upon GABAergic transmission. Curiously, the magnitude of the

enhancement of dopamine release caused by GDNF in synaptosomes

(where a crosstalk between different neuronal structures might be

virtually absent) was similar to that observed in slices upon inhibition of

GABAergic transmission. Considering the influence of the circuitry upon

GDNF striatal effects, glutamate release experiments were then carried

out using isolated nerve terminals, where GDNF (10 ng/ml) also

facilitated glutamate release in a manner dependent on A2A receptor

activation. These observations, which provide a first evidence for a

crosstalk between adenosine A2A receptors and GDNF in the striatum,

were further supported by in vivo studies. Thus, in vivo electrical

stimulation of the corticostriatal pathway induced phosphorylation of

GDNF receptor, and this phosphorylation was prevented by A2A receptor

antagonism, indicating that stimulation of cortical afferents promotes

activation of the GDNF receptor effector in the striatum, a process that

requires co-activation of A2A receptors.

In order to further clarify the role of GDNF as a presynaptic

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modulation system, another goal proposed for this thesis was to

determine which types of striatal nerve terminals are equipped with

GDNF receptor components, Ret (rearranjed during transfection) and

GFRα1 (GDNF family receptor alpha 1), their subsynaptic localization, in

particular their localization in the active zone, where neurotransmitter

release takes place and to investigate whether Ret and GFRα1 were co-

located with A2A receptors. Morphological studies revealed that a high

proportion of the nerve endings at the striatum possess Ret and GFRα1,

mainly located in the presynaptic active zone and in the postsynaptic

density, with a lower relative abundance in the extrasynaptic fraction of

nerve terminals. Ret and GFRα1 were mostly located in dopaminergic

and glutamatergic nerve terminals, where they were found to be co-

located with A2A receptors, in accordance with the functional interplay

between GDNF and adenosine. Thus, these results unequivocally

showed that A2A receptors are present in striatal dopaminergic nerve

terminals, challenging previous ideas that these receptors were absent

from these terminals. That concept arose from the observation that A2A

receptors do not directly interfere with dopamine release. However, by

performing morphological and functional studies, it was possible to show

in the present work that A2A receptors are present in striatal

dopaminergic nerve terminals, where they modulate the action of GDNF

on dopamine release, in spite of the lack of direct influence upon

dopamine release.

In conclusion, these observations provide the first functional and

morphological evidence that GDNF controls nigrostriatal dopaminergic

and corticostriatal glutamatergic pathways in a manner largely

dependent on the co-activation of adenosine A2A receptors, which further

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confirms the role of adenosine as a modulator of modulators.

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RESUMO

As alterações motoras características de algumas doenças

degenerativas resultam da disfunção da neurotransmissão em

determinadas áreas do cérebro. Alterações da neurotransmissão

glutamatérgica ou dopaminérgica no estriado (aferências provenientes

do córtex e da substantia nigra, respectivamente), área cerebral

fundamental no controlo do movimento, estão envolvidas na

fisiopatologia das doenças de Huntington e de Parkinson,

respectivamente.

O neuromodulador adenosina, bem como o factor neurotrófico

derivado da glia (GDNF, do inglês glial cell line-derived neurotrophic

factor) exercem um papel relevante na fisiologia do estriado e

constituem potenciais estratégias terapêuticas em diferentes doenças

neurológicas. Os receptores A2A da adenosina assumem particular

importância no estriado e, de modo consonante, dos quatro subtipos de

receptores que medeiam as acções do modulador - A1, A2A, A2B e A3, a

sua expressão é quantitativamente preponderante nesta área do

cérebro. Os antagonistas destes receptores constituem uma estratégia

terapêutica promissora na doença de Parkinson e os resultados da sua

manipulação em modelos animais da doença de Huntington sugerem

potencial terapêutico nesta patologia. Por seu turno, a administração de

GDNF induziu respostas benéficas em modelos animais das doenças

de Huntington e de Parkinson e, apesar de um intenso debate em torno

de benefícios e desvantagens, vários ensaios clínicos reforçam o

interesse terapêutico da molécula na doença de Parkinson. Atendendo

a que uma das acções mais importantes da adenosina - por activação

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dos receptores A2A - consiste na modulação dos efeitos de diversos

factores de crescimento, esta dissertação teve como objectivo principal

clarificar as acções do GDNF na transmissão dopaminérgica da nigra e

glutamatérgica cortical ao estriado (controlo pré-sináptico da libertação

de dopamina e glutamato), bem como a influência moduladora dos

receptores A2A da adenosina sobre as referidas acções.

Os estudos de quantificação da libertação de dopamina

conduzidos em fatias ou em terminais nervosos isolados de estriado

mostraram que o GDNF (10 ng/ml) aumenta a libertação do

neurotransmissor de modo dependente da activação dos receptores

A2A. Em fatias de estriado, o bloqueio da transmissão GABA (do inglês,

-aminobutyric acid)érgica aumenta a libertação evocada de dopamina,

apesar de atenuar o efeito do GDNF na libertação da mesma, o que

sugere que parte do efeito excitatório do GDNF depende da

transmissão GABAérgica. Curiosamente, o aumento da libertação de

dopamina induzido pelo GDNF em sinaptosomas (onde, virtualmente,

as diferentes estruturas neuroniais não interagem) foi semelhante ao

observado em fatias após inibição da transmissão GABAérgica.

Considerando a influência dos circuitos neuroniais nos efeitos do GDNF,

as experiências de libertação de glutamato foram conduzidas apenas

em terminais nervosos isolados, preparação biológica em que o GDNF

(10 ng/ml) aumentou a libertação de glutamato de modo dependente

dos receptores A2A. Estas observações, que constituem a primeira

indicação de que os receptores A2A da adenosina modulam efeitos do

GDNF no estriado, foram confirmadas por estudos in vivo: a

estimulação eléctrica da via corticoestriatal induziu a fosforilação do

receptor do GDNF no estriado, e esta fosforilação foi inibida por

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antagonismo dos receptores A2A da adenosina.

Com o objectivo de confirmar as acções do GDNF enquanto

sistema modulador pré-sináptico no estriado, considerou-se relevante

identificar os tipos de terminais nervosos que expressam as duas

proteínas que compõem o complexo-receptor do GDNF, Ret (do ingês

rearranjed during transfection) e GFR1 (do inglês GDNF family receptor

alpha 1), bem como analisar a sua localização subsináptica (em

particular na zona activa, onde ocorre a libertação de

neurotransmissores) e possível co-localização com os receptores A2A da

adenosina. As observações morfológicas revelaram que uma elevada

proporção de terminais nervosos expressam as proteínas Ret e GFR1,

que estas proteínas estão principalmente localizadas nos terminais

nervosos dopaminérgicos e glutamatérgicos, onde co-localizam com

receptores A2A, o que reforça a relação funcional estabelecida entre

GDNF e adenosina. Como os ligandos dos receptores A2A não

influenciam directamente a libertação de dopamina no estriado,

estabeleceu-se que estes receptores não seriam expressos nos

terminais nervosos dopaminérgicos. Esta ideia é contrariada pelos

resultados morfológicos e funcionais desenvolvidos neste trabalho, que

permitem concluir que os receptores A2A são expressos no terminal

nervoso dopaminérgico estriatal, onde modulam a acção do GDNF na

libertação de dopamina, ao invés de exercerem acção directa na

libertação do neurotransmissor.

Concluindo, as observações decorrentes dos estudos funcionais

e morfológicos descritos nesta dissertação mostram que o GDNF

controla as vias dopaminérgica nigroestriatal e glutamatérgica

corticoestriatal de modo dependente da co-activação dos receptores A2A

22

da adenosina, o que confirma o papel da mesma como modulador de

moduladores.

23

FIGURE INDEX

1. INTRODUCTION ...............................................................................................29

Figure 1.1.1 - Simplified representation of neural circuitry in the basal ganglia .. .....331

Figure 1.1.2 - Schematic representation of the striatal medium spiny neuron

(MSN). .. .............................................................................................................33

Figure 1.3.1 - Pathways of adenosine production, metabolism and transport. 42

Figure 1.3.2 - Major signal transduction pathway used by adenosine A2A

receptor: cAMP-PKA cascade. ...........................................................................44

Figure 1.3.3 - Schematic representation of distribution of pre- and postsynaptic

A2A receptor-containing heteromeric receptor complexes in the modulation of

striatal glutamatergic transmission in the GABAergic enkephalinergic neuron. 47

Figure 1.4.1 - Mechanisms of GDNF-GFR1-Ret receptor complex activation. 53

2. MATERIALS AND METHODS ...........................................................................61

Figure 2.1.1 - Preparation of striatal slices for neurotransmitter release

experiments. .......................................................................................................62

Figure 2.1.2 - Striatal punches extraction site. . ................................................63

Figure 2.2.1 - [3H]neurotransmitter release from striatal synaptosomes. . ........70

Figure 2.2.2 - [3H]dopamine release from striatal slices. ..................................73

Figure 2.2.3 - Electrode implantation site. . .......................................................75

3. RESULTS ........................................................................................................85

Figure 3.1.1 - GDNF enhances K+-evoked [

3H]dopamine release from striatal

synaptosomes ......................................................................................................89

GDNF Effects Modulation by A2A Receptors in the Rat Striatum

24

Figure 3.1.2 - Ability of the selective A2A receptor agonist, CGS 21680 (10 nM),

and of the A2A receptor antagonist, SCH 58261 (50 nM), to modify the action of

GDNF (10 ng/ml) on [3H]dopamine release from striatal synaptosomes. ........ 89

Figure 3.1.3 - GDNF enhances electrically evoked [3H]dopamine release from

striatal slices. .. .................................................................................................. 91



GDNF (10 ng/ml) on [3H]dopamine release in striatal slices. ............................ 92



GDNF (10 ng/ml) on [3H]dopamine release in striatal slices in the presence of

the GABAA and GABAB receptor antagonists, bicuculline (10 μM) and CGP

55845 (1 μM). .. ................................................................................................. 95

Figure 3.2.1 - Immunohistochemical localization of striatal ERK1/2

phosphorylation induced by cortical electrical stimulation. . ............................ 102

Figure 3.2.2 - Striatal ERK1/2 phosphorylation induced by cortical electrical

stimulation. . ..................................................................................................... 103

Figure 3.2.3 - Striatal GluR1 phosphorylation induced by cortical electrical

stimulation. . ..................................................................................................... 104

Figure 3.3.1 - GDNF enhances electrically evoked [3H]glutamate release from

striatal synaptosomes. .. .................................................................................. 110

Figure 3.3.2 - Blockade of the effect of GDNF by the A2A receptor antagonist,

SCH 58268, and its potentiation by the A2A receptor agonist, CGS 21680. . .. 111

Figure 3.3.3 - Striatal phosphorylation of the GDNF receptor component is

induced by cortical electrical stimulation and depends on A2A receptor

activation. . ...................................................................................................... 113

Figure 3.4.1 - GDNF receptor protein (Ret and GFRα1) are present in

25

synaptosomal membranes with a similar density to that found in total

membranes from the whole striatum. . .............................................................120

Figure 3.4.2 - GDNF receptor proteins (Ret and GFRα1) are located in

synapses, namely in the presynaptic active zone of striatal nerve terminals. ..

..........................................................................................................................121

Figure 3.4.3 - Ret and GFRα1 are located in more thn half of striatal nerve

terminals.). ........................................................................................................122

Figure 3.4.4 - Ret protein is mainly located in dopaminergic and glutamatergic,

rather than GABAergic and cholinergic, nerve terminals in the rat striatum. ...124

Figure 3.4.5 - GFR1 protein is mainly located in dopaminergic and

glutamatergic, rather than GABAergic and cholinergic, nerve terminals in the rat

striatum. . ..........................................................................................................125

Figure 3.4.6 - GDNF receptor protein (Ret and GFRα1) are co-located with

adenosine A2A receptors in glutamatergic nerve terminals of the rat striatum.

..........................................................................................................................127


adenosine A2A receptors in dopaminergic nerve terminals of the rat striatum.

..........................................................................................................................128

Figure 3.4.8 - Adenosine A2A receptors are localized at striatal dopaminergic

nerve terminals. .. .............................................................................................129

4. CONCLUSION ...............................................................................................135

Figure 4.1 - Hypothetical model for a new role of adenosine A2A receptor in

striatal physiology - focus on the functional interaction and receptor co-

localization with GDNF. ...................................................................................139


26

27

INT

RO

DU

CT

ION

Introduction

29

1. INTRODUCTION

The primary focus of neuropharmacology is the development of

new molecules with ever-improving efficacy and safety for diseases of

the nervous system. The control of normal movement is maintained by

multiple, highly interconnected circuits between different brain areas,

with the striatum playing a key role in the process. The imbalance of

synaptic transmission to this brain area underlies the pathophysiology of

neurodegenerative motor diseases, such as Parkinson´s and

Huntington´s diseases, involving a dysfunction of the main inputs to the

striatum: dopaminergic innervation from the nigra and glutamatergic

inputs from the cortex, respectively. Thus, most drugs target synaptic

activity and the therapeutic potential of such molecules largely depends

on their ability to reach this limited brain area. A great challenge in

pharmacological research is to find or re-design molecules able to cross

the blood-brain barrier or, in alternative, more sophisticated approaches

to change synaptic activity or physiological processes related to synaptic

activity.

Adenosine is an endogenous neuromodulator, which controls

and integrates a wide range of brain functions through the activation of

four receptor subtypes - A1, A2A, A2B and A3 (Fredholm et al., 2001a).

Dysfunction of the adenosine system is involved in different pathologies,

namely neurodegenerative disorders and the molecule and/or ligands for

its receptors have been recognized as prime targets for the development

of new therapeutics for neurological diseases. Adenosine A2A receptor

antagonists are currently promising anti-Parkinsonian drugs, which

already underwent phase III clinical trials (LeWitt et al., 2008). The


30

trophic factor GDNF is also crucial to striatal pathophysiology, affording

beneficial effects in animal models of Parkinson‟s disease (e.g. Gash et

al., 1996; Tomac et al., 1995a) and in humans (Patel et al., 2005). The

manipulation of A2A receptors (reviewed in Popoli et al., 2007), as well as

the administration of GDNF (reviewed in Alberch et al., 2004) afford

beneficial effects in animal models of Huntington‟s disease. Thus, both

adenosine and GDNF influence striatal function at the same circuitry

level. Considering that an important role of A2A receptors is their ability to

tightly control the action of different growth factors (Diógenes et al.,

2004, 2007; Mojsilovic-Petrovic et al., 2006; Pousinha et al., 2006;

Wiese et al., 2007; Fontinha et al., 2008; Fernandes et al., 2008), they

are also good candidates to modulate GDNF effects.

The main goal of the work reported in this thesis was to

investigate the influence of GDNF on dopaminergic and glutamatergic

striatal nerve endings and the ability of adenosine A2A receptors to

regulate those actions.

1.1. The striatum: neuronal organization

The term striatum includes both the neostriatum (also called the

dorsal striatum, composed of caudate and putamen) and more ventral

extensions referred to as ventral striatum (which main component is the

nucleus accumbens). This brain area is integrated in a group of bilateral

subcortical nuclei, the basal ganglia, whose major components are the

neostriatum and the globus pallidus (GP). Figure 1.1.1 shows a

schematic representation of neural circuitry in the basal ganglia.

Introduction

31

Figure 1.1.1 - Simplified representation of neural circuitry in the basal ganglia.

Glutamatergic neurons from the cortex innervate two major types of GABAergic neurons

in the striatum. One type, which coexpresses the neuropeptides dynorphin (Dyn) and

substance P (SP), dopamine D1 receptor (D1R) and adenosine A1 receptor (A1R)

subtypes projects directly to the globus pallidus pars interna (GPi) and substantia nigra

pars reticulata (SNr) and give rise to the direct pathway. The other type of GABAergic

neuron gives rise to the indirect pathway, coexpressing enkephalin (Enk), dopamine D2

receptor (D2R) and adenosine A2A receptor (A2AR) subtypes and projecting indirectly to

the GPi and SNr by means of the globus pallidus pars externa (GPe) and subthalamic

nucleus (STn). Dopaminergic neurons in the substantia nigra pars compacta (SNc)

modulate these striatal outputs.

CEREBRAL CORTEX STRIATUM

GPe

SNc /VTA

GPi SNr

STn

THALAMUS

GABAergic

Glutamatergic

Dopaminergic

DIRECT PATHWAY

INDIRECT PATHWAY

Dyn SP

A 1 R D 1 R

D 2 R Enk

CEREBRAL CORTEX STRIATUM

GPe

SNc /VTA

GPi SNr

STn

THALAMUS

DIRECT PATHWAY

INDIRECT PATHWAY

Dyn SP

A 1 R D 1 R

D 2 R Enk

A 2A R


32

Two other subcortical nuclei, the subthalamic nucleus (STn) and

substantia nigra (SN), are functionally and anatomically interconnected

with the neostriatum. The substantia nigra is located in the midbrain and

has two main parts: the pars compacta (SNc), which contains

dopaminergic neurons and the pars reticulate (SNr), which contains

GABAergic neurons. The basal ganglia and associated nuclei are part of

a neural circuit that receives and processes input from the cerebral

cortex, and subsequently provides feedback to the cortex (for a review

see e.g. Bolam et al., 2000).

More than 90% of striatal neurons are medium-sized GABAergic

neurons that project out of the striatum and have dense dendritic spines.

Thus, they are termed medium-size spiny neurons (MSNs). In the dorsal

striatum, MSNs can be classified into two major subpopulations

according to their peptide expression: enkephalinergic and

dynorphinergic neurons (Gerfen, 2004). These neurons give rise to two

efferent pathways, which connect the striatum with the output structures

of the basal ganglia, the SNr and the internal segment of the globus

pallidus (GPi, entopeduncular nucleus in rodents) (Gerfen, 2004). These

are called direct and indirect pathways, with the direct pathway being

made of GABAergic dynorphinergic neurons that coexpress substance P

(SP) directly connecting the striatum with the output structures and the

indirect pathway consisting of enkephalinergic neurons, which connect

the striatum with the external segment of the globus pallidus (GPe,

globus pallidus in rodents), GABAergic neurons that connect the GPe

with the STn and glutamatergic neurons which connect the STn with the

output structures (Figure 1.1.1). GPe GABAergic neurons also project

directly to the output structures without using the STn relay (Gerfen,

Introduction

33

2004).

Dendritic spines of the MSNs are the local of convergence of the

two main striatal inputs, glutamatergic (from cortical, limbic and thalamic

areas) and dopaminergic (from the mesencephalon, either the SNc or

the VTA, ventral tegmental area) afferents (see Figure 1.1.2).

Cortico-limbic-thalamic

glutamatergic input

Mesencephalic

dopaminergic input

Cortico-limbic-thalamic

glutamatergic input

Mesencephalic

dopaminergic input

Figure 1.1.2 - Schematic representation of the striatal medium spiny neuron (MSN).

This neuron receives two main inputs: glutamatergic afferents from cortical, limbic and

thalamic areas and dopaminergic afferents from the mesencephalon. The magnified

scheme shows a dendritic spine of the MSN and glutamatergic and dopaminergic

terminals, which make synaptic contact with the head and neck of the dendritic spine,

respectively.

The morphological organization of the main striatal afferents, with

glutamatergic and dopaminergic terminals making synaptic contacts with

the head and the neck of the dendritic spine, respectively, determines

functional differences: glutamatergic terminals trigger striatal circuits;

dopaminergic terminals, unable to trigger electrical responses in MSNs


34

in the absence of glutamatergic inputs, exert a crucial modulatory action

(Gerfen, 2004). Cholinergic interneurons and GABAergic neurons

constitute the remaining striatal neurons (for a review see e.g. Tepper

and Bolam, 2004). Striatal sources of GABA (-aminobutyric acid)

include interneurons (Kawagushi et al., 1995), afferents from other basal

ganglia structures, such as GPe or SNr (Kita, 1993) and axon collaterals

from the MSNs themselves (Kita, 1993; Park et al., 1980).

1.2. Dopaminergic and glutamatergic neurotransmission in

the dendritic spines of the MSNs

Considering that dopamine and glutamate afferents (onto the

dendritic spines of the MSNs) constitute the major inputs to the striatum

and are also the focus of the present work, dopaminergic and

glutamatergic neurotransmission is discussed in detail in the current

section (1.2). Adenosine is a key element in the control of both

transmission systems and the presence of its receptors at dopaminergic

and glutamatergic terminals is briefly referred in this section (1.2).

However, given its central role in the present thesis, adenosinergic

transmission will be further described in detail in a separate section

(1.3). Besides adenosine, several other modulators/transmitters are able

to control glutamatergic and dopaminergic transmission in the striatum,

whose study is clearly out of the scope of the present work. However, in

this context and because this issue will not be discussed anywhere else

in this dissertation, it is important to notice that: (1) cholinergic receptors

in the striatum are of both the metabotropic (muscarinic acetylcholine

receptor, mAChR) and ionotropic families (nicotinic acetylcholine

Introduction

35

receptors, nAChR) (Exley and Cragg, 2008) and modulate both

glutamatergic (Pakhotin and Bracci, 2007) and dopaminergic

transmission (Harsing and Zigmond, 1998, Hersch et al., 1994, Gomeza

et al., 1999, Jones et al., 2001, Zhou et al., 2001; Rice and Cragg, 2004;

Zhang and Sulzer, 2004) and that (2) GABA exerts its action via

ionotropic GABAA and metabotropic GABAB receptors (Olsen and

Sieghart, 2008) and control dopaminergic transmission (Tepper and Lee,

2008).

1.2.1. Dopaminergic neurotransmission

In 1978 it was proposed that dopamine acts through the

activation of two populations of receptors, one coupled to and the other

independent of adenylyl cyclase (AC) (Spano et al., 1978). In 1979,

Kebabian and Calne suggested to call D1 to the receptor positively

coupled to AC and D2 to the receptors independent of AC.

Subsequently, three novel receptors were characterized - D3 (Sokoloff et

al., 1990), D4 (van Tol et al., 1991) and D5/D1b (Sunahara et al., 1991;

Tiberi et al., 1991) - but all subtypes fall into one of the initially proposed

classification, D1 or D2, according to their AC coupling ability. All these

receptors are GPCRs (G protein-coupled receptors). D1 and D5/D1b are

classified as D1-like and couple with Gs/olf proteins, which activate AC

and whose main signalling pathway is PKA (cAMP-dependent protein

kinase A); D2, D3 and D4 receptor subtypes are D2-like and coupled to Gi

protein, which inhibit AC (for a review see, for e.g. Missale et al., 1998).

As mentioned above (1.1), MSNs can be classified as

enkephalinergic and dynorphinergic neurons (Gerfen, 2004), but the

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18037926#bib120


36

differences between these two subpopulations of neurons are not limited

to their peptide expression. The expression of GPCRs, dopamine and

adenosine receptors, is also different: GABAergic enkephalinergic

neurons express predominantly D2 dopamine receptors (and A2A

adenosine receptors) and dynorphinergic neurons express

predominantly D1 dopamine receptors (and A1 adenosine receptors)

(see Figure 1.1.1) (Ferré et al., 1991, 1994, 1997; Fink et al., 1992;

Gerfen, 2004; Schiffmann et al., 1991a; Schiffmann and

Vanderhaeghen, 1993; Svenningsson et al., 1999). Relevant for striatal

pathophysiology are the antagonistic interactions between A1 and D1

receptors and A2A and D2 receptors, which underlie GABAergic

dynorphinergic and enkephalinergic neuronal function (Ferré et al.,

1997, 2005). Furthermore, A2A and A1 receptors form heteromers with D2

and D1 receptors (Canals et al., 2003; Ciruela et al., 2004; Ginés et al.,

2000; Hillion et al., 2002), mainly localized postsynaptically on dendritic

spines of GABAergic enkephalinergic and dynorphinergic neurons,

respectively (Ferré et al., 2005). This tight interaction between dopamine

and adenosine receptors has profound implications in striatal

pathophysiology (see 1.3.4).

The typical neuronal arrangement of the striatum, with

dopaminergic and glutamatergic afferents making synaptic contacts with

the neck and the head of dendritic spines, constitutes a functional unit

which allows dopamine to modulate glutamatergic excitatory inputs

through synaptic transmission to postsynaptic receptors on the MSNs.

However, dopamine also acts through the stimulation of extra-synaptic

receptors. D2/4 receptors are commonly found presynaptically at

glutamatergic (Tarazi et al., 1998a; Bamford et al., 2004b; Brady and

Introduction

37

O´Donnell, 2004) and dopaminergic terminals (Sesack et al., 1994;

Usiello et al., 2000) on enkephalin MSNs, where they provide inhibitory

modulation of glutamatergic inputs (Bamford et al., 2004a; Brady and

O´Donnell, 2004) and act as auto-receptors (Sesack et al., 1994; Usiello

et al., 2000), respectively. One of the mechanisms proposed to be

involved in dopamine release modulation by auto-receptors is their

heteromerization with ionotropic nicotinic acethylcholine receptors

(Quarta et al., 2007).

Besides the negative feedback mechanism of regulation of

dopamine transmission operated by D2 receptor activation, the

transmitter release is also regulated by adenosine A1 receptors (Borycz

et al., 2007) and glutamate receptors presynaptically expressed at the

dopaminergic nerve terminal (see e.g. Quarta et al., 2004 and also

1.2.2).

1.2.2. Glutamatergic neurotransmission

Glutamate is the main excitatory neurotransmitter in the central

nervous system (CNS), where it is involved in synaptic transmission and

excitotoxic neuronal cell death underlying several neurological disorders.

Glutamate-mediated actions depend upon the activation of both

ionotropic and metabotropic receptors. AMPA (alpha-amino-3-hydroxy-

5-methyl-4-isoxazole propionic acid) and NMDA (N-methyl-D-aspartate),

ionotropic receptors mediate rapid excitatory synaptic transmission and

are mainly localized at the postsynaptic density (PSD) (Bernard and

Bolam, 1998) on the MSNs. PSD is a thickening of the postsynaptic

membrane also composed of a protein lattice which mediates changes


38

underlying synaptic plasticity, namely the phosphorylation and

redistribution of AMPA receptors (Lee et al., 2003). As it occurs with

dopamine, glutamate can also activate extrasynaptic receptors at

glutamatergic and dopaminergic synapses (Gracy and Pickel, 1996;

Tarazi et al., 1998b; Paquet and Smith, 2003; Galvan et al., 2006).

Glutamate metabotropic receptors (mGluRs) are GPCRs divided

into three groups - group I (with two subtypes, mGluR1 and mGluR5),

coupled to Gq proteins and mainly located postsynaptically in

glutamatergic terminals (Smith et al., 2000), and groups II and III, Gi/o-

coupled receptors, usually found presynaptically, where they function as

auto-receptors (Schoepp, 2001). Group I metabotropic receptors are

also found at dopaminergic synapses, with mGlu5 receptors localized

peri- and postsynaptically and mGlu1 receptors localized presynaptically

(Paquet and Smith, 2003), modulating dopamine release (Zhang and

Sulzer, 2003).

Besides glutamate receptors, which are expressed at the

glutamatergic nerve terminal and control neurotransmission, there are

several receptors involved in the regulation of glutamate release.

Adenosine A2A receptors are co-localized and exert a synergistic effect

with mGlu5 receptors in the facilitation of glutamate release (Rodrigues

et al., 2005). On the other hand, the same receptors co-localize and

interact negatively with adenosine A1 receptors to modulate glutamate

release (Ciruela et al., 2006; Quarta et al., 2004).

Introduction

39

1.3. Adenosine, a modulator of striatal physiology

1.3.1. Overview as a modulator

Adenosine belongs to a family of compounds - purines - that

have important effects on many biological processes. Ubiquitous in

nature, it is essential to living cells as a component of nucleic acids

(thus, determining the genetic code) and as a sensor/regulator of energy

metabolism, in a way that its formation is closely related to the energy

consumption of each cell (see Dunwiddie and Masino, 2001; Ribeiro et

al., 2003). In 1929, when Drury and Szent-Gyorgyi showed that

adenosine extracted from heart muscle, has pronounced biological

effects, including heart rate and blood pressure decrease, arterial

dilatation and inhibition of intestinal contraction, began an intense

research on purine effects in several biological preparations, including

neurotransmission studies in the peripheral and central nervous system

(Burnstock, 2007). Early studies on the electrically-evoked release of

nucleotides and nucleosides suggested their involvement in intercellular

transmission (Kuroda and McIlwain, 1973; Pull and McIlwain, 1972;

Sulakhe and Phillis, 1975) and, just to mention a couple of examples, it

was shown that adenosine depressed cortical neurons excitability (Phillis

et al., 1974) and the firing of cerebellar Purkinje cells (Kostopoulos et al.,

1975). Since these first observations on the role of adenosine in the

nervous system, several studies report and reinforce adenosine actions

as a modulator of neurotransmission. Besides fast synaptic actions,

there is increasing interest in adenosine long-term (trophic) role, namely

in its synergism with trophic factors. In 1996, Hopker et al. showed that


40

adenosine leads to increased striatal neurite outgrowth in co-culture with

myenteric plexus explants and proposed that it does so either by

triggering the release of a neuritogenic factor or by acting synergistically

with the growth factor. Indeed, another important feature of adenosine is

the ability to control the effect of neurotrophic factors, an action

mediated by adenosine A2A receptors in the peripheral and central

nervous systems (Diógenes et al., 2004, 2007; Mojsilovic-Petrovic et al.,

2006; Pousinha et al., 2006; Potenza et al., 2007; Wiese et al., 2007;

Fontinha et al., 2008; Fernandes et al., 2008). It was observed that

treatment with adenosine is capable of activating neurotrophic factor

receptors (activation of tyrosine kinases), even in the absence of their

ligand (Lee and Chao, 2001).

Adenosine is now widely recognized as key molecule in the

nervous system, where it claims a role of modulator of modulators,

acting through combined presynaptic, postsynaptic and non-synaptic

actions (Sebastião and Ribeiro, 2000).

1.3.2. Pathways of production, metabolism and transport

The effect of adenosine is mediated by specific cell-surface

receptors (see 1.3.3.) with variable affinity for adenosine. Their activation

depends on adenosine concentration, which is mainly regulated by

metabolism and transport across the plasma membrane.

Adenosine is a nucleoside whose formation in the CNS occurs

both intra- and extracellularly by the metabolism of nucleotides through

the activity of 5´-nucleotidases. The adenosine produced intracellularly is

released by bidirectional nucleoside transporters, which regulate

Introduction

41

extracellular adenosine levels. Two major classes of nucleoside

transport systems have been described in mammalian cells: the

concentrative system, independent of nucleoside concentration and the

equilibrative system, which depends on the nucleoside concentration

gradient across the plasma membrane and appears to dominate in the

CNS. Several factors determine the intra- or extracellular origin of

adenosine, namely the neuronal preparation used and the method used

to induce adenosine release (for a review see Latini and Pedata, 2001).

For example, adenosine can be produced postsynaptically by MSNs,

being released by NMDA stimulation (Delaney and Geiger, 1998), or

metabolized by ecto-nucleotidases from ATP (adenosine 5´-

triphosphate) co-released with glutamate, in situations of increased

frequency of impulses arriving at the glutamatergic nerve terminal

(Cunha, 2001). The relevant point is that, as a product of degradation of

nucleotides, which are the basis of intracellular energy transfer (Lipman,

1941), adenosine concentration increases in response to an imbalance

between cellular energy use and supply (see Dunwiddie and Masino,

2001; Ribeiro et al., 2003).

Adenosine is mainly degraded by phosphorylation to AMP by

adenosine kinase (AK) or deamination to inosine by adenosine

deaminase (ADA) after uptake across the cell membrane of neurons or

neighbouring cells. Another possible pathway for adenosine inactivation

is the reversible reaction catalysed by SAH (S-adenosyl homocysteine)

hydrolase, forming SAH from adenosine and L-homocysteine, but this

constitutes only a minor pathway of adenosine metabolism in brain

tissue under basal conditions (Reddington and Pusch, 1983; Latini and

Pedata, 2001). Figure 1.3.1 shows a schematic representation of the


42

pathways of adenosine formation, metabolism and transport.

ADENOSINE

5´-AMP SAH

cAMP

PDE

5´-nucleotidase SAH hydrolase

INOSINE

ADA

ET

5´-AMPADENOSINEecto-5´-nucleotidase

cAMP

AK

ATP

ADP

NUCLEOTIDES

ADENOSINE

5´-AMP SAH

cAMP

PDE

5´-nucleotidase SAH hydrolase

INOSINE

ADA

ET

5´-AMPADENOSINEecto-5´-nucleotidase

cAMP

AK

ATP

ADP

NUCLEOTIDES

Figure 1.3.1 - Pathways of adenosine production, metabolism and transport.

Abbreviations: ADA, adenosine deaminase; AK, adenosine kinase; ET, equilibrative

transporter; PDE, phosphodiesterase; SAH, S-adenosyl homocysteine (adapted from

Latini and Pedata, 2001).

1.3.3. Adenosine receptors - an overview from subtypes and

signalling to distribution, heteromerization and concentration-

dependent function

In 1978 Burnstock proposed the first formal classification of

“purinergic” receptors, with adenosine as the main natural ligand at the

proposed P1 class of receptors. It is now established that adenosine

actions are mediated by four subtypes of GPCRs, A1, A2A, A2B and A3

Introduction

43

(Fredholm et al., 2001a) and its central effects are primarily attributed to

A1 and A2A receptors (Dunwiddie and Masino, 2001). These receptors

can be differentiated based on different pharmacological criteria, namely

the respective signalling pathway: the A1 receptor mediates a decrease

in cAMP associated to its coupling to pertussis toxin-sensitive G proteins

of the Gi and Go family, which inhibit AC (Akbar et al., 1994; Freissmuth

et al., 1991; Jockers et al., 1994), whereas A2A receptors associate with

Gs or Golf proteins, which stimulate AC activity and increase cAMP (cyclic

adenosine 5´-monophosphate) formation (Corvol et al., 2001; Hervé et

al., 2001; Kull et al., 2000; Olah, 1997). A2A receptors are concentrated

in the different parts of the striatum in different mammalian species

(Schiffmann et al., 1991a,b; Schiffmann and Vanderhaeghen, 1993,

Svenningsson et al., 1999; Rosin et al., 1998, 2003) and constitute the

main focus of the present thesis. Figure 1.3.2 shows the preferential

signalling pathway of the adenosine A2A receptor, the cAMP-PKA

(cAMP-dependent protein kinase) pathway. cAMP activates a PKA and

A2A receptor-mediated PKA activation leads to changes that have

implications for neurotransmitter release - PKA phosphorylates different

proteins of the presynaptic machinery involved in vesicular fusion

(Leenders and Sheng, 2005), gene expression - by phosphorylating

CREB (cAMP response element-binding) (Mayr and Montminy, 2001) -

and synaptic plasticity - for instance, by phosphorylating GluR1 subunit

of the postsynaptic AMPA receptor at Ser845 (Wolf et al., 2003).

In addition to this preferential pathway, A2A receptors have also

been shown to signal through PLC (phospholipase C) (Wirkner et al.,

2000), phosphatidylinositol 3-kinase (PI3K) (Lee and Chao, 2001) and

MAPK (mitogen-activated protein kinase) pathways (Ferré et al., 2002;


44

Klinger et al., 2002a,b; Seidel et al., 1999; Sexl et al., 1997).

Neurotransmitter

release

A2AR

Gs/olf

s/olf

A cyclase

cAMP

PKA

MAPKCREB

GluR1

(Ser845)Plasticity

Gene

expression

VDCC

Ca2+

Nucleus

Synaptic

vesicle

PMVF

Neurotransmitter

release

A2AR

Gs/olf

s/olf

A cyclase

cAMP

PKA

MAPKCREB

GluR1

(Ser845)Plasticity

Gene

expression

VDCC

Ca2+

Nucleus

Synaptic

vesicle

PMVF

Figure 1.3.2 - Major signal transduction pathway used by adenosine A2A receptor:

cAMP-PKA cascade. Abbreviations: A cyclase, adenylyl cyclase; PKA, protein kinase A;

MAPK, mitogen-activated protein kinases; CREB, cAMP response element binding

protein; PMVF, protein machinery involved in vesicular fusion; VDCC, voltage-dependent

Ca2+

channel.

In the striatum, A2A receptors are preferentially located both pre-

and postsynaptically at glutamatergic synapses, in a strategic position to

modulate corticostriatal input to the GABAergic enkephalinergic neurons

(Hettinger et al., 2001; Ciruela et al., 2006). A2A receptors

presynaptically expressed (Hettinger et al., 2001; Rosin et al., 2003),

facilitate excitatory glutamatergic (Ciruela et al., 2006; Rodrigues et al.,

2005), inhibitory GABAergic and cholinergic striatal transmission (Jin

and Fredholm, 1997; Kirk and Richardson, 1994). In contrast with what

Introduction

45

happens with glutamate, GABA or acetylcholine neurotransmission, the

release of dopamine from striatal nerve endings is not under the direct

control of adenosine A2A receptors (Jin et al., 1993; Jin and Fredholm,

1997; Lupica et al., 1990), which led to the concept that these receptors

might be absent from striatal dopaminergic nerve endings.

A2A receptors have a particular pattern of cellular and subcellular

distribution at the striatum, exhibiting an efficient interplay between its

own receptors and the receptors for other neurotransmitters,

neuromodulators or both (Sebastião and Ribeiro, 2000). During

decades, in the field of adenosine neuromodulation, A2A receptors were

considered as isolated entities. However, the ability of these receptors to

form heteromers with other GPCRs, such as dopamine and glutamate

receptors and heteromers constituted by different adenosine receptor

subtypes (Ferré et al., 2007a,b) is now established at the anatomic,

biochemical and functional levels. For instance, at the presynaptic site of

the glutamatergic synapse, A2A receptors form heteromeric complexes

with adenosine A1 receptors (Ciruela et al., 2006), which modulate

glutamate release, triggering the functioning of striatal circuits. At the

postsynaptic site, A2A receptors form heteromeric complexes with

dopamine D2 receptors and metabotropic glutamate mGlu5 receptors,

important in the control of MSNs responsiveness and in the regulation of

synaptic plasticity (Ferré et al., 2005).

The activation of a specific adenosine receptor subtype is

determined by the concentration of adenosine, which varies as a

function of pathophysiological conditions and neuronal activity in

general. Under basal conditions, the relatively low extracellular levels of

adenosine preferentially stimulate A1 receptor, which show a higher


46

affinity for adenosine than A2A receptors (Fredholm et al., 2001b). At the

presynaptic level, adenosine A1 receptor stimulation in the A1-A2A

heteromer inhibits glutamatergic neurotransmission (Ciruela et al.,

2006); at the postsynaptic level endogenous dopamine exerts a strong

tonic activation of D2 receptors in the striatum, which impair the ability of

A2A receptors to signal (Kull et al., 1999). Thus, there is a preferential D2

receptor-mediated modulation of the A2A-D2-mGlu5 heteromeric receptor

complex, which inhibits A2A receptor-induced activation of AC (Hillion et

al., 2002) (see Figure 1.3.3A). Under conditions of stronger adenosine

release (see Figure 1.3.3B) is possible to observe a tonic activation of

A2A receptors. At the presynaptic level, A2A receptor activation in the

heteromer would block A1 receptor-mediated function and the overall

result will be a facilitation of glutamate release and, postsynaptically, the

dominant A2A receptor activation in the A2A-D2-mGlu5 receptor heteromer

allows gene expression, protein synthesis and plastic changes

(Schiffmann et al., 2007). In conclusion, A2A receptors, which are

localized pre- and postsnaptically taking part of different heteromeric

complexes, play a key role in the functional changes of glutamatergic

synapses of the enkephalinergic striatal neurons.

Introduction

47

ATP

Glutamate

ATP

A1R A2AR

Adenosine

Glutamate ATP

Dopamine

D2R

Glutamate

ATP

A1R A2AR

Adenosine

Glutamate

Dopamine

D2R

A. B.

mGlu5RmGlu5R

ATP

Glutamate

ATP

A1R A2AR

Adenosine

Glutamate ATP

Dopamine

D2R

Glutamate

ATP

A1R A2AR

Adenosine

Glutamate

Dopamine

D2R

A. B.

mGlu5RmGlu5R

Figure 1.3.3 - Schematic representation of distribution of pre- and postsynaptic

A2A receptor-containing heteromeric receptor complexes in the modulation of

striatal glutamatergic transmission in the GABAergic enkephalinergic neuron. A.

During weak cortico-limbic-thalamic input adenosine favors a decrease in the probability

of glutamate release, due to a preferential stimulation of A1 receptors. At the post-

synaptic site, D2 receptor signalling is favored, reducing neuronal excitability and plastic

changes. B. During strong cortico-limbic-thalamic input, which facilitates A2A and mGlu5

synergistic activation, the probability of glutamate release increases due to inhibition of

A1 receptor signalling. At the post-synaptic site, a synergistic A2A–mGlu5 receptor

interaction counteracts D2 receptor signaling and favors neuronal excitability and

synaptic plasticity (Adapted from Schiffmann et al., 2007).


48

1.3.4. Pathophysiological involvement and therapeutic potential of

A2A receptors in the striatum

The particular cellular localization and assembly of A2A receptors

in specific heteromers (see 1.3.3) support a plethora of actions

influencing the physiology and dysfunction of the striatum, the rationale

for their therapeutic potential in some neurodegenerative diseases.

Parkinson´s disease hallmark is the loss of dopaminergic input to striatal

output neurons, resulting from a progressive degeneration of

nigrostriatal neurons, which determines the enhancement of the

striatopallidal GABAergic signalling and the motor disturbances

characteristic of the disease. The indirect striatal output pathway

appears overactive as a result of removal of inhibitory dopaminergic

tone. After the first studies demonstrating that the administration of

methylxanthines, which have been later recognized as adenosine

receptor antagonists, can exert the same behavioral effects as

dopamine receptor agonists (Fuxe and Ungerstedt, 1974) and the

colocalization of D2 and A2A receptors (Hillion et al., 2002), physically and

biochemically involved in adenosine-dopamine antagonistic interactions

(Ferré et al., 2004), attention was focused on blocking A2A receptors as a

potential mean of control of the motor imbalance in Parkinson´s disease.

Motor dysfunction can also result from the imbalance of the

corticostriatal glutamatergic inputs, as occurs in Huntington´s disease,

which is characterized by the selective death of the MSNs, resulting in

the appearance of generalized involuntary movements, the main

phenotypic alteration of the disease. Since A2A receptors are mainly

localized on the neurons that degenerate early in Huntington´s disease

Introduction

49

and since the receptor (density and distribution) pattern changes in the

early stages of the disease (Glass et al., 2000), they may also be

involved in its pathogenesis and A2A receptor antagonism can be

neuroprotective (Popoli et al., 2007).

The preferential localization of adenosine A2A receptors at pre-

and postsynaptic levels of the corticostriatal glutamatergic synapses also

suggests that A2A receptors may play an important role in the regulation

of synaptic plasticity (Ferré et al., 2005). Presynaptic A2A receptors

localized in glutamatergic nerve terminals modulate striatal glutamate

release (Ciruela et al., 2006) and evidence exists for the involvement of

fluctuations of glutamate release in LTP (long-term potentiation) and

LTD (long-term depression), prolonged changes in synaptic strength

(Ronesi et al., 2005; Schulz, 1997; Sola et al., 2004). Instrumental in the

establishemment of plastic changes in excitatory synapses is the

recruitment of ionotropic glutamate receptors AMPA to the synapse

(Luscher et al., 1999; Malinow and Malenka, 2002; Soderlink and

Derkach, 2000). In the GABAergic dynorphinergic neuron, this

mechanism involves two initial critical steps: D1 receptor-mediated PKA

activation, which induces phosphorylation at Ser 845 of the GluR1

subunit of the AMPA receptor (Wolf et al., 2003) and phosphorylation of

the MAPK-ERK1/2 (Malinow and Malenka, 2002; Thomas and Huganir,

2004). However, this mechanism cannot operate in the GABAergic

enkephalinergic neurons, where dopamine, by acting on D2 receptors,

inhibits the cAMP-PKA cascade and MAPK (Ferré et al., 2004, 2005;

Gerfen et al., 2002). In this case, adenosine, by acting on A2A receptors,

mimics the role of dopamine on D1 receptors.

Both enkephalinergic and dynorphinergic neurons receive


50

corticostriatal afferents and display excitatory postsynaptic responses to

corticostriatal stimulation (Kawaguchi et al., 1990). However, cortical

stimulation selectively activates MAPK in the enkephalinergic neurons

without activation in dynorphinergic neurons (Berretta et al., 1997;

Gerfen et al., 2002), further supporting a central role for A2A receptors in

striatal plastic changes.

1.4. GDNF, another modulation system in the striatum

1.4.1. Brief description and overview as a trophic factor in the CNS

It was some decades ago that Levi-Montalcini and Hamburger

discovered the first growth factor, NGF (nerve growth factor), a protein

required for the survival of certain sympathetic and sensory neurons.

The discovery represents an important milestone in neurobiology and

since then on, several other trophic factors were identified. In 1993, Lin

and coworkers purified and characterized GDNF from the supernatant of

a rat glial cell line as a potent growth factor, as well as survival factor for

embryonic SN dopaminergic neurons. Its trophic effects on

dopaminergic neurons in the adult brain were demonstrated by the

retrograde axonal transport from dopaminergic terminals to the SN

(Tomac et al.,1995b). GDNF also promotes the survival, induces

differentiation and increases the high affinity dopamine uptake of

dopaminergic neurons from SN in vivo (Hudson et al., 1995; Tomac et

al., 1995a) and exhibited the ability to prevent neurotoxic insult-induced

neuronal degeneration (Lapchak et al., 1997a).

In addition to the well described effects of GDNF on

Introduction

51

dopaminergic neurons from the nigra, early studies were also extended

to other neuronal populations, namely to noradrenergic (Arenas et al.,

1995), cholinergic (Lapchak et al., 1997b), Purkinje cells (Mount et al.,

1995) and striatal MSNs (Perez-Navarro et al., 1996). It was also shown

that GDNF exerts a dose-dependent effect on the survival of

motoneurons (Henderson et al., 1994; Oppenheim et al., 1995; Houenou

et al., 1996).

Yet in 1993 (Scharr et al.), GDNF mRNA (messenger ribonucleic

acid) expression was detected both in developmental and neonatal rat

brain areas and it was find that low GDNF expression is maintained

throughout life, particularly in the brain (Stromberg et al.).

GDNF also exerts important roles outside the nervous system.

For instance, it acts as a morphogen in kidney development and GDNF-

deficient mice do not develop kidneys (Moore et al., 1996; Pichel et al.,

1996; Sanchèz et al., 1996). GDNF also regulates the differentiation of

spermatogonia. It is expressed in the developing testes, where it

regulates cell fate of undifferentiated spermatogonial cells (Meng et al.,

2000). However, a steady stream of positive effects of GDNF in the CNS

and, in particular at the nigrostriatal pathway, has sustained its potential

as a therapeutic approach to neurodegenerative diseases, with

Parkinson´s disease receiving much the attention either in pre-clinical

and clinical trials involving the trophic factor.


52

1.4.2. GDNF receptor-complex - characterization, signalling and

distribution

GDNF is the first member of a family of proteins termed GDNF

family ligand (GFL), consisting of GDNF (Lin et al., 1993), neurturin

(Kotzbauer et al., 1996), artemin (Baloh et al., 1998), and persephin

(Mildbrandt et al., 1998). The activation of intracellular signalling

pathways by each ligand requires the presence of two receptor

components on the target cell: a surface receptor, a small protein

anchored by a glycosylphosphatidylinositol (GPI)-link to the outer

plasma membrane, termed GDNF family receptors α-1 to -4; and Ret,

the transmembrane tyrosine kinase receptor (Bespalov and Saarma,

2007). All ligands share the same transmembrane receptor Ret, but

each ligand binds a preferential GFRα surface receptor. However,

crosstalk between the different ligands and receptors has been

described (Baloh et al., 1997; Sanicola et al., 1997; Trupp et al., 1998).

For instance, GDNF binds with high affinity to GFRα1 and with low affinity

to either GFRα2 or GFRα3 (Airaksinen et al., 1999). Neurturin and artemin

may crosstalk weakly with GFRα1, while persephin, on the other hand,

has only been reported binding to GFRα4 (Masure et al., 2000). Durbec

et al. (1996) showed that GDNF is a ligand for the tyrosine kinase

receptor Ret and Treanor et al. (1996) and Jing et al. (1996) showed that

GDNF acts through the GFR1 and further demonstrated that GDNF

promotes the formation of a physical interaction between GFR1 and

Ret, with the subsequent phosphorylation of the latest. The following

signaling model was proposed: first, a homodimeric member of the

GDNF family binds to the corresponding surface receptor GFRα-(1 to 4),

Introduction

53

allowing the subsequent interaction of the GFL/GFRα-(1 to 4) complex

with two molecules of Ret and inducing its homodimerization,

autophosphorylation and further activation of signaling pathways

(Treanor et al., 1996; Trupp et al., 1996). Recently, a novel concept of

GFR-Ret complex activation was proposed, based on the pre-existence

of monomeric receptor subunits, GFR1 and Ret, in dynamic equilibrium

with homodimers, heterodimers or heterotetramers (Figure 1.4.1).

A. B. C.

Active

heterotetramer

Ret

GDNF

GFR1

Signalling

A. B. C.

Active

heterotetramer

Ret

GDNF

GFR1

Signalling

Figure 1.4.1 - Mechanisms of GDNF-GFR1-Ret receptor complex activation. There

are three proposed mechanisms of Ret activation by its ligands. A. GDNF first binds to

GFR1, then engages a second co-receptor, and the GDNF-(GFR1)2 complex recruits

two molecules of Ret. Alternatively, following binding to to GFR1, the first molecule of

Ret is engaged and then additional monomeric GFR1 and Ret are recruited to the

GDNF-GFR1-Ret complex (Schlee et al., 2006). B. GFR1 and Ret are in a preform

GFR1-Ret pair by „crosslinking‟ them. C. The GFR1-Ret complex might exist as a

preformed (GFR1)2(Ret)2 heterotetramer, which acquires a conformational change in

response to GDNF, becoming competent for signalling (Adapted from Bespalov and

Saarma, 2007).


54

Thus, three mechanisms of Ret activation by GDNF are

proposed: (a) GDNF binds to GFR1, a second molecule of GFR1 is

engaged and this complex recruits two molecules of Ret; alternatively,

following binding to GFR1, the first molecule of Ret is engaged and then

additional monomeric GFR1 and Ret are recruited to the GDNF-GFR1-

Ret complex (Schlee et al., 2006); (b) GDNF binds to a preformed

heterodimer consisting of GFR1 and Ret and recruits another GFR1-

Ret pair; (c) GDNF impinges a conformational change to a preformed

heterotetramer composed of two molecules GFR and two molecules of

Ret.

GDNF induces different signaling pathways, both in Ret-

dependent or Ret-independent manners. When acting through the

GFRα1-Ret receptor complex (Trupp et al., 1999; Pezeshki et al., 2001),

these pathways include MAPK-ERK (inducing phosphorylation of CREB)

(Airaksinen et al., 1999). In the absence of Ret, GFRα1 fails to activate

the ERK cascade, but allowed ERK-independent phosphorylation of

CREB (Pezeshki et al., 2001).

There are several studies at the mRNA expression level, both in

the developmental and adult brain pointing out a wide distribution for

both GDNF and its receptors within the striatum. Particularly at the nigral

dopaminergic pathway to the striatum, most studies show a

complementary distribution and expression pattern of GDNF and its

receptor components, with mRNA for GDNF in the striatum and for

GFR1 and Ret in dopaminergic cells in the adult brain (Trupp et al.,

1996; Golden et al., 1998; Sarabi et al., 2001), consistently with the

target-derived nature of GDNF. At the protein level in the adult brain,

Matsuo et al. (2000), found positive neurons for GFR1 widely distributed

Introduction

55

in several brain areas, but these neurons were particularly abundant in

areas such as substantia nigra and Wang and coworkers (2008)

demonstrated the presence of Ret protein in the substantia nigra pars

compacta. Concerning the corticostriatal pathway, attention has been

paid to the location at the target striatal cells, reflecting a particular

interest in GDNF system modulation as an endogenous trophic

response of striatal cells to excitotoxic insults, accordingly to the well

described regulation of GDNF and its receptors expression by excitatory

amino acids (Ho et al., 1995; Marco et al., 2002; Alberch et al., 2002).

Matsuo et al. (2000), also described a high density of positive neurons

for GFR1 protein in the motor cortex.

1.4.3. GDNF - pathophysiological involvement and therapeutic

potential in the striatum

GDNF was originally identified as a potent trophic factor for

cultured midbrain dopaminergic neurons and exerts an essential role in

their development and maintenance (Lin et al., 1993). Later, pronounced

effects on other neuronal subpopulations, namely on the MSNs, the

striatal neuronal population most vulnerable in Huntington´s disease,

were also reported (Pérez-Navarro et al., 1996).

In rodent and primate models of Parkinson's disease involving

selective degeneration of dopaminergic neurons, GDNF has been

shown to be neuroprotective, to promote fiber outgrowth and to improve

motor function when delivered into the cerebral ventricles or directly into

the striatum or substantia nigra (Stanford et al., 2007; Björklund et al.,

1997; Gash et al., 1996; Tomac et al., 1995a), as well as


56

microencapsulated in secreting cells implanted in the striatum

(Grandoso et al., 2007). In a primate model of the disease, it was

recently shown that GDNF delivered by a viral vector is important to

protect and maintain grafted fetal dopamine neurons to depleted areas

(Elsworth et al., 2008). Neural transplantation offers the potential of

treating Parkinson's disease, but clinical studies have produced minor

improvements, mainly because transplanted neurons have low survival

rates, a problem that may be solved by the adequate supply of trophic

factors. It was recently proposed that the impairment of striatal plasticity

after dopamine denervation plays a role in Parkinson's disease

pathology and that viral expression of GDNF may be protective against

this neurobiological deficit (Chen et al., 2008). Results obtained either in

an open clinical trial with intraputamenal infusion of GDNF (Gill et al.,

2003; Patel et al., 2005) as well as with postmortem analysis of a patient

of the same clinical trial (Love et al., 2005) pointed out towards a

therapeutic potential of GDNF in Parkinson's disease. Even after

treatment withdrawn, patients still benefit from GDNF administration

(Slevin et al., 2007). However, an intense debate of the therapeutic

potential of GDNF in this neurodegenerative disease (Barker, 2006;

Kotzbauer and Holtzman, 2006) started recently due to the negative

outcome of a double blind randomized clinical trial also with GDNF

infusion in the putamen (Lang et al., 2006). Furthermore, GDNF

triggered an immune response in subjects receiving recombinant human

glial-derived neurotrophic factor (Tatarewicz et al., 2007). This

controversy clearly pointed out the need of further laboratory studies to

clarify GDNF actions on dopaminergic neurons.

In addition to its potent effects on dopaminergic midbrain

Introduction

57

neurons, GDNF has also been shown to protect striatal MSNs (Perez-

Navarro et al., 1996), the neuronal population most affected in

Huntington´s disease. Excitotoxicity has been related with the

pathogenesis of the disorder (Estrada-Sanchez et al., 2008) and up-

regulation of GDNF and its receptors was observed after excitotoxic

insults to the striatum (Alberch et al., 2002). In rodent models of

Huntington´s disease, GDNF given intraventricularly (Araújo et al., 1997)

or overexpressed by transplanted cells (Pérez-Navarro et al., 1996;

1999) had been shown to be neuroprotective in behavioral and

neurochemical parameters deficits characterizing the disease. The

referred GDNF actions can be related to its ability to regulate

neurotransmitter release in the striatum. If there is a large body of

evidence showing the ability of GDNF to mediate dopaminergic

transmission, its role upon glutamate release was not studied and a

direct evaluation of GDNF actions on isolated glutamatergic nerve

endings has not yet been reported so far.

1.5. Aim

The main goal of the present work was to explore the ability of

adenosine A2A receptors to modulate striatal physiology by regulating

GDNF effects upon the main inputs - dopaminergic and glutamatergic -

to the striatum, as well as to reinforce this functional information with

morphological data on the distribution, localization and colocalization of

the receptors of both modulation systems, GDNF and adenosine. Thus,

the specific objectives of the work are:


58

1. Investigation of the presynaptic role of GDNF in the release of

dopamine and glutamate from the rat striatum.

2. Investigation of adenosine A2A receptor modulation of GDNF effects.

3. Study of GDNF receptor components distribution among the main

striatal nerve terminals.

4. Analysis of adenosine A2A receptor and GDNF receptor components

colocalization at striatal dopaminergic and glutamatergic nerve endings.

The functional interaction between adenosine A2A receptors and

GDNF (points 1 and 2) was studied using in vitro and in vivo

approaches. In order to clarify the presynaptic nature of the A2A

receptor-mediated effect of GDNF upon neurotransmitter release, in vitro

studies were carried out by using striatal purified nerve terminals (or

synaptosomes) and slices. Additionally, it was considered relevant to

verify if the cortical stimulation, which triggers striatal circuitry, is able to

recruit GDNF signalling and to investigate the influence of adenosine A2A

receptors upon this in vivo measurable physiological parameter - GDNF

Ret receptor phosphorylation. Observations on the distribution of GDNF

among different striatal terminals, their synaptic and subsynaptic

localization and colocalization with adenosine A2A receptors (points 3

and 4) were carried out by performing western blot and

immunocytochemical analysis.

MA

TE

RIA

LS

AN

D M

ET

HO

DS

Material and Methods

61

2. MATERIALS AND METHODS

2.1. Animal handling and biological research preparations

2.1.1. Animals used

Adult (3-4 to 6-8 weeks-old) rats (Wistar or Sprague-Dawley)

were purchased from Harlam Interfauna Iberica, SL (Barcelona, Spain)

or Charles River Laboratory (Wilmington, MA). The animals were

handled according to the European guidelines, the Portuguese law and

the guidelines of the Institutional Care and Use Committee of the

Intramural Research Program, National Institute on Drug Abuse, NIH. In

the in vitro studies, the animals were deeply anesthetized (no skin

pinched reaction while still breathing) with halothane before decapitation.

In the in vivo experiments, animals were kept anesthetized with

equithesin during all surgical procedures.

2.1.2. Dissection and preparation of striatal synaptosomes and

slices for neurotransmitter release experiments

The striatum was dissected free within ice-cold Krebs solution

containing pargyline (10 μM), composed of (mM): NaCl 124; KCl 3;

NaH2PO4 1.25; NaHCO3 26; MgSO4 1; CaCl2 2; and glucose 10,

previously gassed with 95% O2 and 5% CO2, pH 7.4. A synaptosomal

fraction of the striatum was prepared with slight modifications of the

technique described elsewhere (Kirk and Richardson, 1994). The

removed striata were homogenized in ice-cold 0.32 M sucrose solution


62

(containing 1 mM EDTA, 1 mg/ml BSA (bovine serum albumin) and 10

mM N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid (HEPES), pH

7.4) at 4°C, and centrifuged at 3000 g for 10 min. The supernatant was

centrifuged at 14000 g for 12 min. The pellet was resuspended in ice-

cold 45% (v/v) Percoll in Krebs-HEPES-Ringer (KHR) solution (NaCl 140

mM, EDTA 1 mM, HEPES 10 mM, KCl 5 mM, glucose 5 mM, pH 7.4)

and centrifuged at 14000 g for 2 min to eliminate free mitochondria and

glial debris. The top layer was washed twice at 14000 g for 2 min in KHR

solution at 4°C. When striatal slices were used, these were cut (400 μm

thick) perpendicularly to the longer axis of the striatum with a McIlwain

tissue chopper and allowed to recover functionally and energetically for

at least 1 h in a resting chamber, filled with the same solution, at room

temperature (22-25°C) (Figure 2.1.1).

A.

C.

B.

A.

C.

B.

Figure 2.1.1 - Preparation of striatal slices for neurotransmitter release

experiments. A. Photograph of a rat brain. B. Photograph of a McIlwain tissue chopper.

C. Photograph of a resting chamber for brain slices.


63

2.1.3. Preparation of striatal punches for western blot analysis

After the cortical stimulation procedure (see 2.2.2) the brains

were rapidly extracted, frozen in dry ice-cold isopentane, and stored at -

80°C. Subsequently, unilateral tissue punches of the lateral striatum (16

gauge) at the AP (anterioposterior) level of bregma 0.0 were obtained

from 1 mm-thick coronal sections cut in the cryostat at -20°C. The rostral

side of the coronal sections was localized approximately at bregma 0.5

mm and the caudal side at bregma -0.5 mm (Figure 2.1.2). The tissue

punches were sonicated for 10-15 s in homogenization buffer (200 mL

1% sodium dodecyl sulphate (SDS) in deionized ultrapure sterile water)

to subsequent western blot analysis.

Figure 2.1.2 - Striatal punches extraction site. Schematic drawing based on the atlas

Paxinos and Watson (2004).


64

2.1.4. Preparation of striatal slices for immunochemical analysis

Immediately after the offset of cortical stimulation (see 2.2.2), rats

were deeply anesthetized with equithesin and perfused transcardially

with 0.1 M PBS (phosphate buffer saline), followed by 4% formaldehyde

in 0.1 M sodium phosphate monobasic buffer, pH 7.4. Brains were

postfixed in the same fixative for 2 h and immersed in 20% sucrose/0.1M

sodium phosphate, pH 7.4, solution for 48h at 4°C. 20 m-thick coronal

sections were cut at the AP level of bregma 0.0 1.0 mm in a Leica

(Nussloch, Germany) CM3050S cryostat at -20°C, collected in PBS, and

stored in anti-freeze-buffered solution (20% ethylene glycol, 10%

glycerol, and 10% sucrose in PBS) at -80°C until processing (see 2.2.3).

2.1.5. Preparation of total membranes and synaptosomes for

synaptic localization studies of Ret and GFRα1

To evaluate the synaptic localization of GDNF receptors, the

density of the two proteins was compared in total membranes from the

whole striatum and in membranes from purified striatal nerve terminals,

a strategy previously used to allocate receptors to nerve terminals

(Rebola et al., 2005). Total membranes from the striatum and Percoll-

purified striatal synaptosomes were prepared as in previous reports (e.g.

Rebola et al., 2005). Briefly, the two striata from one rat were

homogenized at 4°C in sucrose solution (0.32 M) containing 50 mM Tris

(tris(hydroxymethylaminoethane)-HCl, 2 mM EGTA (ethylene glycol

tetraacetic acid) and 1 mM dithiothreitol, pH 7.6. The resulting

homogenates were centrifuged at 3000 g for 10 min at 4°C, the


65

supernatants collected and centrifuged at 14000 g for 20 min at 4°C.

The pellet of one of the samples was taken as the total membrane

fraction and was resuspended in SDS-PAGE (sodium dodecyl sulphate

polyacrylamide gel electrophoresis) buffer for Western blot analysis (see

2.2.5). The pellet of the other sample was resuspended in 1 ml of a 45%

(v/v) Percoll solution made up in a Krebs solution (composition 140 mM

NaCl, 5 mM KCl, 25 mM HEPES, 1 mM EDTA (ethylenediamine

tetraacetic acid) 10 mM glucose, pH 7.4). After centrifugation at 14000 g

for 2 min at 4ºC, the top layer was removed (synaptosomal fraction),

washed in 1 ml Krebs solution and resuspended in 10 ml of incubation

buffer. This mixture was centrifuged at 14000 g for 20 min at 4ºC and the

pellet corresponded to the synaptosomes. The membranes were

obtained by resuspension in SDS-PAGE buffer for Western blot analysis

(see 2.2.5).

2.1.6. Subcellular fractionation of nerve terminals for subsynaptic

localization studies of Ret and GFRα1

The extrasynaptic, presynaptic active zone and postsynaptic

fractions from rat striatal synaptosomes were separated as in previous

reports (Rodrigues et al., 2005). It was previously confirmed that this

subsynaptic fractionation method allows an over 90% effective

separation of active zone (synaptosome-associated protein of 25000

daltons, SNAP25), postsynaptic density (PSD95) and non-active zone

fraction (synaptophysin) markers and can be used to access the

subsynaptic distribution of receptors (see Rodrigues et al., 2005). Briefly,

striata from 10 rats were homogenized at 4ºC in 15 ml of isolation


66

solution (0.32 M sucrose, 0.1 mM CaCl2, 1 mM MgCl2, 0.1 mM

phenylmethylsulfonylfluoride, PMSF). The concentration of sucrose was

raised to 1.25 M by addition of 75 ml of 2 M sucrose and 30 ml of 0.1

mM CaCl2 and the suspension divided into 10 ultracentrifuge tubes. The

homogenate was overlaid with 8 ml 1.0 M sucrose, 0.1 mM CaCl2 and

with 5 ml of homogenization solution and centrifuged at 100000 g for 3 h

at 4ºC. Synaptosomes were collected at the 1.25/1.0 M sucrose

interface, diluted 1:10 in cold 0.32 M sucrose with 0.1 mM CaCl2 and

pelleted (15000 g for 30 min at 4ºC). Pellets were resuspended in 1 ml of

0.32 M sucrose with 0.1 mM CaCl2 and a small sample taken for gel

electrophoresis. Then, synaptosomal suspension was diluted 1:10 in

cold 0.1 mM CaCl2 and an equal volume of 2x solubilization buffer (2%

Triton X-100, 40 mM Tris, pH 6.0) was added to the suspension. The

membranes were incubated for 30 min on ice with mild agitation and the

insoluble material (synaptic junctions) pelleted (40000 g for 30 min at

4ºC). The supernatant (extrasynaptic fraction) was decanted and

proteins precipitated with 6 volumes of acetone at 20ºC and recovered

by centrifugation (18000 g for 30 min at 15ºC). The synaptic junctions

pellet was washed in pH 6.0 solubilization buffer, resuspended in 10 ml

of 1% Triton X-100 and 20 mM Tris (pH 8.0), incubated for 30 min on ice

with mild agitation, centrifuged (40000 g for 30 min at 4ºC) and the

supernatant (presynaptic active zone fraction) processed as above.

PMSF (1 mM) was added to the suspension in all extraction steps. The

pellets from the supernatants and the final insoluble pellet (postsynaptic

density fraction) were solubilized in 5% SDS, the protein concentration

determined and the samples added to a 1/6 volume of 6x SDS-PAGE

sample buffer for Western blot analysis (see 2.2.5).


67

2.1.7. Preparation of striatal nerve terminals for localization and

colocalization studies

The double or triple labelling immunocytochemical analysis to

quantify the localization of Ret and GFR1 receptors in striatal nerve

terminals or their co-localization with A2A receptor was performed

essentially as in previous reports (e.g. Rodrigues et al., 2005). Briefly,

striatal synaptosomes were obtained through a discontinuous Percoll

gradient. This procedure for preparation of the synaptosomes is crucial

to reduce the amount of postsynaptic density material. In fact,

immunocytochemical analysis of the synaptosomes obtained with this

discontinuous Percoll gradient showed that less than 1% of the

synaptophysin-positive elements were labelled by an anti-PSD95

antibody (Rebola et al., 2005). Briefly, striatal tissue was homogenized

in a medium containing 0.25 M sucrose and 5 mM TES (pH 7.4). The

homogenate was spun for 3 min 2000 g at 4ºC and the supernatant

spun again at 9500 g for 13 min. Then, the pellets were re-suspended in

8 mL of 0.25 M sucrose and 5 mM TES (pH 7.4) and 2 mL were placed

onto 3 mL of Percoll discontinuous gradients containing 0.32 M sucrose,

1 mM EDTA, 0.25 mM dithiothreitol and 3, 10, or 23% Percoll, pH 7.4.

The gradients were centrifuged at 25000 g for 11 min at 4ºC. The

synaptosomes were collected between the 10 and 23% Percoll bands,

they were washed in 15 mL of HEPES buffered medium (140 mM NaCl,

5 mM KCl, 5 mM NaHCO3, 1.2 mM NaH2PO4, 1 mM MgCl2, 10 mM

glucose, and 10 mM HEPES, pH 7.4) and recovered by centrifugation at

22000 g for 11 min at 4ºC.


68

2.2. Experimental procedures

2.2.1. Neurotransmitter release from striatal synaptosomes and

slices

The release of exogenous, radiolabelled neurotransmitters,

dopamine and glutamate, was monitored by a continuous superfusion

technique. Purified nerve terminals (or synaptosomes) are the most

adequate in vitro experimental model to study presynaptic phenomena,

since they enable the direct evaluation of nerve terminal function (Raiteri

and Raiteri, 2000). Some experiments were also performed in striatal

slices, which are more complex biological preparations, but are also

adequate to the study of presynaptic events taking place in the

dopaminergic nerve terminals from neurons with cell bodies localized in

the nigra, therefore absent from the striatum. Slices were used for

comparison with isolated nerve endings, since they retain local

anatomical integrity, allowing additional modulation of

neurotransmission.

[3H]dopamine release from synaptosomes was performed

according to previous reports with slight modifications (Kirk and

Richardson, 1994). The synaptosomes were diluted and equilibrated

with careful oxygenation (95% O2, 5%CO2) at 37°C for 5 min, after which

5 μCi of [3H]dopamine (Amersham Biosciences) was added to

synaptosomes for 20 min (loading period). Preloaded synaptosomes

were transferred into four parallel 900 μl perfusion chambers, kept

layered over Whatman® GF/C filters and superfused continuously at a

rate of 0.8 ml/min at 37°C until the end of the experiment. After


69

termination of the 30 min washout period, 2 min samples were

continuously collected for liquid scintillation assay. The release of

[3H]dopamine was stimulated by perfusing 20 mM KCl (isomolar

substitution of Na+ by K+ in the buffer) for 2 min, at 6 min (S1) and 24 min

(S2) after starting sample collection.

The release of [3H]glutamate from rat striatal nerve terminals

prepared using a combined sucrose/Percoll centrifugation protocol was

performed according to previous reports (see e.g. Rodrigues et al.,

2005). The nerve terminals were equilibrated at 37ºC for 10 min, loaded

with 0.2 µM [3H]glutamate for 5 min at 37ºC, washed, layered over

Whatman GF/C filters and superfused (flow rate: 0.8 ml/min) with Krebs

solution for 20 min before starting collection of the superfusate. The

synaptosomes were stimulated with 20 mM KCl (isomolar substitution of

NaCl by KCl in the Krebs solution) at 3 and 9 min after starting sample

collection (S1 and S2). GDNF was added before S2 onwards and its

effect quantified by the modification of S2/S1 ratio versus control (i.e.

absence of GDNF). When testing the action of GDNF in the presence of

other drugs (CGS 21680, SCH 58261), these drugs were added to the

perfusion 15 min before starting sample collection and remained in the

bath up to the end of the experiment; hence, they were present both in

S1 and S2, and neither drugs modified the S2/S1 ratio versus control.


70

S1 S2

0 5 10 15 20 25 30 35 40

Time (min)

GDNF (10 ng/ml)

S1 S2

0 5 10 15 20 25 30 35 40

Time (min)

Control Test

A.

B.

C.

S1 S2

0 5 10 15 20 25 30 35 40

Time (min)

GDNF (10 ng/ml)

S1 S2

0 5 10 15 20 25 30 35 40

Time (min)

Control TestControlControl TestTest

A.

B.

C.

Figure 2.2.1 - [3H]neurotransmitter release from striatal synaptosomes. A.

Photograph of the experimental setup (a cortesy by Sandra H. Vaz). B. Schematic

representation of the superfusion setup (chambers and collection system). C. Averaged

time course of [3H] dopamine release experiments. Striatal synaptosomes were labelled

with [3H]dopamine as described in Experimental procedures. Release was evoked twice,

S1 and S2, by chemical stimulation (20 mM KCl, 2 min). GDNF was added to the test

chambers before S2, as indicated by the horizontal bar (filled circles), whereas it was not

added to the parallel control chambers (open circles). Time in the abscissa represents

ending of collecting period for each sample.


71

[3H]dopamine release from striatal slices was assessed as

previously (Milusheva et al., 1996) with slight modifications. Striatal

slices prelabeled with [3H]dopamine (5 μCi for a 30 min incubation

period) were placed into 4 superfusion chambers (1 slice per chamber)

and superfused (0.6 ml/min) with Krebs solution for 1 hr (washing

period) before the collection of 1.8 ml (3 min) fractions was begun. Two

electrical stimulation periods (supramaximal 2ms rectangular pulses at 2

Hz, for 2 min), were delivered at 9 min (S1) and 42 min (S2) after starting

sample collection. Whenever testing the effect of GDNF or of any other

drug on dopamine release, these test drugs were only added before S2

(at 27 min) after starting sample collection and remained in the bath up

to the end of the experiment. When testing the action of GDNF in the

presence of other drugs (CGS 21680, SCH 58261, CGP 55845 and

bicuculline), these drugs were added to the perfusion 15 min before

starting sample collection and remained in the bath up to the end of the

experiment; therefore, they were present both in S1 and S2. The

radioactivity released from the preparations was measured with a

Packard 1600 CA liquid scintillation spectrometer and expressed in

terms of disintegrations per second per milligram of protein (Bq/mg) in

each chamber. In order to determine the amount of tritium retained by

synaptosomes and slices, the filters and the homogenized slices (in Tris-

HCl 50 mM with MgCl2 10 mM, pH 7.4) were also kept for scintillation

counting. The amount of [3H]neurotransmitter release in each sample

was expressed as a percentage of the total radioactivity present in the

preparation at the point of sample collection (fractional release). The

amount of radioactivity released by stimulation was calculated by

integration of the area under the peak of evoked tritium release upon


72

subtraction of basal tritium release. Effects (taken as % change) were

evaluated by modification of the S2/S1 ratios in test (GDNF added before

S2) and control (no GDNF added) conditions.

Values are presented as mean ± SEM from n observations, and

the significance of the means was calculated by the Student´s t test.

When comparing the effect of GDNF in more than two experimental

conditions, one way analysis of variance (ANOVA) followed by

Bonferroni´s test was used; p values of 0.05 or less were considered to

represent significant differences. Whenever a statistically significant

difference between the effects of GDNF under two different experimental

conditions was found, the difference was confirmed by comparing the

effect of GDNF under those experimental conditions in synaptosomes

from the same animal.


73

S1 S2

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

S1 S2

0 5 10 15 20 25 30 35 40 45 50 55 60 65

GDNF (10 ng/ml)

Time (min)

A. B.

S1 S2

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

S1 S2

0 5 10 15 20 25 30 35 40 45 50 55 60 65

GDNF (10 ng/ml)

Time (min)

A. B.

S1 S2

0 5 10 15 20 25 30 35 40 45 50 55 60 65

GDNF (10 ng/ml)

Time (min)

A. B.

Figure 2.2.2 - [3H]dopamine release from striatal slices. A. Photograph of the

superfusion chambers for brain slices. B. Averaged time course of [3H] dopamine

release experiments. Striatal slices were labelled with [3H]dopamine as described in

Experimental procedures. Release was evoked twice, S1 and S2, by electrical stimulation

(supramaximal, 2ms pulses, 2 Hz, 2 min). GDNF was added to the test chambers before

S2, as indicated by the horizontal bar (filled circles), whereas it was not added to the

parallel control chambers (open circles). Time in the abscissa represents ending of

collecting period for each sample.


74

2.2.2. Electrode implantation and in vivo electrical cortical

stimulation

The animals were implanted unilaterally under Equithesin (NIDA

Pharmacy, Baltimore, MD) anesthesia with bipolar stainless steel

electrodes, 0.15 mm in diameter (Plastics One, Roanoke, VA) into the

orofacial area of the lateral agranular motor cortex (3 mm anterior, 3 mm

lateral and 4.2 mm below to bregma) (Figure 2.2.3). The electrodes and

a head holder (connected to a swivel during stimulation) were fixed on

the skull with stainless steel screws and dental acrylic resin.

Five days after surgery, rats were placed in individual bowl

chambers and the implanted electrodes were attached to an electrical

stimulator (Grass S88K; Grass Instruments Co., W. Warwick, RI). Ten

min before cortical stimulation the animals were given an i.p. (intra-

peritoneal) administration of saline, A2A receptor antagonists, MSX-3 (3

mg/kg) or caffeine (10 mg/kg) or A1 receptor antagonist, CPT (4.8

mg/kg) in a volume of 3 ml/kg of body weight. After 10 min of

habituation, biphasic current pulse trains (pulse 0.1 msec; 150-200 µA,

100 Hz, 160 msec trains repeating once per second) were delivered

using two-coupled constant current units (Grass PSIU6; Grass

Instruments Co.). The intensity was 150 µA for most cases or it was

increased up to 200 µA, until small jaw movements were observed. The

cases that failed to show visible somatic movements were excluded from

additional analysis. In no case did animals display evidence of seizure

activity from the electrical stimulation. Stimulation was applied for 20 min

and the animals were killed immediately after the stimulation offset. The

brains were rapidly extracted, frozen in dry ice-cold isopentane, and


75

stored at -80°C. Subsequently, unilateral tissue punches of the lateral

striatum were obtained from coronal sections and stored at -80°C until

their use in western blot analysis.

In order to verify the correct implantation of electrodes, one slice

per animal was stained using the cresyl violet technique. Briefly,

perfused and fixed slices were mounted on a slide, rehydrated (5 min in

distilled water) and exposed to increasing concentrations of alcohol (50,

70, 90 and 100%, 5 min each), then to decreasing concentrations of

alcohol (100, 90, 70, 50% and water, 5 min each) and finally to the stain

cresyl violet solution (5-10 min). The slices were then dehydrated in

increasing concentrations of alcohol (50, 70, 90 and 100%, 5 min each)

and exposed to xylene (5 min). Using the shaft of a tip, a small drop of

mounting media was applied and a coverslip was slowly lowered onto

the slide, which was allowed to dry sufficiently (air dry, at least 24 hs).

A.

B.

A.

B.

Figure 2.2.3 - Electrode implantation site. A. Schematic drawing based on the atlas

Praxinos and Watson (2004). B. Cresyl violet staining of a brain slice to confirm the

correct implantation of the electrode.


76

2.2.3. Immunocytochemical analysis of striatal slices

The slices (stored at -80ºC) collected after the cortical stimulation

protocol were rinsed with PBS, incubated with 100 mM sodium

borohydride and 0.1% hydrogen peroxide in PBS for 10 min, rinsed, and

incubated in blocking buffer (PBS/0.1% Triton X-100/5% BSA) for 2 h

before incubation with rabbit polyclonal anti-phospho-Thr202/Tyr204

ERK1/2 (1:2000 dilution; Cell Signaling Technology, Danvers, MA) in

blocking buffer for 24 h at 4°C. Sections were washed and incubated in

1:200 biotinylated goat anti-rabbit antibody (Vector Laboratories,

Burlingame, CA) for 2 h, then washed and incubated for 2 h in ABC

reagent (PK-6100; Vector Laboratories), washed again, treated with 0.33

mg/ml 3,3´-diaminobenzidine, 50 mM ammonium nickel sulfate, and

0.003% peroxide until development (5-10 min), transferred to PBS,

mounted onto chrom-alum gelatin-coated slides, air-dried, dehydrated

through graded ethanols, cleared in xylene, and coverslipped with DPX

(mixture of xylene and dibutyl phthalate; Fisher Scientific, Tustin, CA).

Images were acquired using a digital camera (HRC; Zeiss, Gottingen,

Germany) coupled to an Axioimager A1 microscope (Zeiss).

2.2.4. Immunocytochemical analysis of striatal nerve terminals

The striatal synaptosomes were placed onto coverslips

previously coated with poly-L-lysine, fixed with 4% paraformaldehyde for

15 min and washed twice with PBS medium (140 mM NaCl, 3 mM KCl,

20 mM NaH2PO4, 15 mM KH2PO4, pH 7.4). The synaptosomes were

permeabilized in PBS with 0.2% Triton X-100 for 10 min and then


77

blocked for 1 h in PBS with 3% BSA and 5% normal rat serum. The

synaptosomes were then washed twice with PBS and incubated for 1 h

at room temperature with different mixtures of primary antibodies,

namely: rabbit anti-Ret (1:100) or rabbit anti-GFR1 (1:100) and mouse

anti-synaptophysin (1:200) antibodies; anti-Ret or anti-GFR1 and either

guinea-pig anti-vesicular glutamate transporter type 1 (vGluT1, 1:1000),

anti-vGluT2 (1:1000), mouse anti-tyrosine hydroxylase (TH, 1:500),

guinea-pig anti-vesicular GABA transporter (vGAT, 1:1000) or guinea-

pig anti-vesicular acetylcholine transporter (vAChT, 1:500) antibodies;

anti-Ret or anti-GFR1 together with goat anti-A2A receptor antibody

(1:200) and either anti-vGluT1 and 2 or anti-TH antibodies; anti-A2A

receptor antibody and either anti-TH or rat anti-dopamine transporter

(DAT, 1:500) antibody. For the double immunocytochemical labelling,

the synaptosomes were then washed three times with PBS with 3% BSA

and incubated for 1 h at room temperature with AlexaFluor-598 (red)-

labelled donkey anti-guinea pig or anti-rat or anti-goat or anti-mouse or

anti-rat IgG antibodies (1:200 for each), carefully washed with PBS and

then incubated for 1 h at room temperature with AlexaFluor-488 (green)-

labelled donkey anti-rabbit or anti-goat IgG antibodies (1:200 for each).

For the triple immunocytochemical labelling, the synaptosomes were first

incubated with AlexaFluor-488-labelled donkey anti-goat antibody

(1:200) and then with a mixture of AlexaFluor-598-labelled donkey anti-

mouse or anti-rat or anti-guinea-pig and AlxaFluor-350 (blue)-labelled

goat anti-rabbit antibodies (1:200 for each), to avoid recognition of the

goat anti-rabbit by the donkey anti-goat antibody (see Rodrigues et al.,

2005). It was confirmed that none of the secondary antibodies produced

any signal in preparations to which the addition of the corresponding


78

primary antibody was omitted. Most importantly, it was confirmed that

the individual signals in double-labelled fields are not enhanced over the

signals under single-labelling conditions. After washing and mounting on

slides with Prolong Antifade, the preparations were visualized in a Zeiss

Axiovert 200 inverted fluorescence microscope equipped with a cooled

CCD camera and analyzed with MetaFluor 4.0 software. Each coverslip

(3 to 4 per experiment) was analyzed by counting three different fields

and in each field a minimum of 50 individualized elements. The values

are presented as mean ± SEM of n experiments (i.e. in preparation

obtained from different rats).

2.2.5. Western blot analysis

After determining the amount of protein, each sample was diluted

with 5 volumes of SDS-PAGE buffer containing 30% (v/v) glycerol, 0.6 M

dithiothreitol, 10% (w/v) SDS and 375 mM Tris-HCl pH 6.8, boiled at 95

ºC for 5 min. These diluted samples and the pre-stained molecular

weight markers (Amersham, GE Healthcare, Lisbon, Portugal) were

separated by SDS-PAGE (10% with a 4% concentrating gel) under

reducing conditions and electro-transferred to polyvinylidene difluoride

membranes (0.45 µm, from Amersham) in the absence or presence (for

phosphorylated proteins) of 2% polyvinylpyrrolidone (PVP) and 50 mM

sodium fluoride. After blocking for 2 h at room temperature with 5% milk

in Tris-buffered saline, pH 7.6 containing 0.1% Tween 20 (TBS-T), the

membranes were incubated overnight at 4ºC with the different

antibodies, namely: rabbit anti Ret (1:500), rabbit anti-GFR1 (1:100),

rabbit anti-phosphorylated (Ser696) Ret (1:1000), mouse anti-SNAP25


79

(1:20,000), mouse anti-PSD95 (1:100,000) or mouse anti-synaptophysin

(1:20,000). After four washing periods for 10 min with TBS-T containing

0.5% milk, the membranes were incubated with either alkaline

phosphatase-conjugated anti-rabbit (1:20,000) or anti-mouse secondary

antibody (1:10,000) or horseradish peroxidase-conjugated secondary

goat anti-rabbit antibody in TBS-T containing 1% milk during 90 min at

room temperature. After five 10 min-washes in TBS-T with 0.5% milk the

membranes were incubated with Enhanced Chemi-Fluorescence during

five minutes or with Enhanced Chemi-Luminescence (Amersham

Biosciences, Piscataway, NJ) before being analysed with a VersaDoc

3000 (Biorad, Hercules, CA). The membranes were always re-probed to

confirm the amount of loaded protein by measuring the immunoreactivity

against α-tubulin. Briefly, the membranes were first incubated for 30 min

with 40% methanol, then for 1 hour at room temperature with a 0.1 M

glycine (pH 7.2) solution and then blocked as previously described

before incubation with mouse anti-β-actin antibody (1:20,000 dilution).

The membranes were then washed and incubated with the secondary

antibody as described. Statistical differences were analyzed by one-way

ANOVA followed by Dunnett‟s multiple comparisons test.

2.3. Reagents, drugs, antibodies and solutions

7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-

triazolol[1,5-c]pyrimidine (SCH 58261), was from Sigma (St. Louis, MO)

o a generous from S.Weiss (Vernalis, UK). 2-[p-(2-

carboxyethyl)phenethylamino]-5′-N-ethylcarboxamido adenosine (CGS

21680), 8-cyclopentyl-1,3-dimethylxanthine (CPT), 3,7-dihydro-8-[(1E)-2-


80

(3-ethoxyphenyl)ethenyl]-7-methyl-3-[3-(phosphooxy)propyl-1-(2-

propynil)-1H-purine-2,6-dione (MSX-3) and caffeine (anhydrate base)

were from Sigma (St. Louis, MO). Glial cell linederived neurotrophic

factor (GDNF) was from Promega (Madison, WI) and [3H]dopamine

(specific activity 8.80 Ci/mmol) and [3H]glutamate (specific activity 50

Ci/mmol) were from Amersham (GE Healthcare, Lisbon, Portugal or

Buckinghamshire, UK). 3-N[1-(S)-(3,4-dichlorophenyl) ethyl]amino-2-(S)-

hydroxypropyl-P-benzyl-phosphinic acid (CGP 55845) and bicuculline

were from Tocris.

CGS 21680 and SCH 58261 were made up in 5 and 10 mM

stock solutions, respectively, in DMSO. GDNF stock solution was

prepared in distilled water, at a final concentration of 0.1 mg/ml. CGP

55845 and bicuculline were made up in 10 and 100 mM stock solutions,

respectively, in DMSO. All aliquots of stock solutions were kept frozen at

−20 °C until use, except SCH 58261 aliquots, which were kept at 4 °C

until use. MSX-3, CPT and caffeine were dissolved in sterile saline (with

a few drops of 0.1 N NaOH for MSX-3; final pH 7.4).

The antibodies used were as follows: goat anti-adenosine A2A

receptor, rabbit anti-GFR1 and rabbit anti-Ret antibodies (Santa Cruz

Biotechnology, Santa Cruz CA), rabbit anti-phosphorylated Ret (at

serine 696) antibody (Novus Biologicals, Litlleton CO), guinea-pig anti-

vesicular glutamate transporters type 1 and type 2 (vGluT1,2), guinea-

pig anti-vesicular acetylcholine transporter (vAChT), mouse anti-tyrosine

hydroxilase (TH), rat anti-dopamine transporter (DAT) and mouse anti-

PSD95 antibodies (Chemicon Europe, Southampton, UK), guinea-pig

anti-vesicular GABA transporter (vGAT) antibody (Calbiochem,

Darmstadt, Germany), mouse anti-SNAP25, mouse anti-synaptophysin


81

and mouse anti-β-actin (Sigma-Aldrich Ibérica, Sintra, Portugal), alkaline

phosphatase-labelled goat anti-rabbit or anti-mouse antibodies

(Amersham, GE Healthcare, Lisbon, Portugal), AlexaFluor-598-labelled

donkey anti-guinea pig, anti-rat, anti-goat and anti-mouse, AlexaFluor-

488-labelled donkey anti-rabbit or anti-goat and AlexaFluor-350-labelled

goat anti-rabbit antibodies (Molecular Probes, Alfagene, Lisbon,

Portugal).

RE

SU

LT

S

Results

85

3. RESULTS

3.1. Adenosine A2A receptor modulation of GDNF effect upon

the dopaminergic input to the striatum

3.1.1. Rationale of the study

Evidence has been accumulating that supports a facilitatory role

for GDNF in striatal dopamine release in vivo (Grondin et al., 2003;

Hebert and Gerhardt, 1997; Smith et al., 2005). This neurotrophic factor

also exerts an acute effect on the function of midbrain dopaminergic

neurons in culture, rapidly and reversibly inhibiting A-type potassium

channels, which leads to an increase of their excitability (Yang et al.,

2001). These GDNF actions can secondarily cause an increase in

dopamine release in the striatum, but a direct evaluation of GDNF

actions on isolated dopaminergic nerve endings has not yet been

reported so far. On the other hand, it is well established that adenosine

A2A receptors modulate striatal function. A2A receptors are highly

expressed in the striatum, predominantly in the medium spiny

striatopallidal output neurons (see Svenningsson et al., 1999). Though

A2A receptors modulate the release of neurotransmitters from striatal

nerve endings, namely GABA and acetylcholine (Kirk and Richardson,

1994) as well as glutamate (Rodrigues et al., 2005), these receptors do

not directly influence dopamine release at striatal slices (Jin et al., 1993;

Lupica et al., 1990). It, therefore, seemed of interest to evaluate if GDNF

could presynaptically modulate dopamine release and if so, whether

adenosine A2A receptors could modulate that action of GDNF.


86

3.1.2. GDNF enhanced K+-evoked dopamine release from isolated

nerve terminals

In striatal synaptosomes incubated with [3H]dopamine, the initial

(first three collected samples) basal release of tritium was 1.73±0.02% of

the total tritium incorporated in the synaptosomes (fractional release)

(n=12) (Figure 3.1.1A). The first stimulation period (S1) caused a twofold

increase in the amount of tritium release, whereas the second caused a

smaller increase, so that the S2/S1 ratio obtained was 0.56±0.02 (n=12).

When GDNF (10 ng/ml) was present during S2, the S2/S1 ratio was

increased to 0.80±0.03 (p<0.05 as compared with the control) (Figure

3.1.1C), corresponding to a 42±4.8% (n=8) enhancement of tritium

release (Figure 3.1.2). As shown in Figure 3.1.1B, the facilitation by

GDNF of the K+-evoked [3H]dopamine release increased with GDNF

concentration from 3 ng/ml to 30 ng/ml. For the subsequent studies on

the modulation of the GDNF effect upon dopamine release, the

intermediate concentration of 10 ng/ml was chosen to avoid a ceiling

effect; furthermore, concentrations of GDNF causing more than twofold

increase in dopamine release are not expected to occur endogenously.

Results

87

A.

B.

S1 S2

0 5 10 15 20 25 30 35 40

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

GDNF (10 ng/ml) ()

Time (min)

Fra

cti

on

al R

ele

as

e (

%)

0.0

0.2

0.4

0.6

0.8

1.0

*

GDNF - +(10 ng/ml, S2)

S2/S

1

1 10 100

0

50

100

150

200

[GDNF] (ng/ml)

Fra

cti

on

al R

ele

as

e (

%) C.

A.

B.

S1 S2

0 5 10 15 20 25 30 35 40

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

GDNF (10 ng/ml) ()

Time (min)

Fra

cti

on

al R

ele

as

e (

%)

0.0

0.2

0.4

0.6

0.8

1.0

*


S2/S

1

1 10 100

0

50

100

150

200

[GDNF] (ng/ml)

Fra

cti

on

al R

ele

as

e (

%) C.

Figure 3.1.1 - GDNF enhances K+-evoked [

3H]dopamine release from striatal

synaptosomes. A. Averaged time course of [3H]dopamine release experiments. Striatal

synaptosomes were labeled with [3H]dopamine as described in Experimental

Procedures. Release was evoked twice, S1 and S2, by chemical stimulation (20 mM KCl,

2 min). GDNF was added to the test chambers before S2, as indicated by the horizontal

bar (filled circles), whereas it was not added to the parallel control chambers (open

circles). Time in the abscissa represents ending of collecting period for each sample.

Each point represents the mean SEM of the results obtained in 8–12 experiments

performed in duplicate. B. Concentration-response curve for the enhancement of K+-

evoked [3H]dopamine release caused by GDNF in striatal nerve terminals. The effect of

GDNF was calculated by comparing the S2/S1 ratio obtained in test (presence of GDNF

during S2) and in control conditions in the same experiments. Each point represents the

mean SEM of results obtained in 2 (GDNF 3 and 30 ng/ml) to 8 (GDNF 10 ng/ml)

experiments. C. S2/S1 ratio obtained in the absence (−) or in the presence (+) of GDNF,

as indicated below each bar; *p<0.05 as compared with the control (left column,

Student's t test).


88

3.1.3. GDNF enhancement of K+-evoked dopamine release from

isolated nerve terminals is dependent on adenosine A2A receptor

activation

The ability of adenosine A2A receptors to modulate GDNF-

mediated enhancement of dopamine release was evaluated by

performing experiments where the A2A receptor agonist, CGS 21680,

was present during S1 and S2 (see Materials and Methods) in all

perfusion chambers, at a concentration (10 nM) selective for adenosine

A2A receptors (Jarvis et al., 1989). As before, the effect of GDNF (10

ng/ml) was evaluated by comparing the S2/S1 ratio obtained in the

absence of GDNF with the S2/S1 ratio obtained in parallel chambers

where GDNF was added before S2. Under these conditions, GDNF

enhanced the evoked release of tritium by 65±2.9% (n=5), an effect

significantly (p<0.05) greater than that observed in the absence of CGS

21680 (Figure 3.1.2). We then tested whether blockade adenosine A2A

receptors with the selective antagonist, SCH 58261, affected the GDNF

excitatory action. The protocol used was similar to that described above,

except that SCH 58261 was used instead of CGS 21680. The

concentration of SCH 58261 (50 nM) used in these experiments was

100 times higher its Ki value for adenosine A2A receptors and well below

its Ki for other adenosine receptor subtypes (Zocchi et al., 1996).

As illustrated in Figure 3.1.2, the GDNF-induced enhancement of

dopamine release was prevented (p<0.05, n=4) by the A2A receptor

antagonist, suggesting that the effect of GDNF in striatal dopaminergic

nerve endings requires tonic activation of adenosine A2A receptors. The

presence of CGS 21680 (10 nM) or SCH 58261 (50 nM) during S1 and

Results

89

S2 did not appreciably modify the S2/S1 ratio (p>0.05) as compared with

the ratio obtained in the absence of any drug.

0

25

50

75

GDNF + + +(10 ng/ml, S2)

CGS 21680 - + -(10nM, S1, S2)

SCH 58261 - - +(50nM, S1, S2)

% i

nc

rea

se

in

[3H

]do

pam

ine

re

lea

se

by G

DN

F

Figure 3.1.2 - Ability of the selective A2A receptor agonist, CGS 21680 (10 nM), and

of the A2A receptor antagonist, SCH 58261 (50 nM), to modify the action of GDNF

(10 ng/ml) on [3H]dopamine release from striatal synaptosomes. Zero percent in the

ordinates represents the S2/S1 ratio obtained in the absence of GDNF for each

experimental condition indicated below the bars. Results are mean SEM from n

experiments performed in duplicate.*p<0.05 as compared with 0% (Student's t test);

φp<0.05 as compared with the effect of GDNF (10 ng/ml) in the absence of other drugs

(left column) (one-way ANOVA analysis followed by Bonferroni's test).


90

3.1.4. GDNF enhanced electrically-evoked dopamine release from

striatal slices

In striatal slices incubated with [3H]dopamine, the initial basal

fractional release of tritium was 0.91±0.06% (n=10) (Figure 3.1.3A). The

first stimulation period (S1) caused a nearly threefold increase in the

amount of tritium release, whereas the second one caused a smaller

increase, the S2/S1 ratio being 0.70±0.02 (n=10). When GDNF (10

ng/ml) was present during S2, the S2/S1 ratio increased (p<0.05) up to

1.3±0.12 (Figure 3.1.3B) corresponding to 89±9.3% (n=10)

enhancement of tritium release (Figure 3.1.4).

3.1.5. GDNF effect on dopamine release from striatal slices also

depends upon A2A receptor tonic activation

When A2A receptors were blocked with SCH 58261 (50 nM, in S1

and S2), the effect of GDNF upon dopamine release was prevented (%

change by GDNF: 2.6±1.2%, n=4, p<0.05, as compared with the effect

of GDNF alone) (Figure 3.1.4), indicating that A2A receptors are also

required for the GDNF effect on dopamine release from striatal slices.

Surprisingly, the A2A receptor agonist, CGS 21680 (10 nM) decreased

(n=7, p<0.05) the facilitatory effect of GDNF on dopamine release from

striatal slices (Figure 3.1.4), in clear contrast with what was observed in

striatal synaptosomes (Figure 3.1.2).

Results

91

S1 S2

0 5 10 15 20 25 30 35 40 45 50 55 60 65

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

GDNF (10 ng/ml) ()

Time (min)

Fra

cti

on

al

Re

lea

se

(%

)

0.0

0.5

1.0

1.5 *


S2/S

1

A.

B.

S1 S2

0 5 10 15 20 25 30 35 40 45 50 55 60 65

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

GDNF (10 ng/ml) ()

Time (min)

Fra

cti

on

al R

ele

as

e (

%)

0.0

0.5

1.0

1.5 *


S2/S

1

A.

B.

Figure 3.1.3 - GDNF enhances electrically evoked [3H]dopamine release from

striatal slices. A. Averaged time course of [3H]dopamine release experiments. Striatal

slices were labelled with [3H]dopamine as described in Experimental Procedures.

Release was evoked twice, S1 and S2, by electrical stimulation (supramaximal, 2ms

pulses, 2 Hz, 2 min). GDNF was added to the test chambers before S2, as indicated by

the horizontal bar (filled circles), whereas it was not added to the parallel control

chambers (open circles). Time in the abscissa represents ending of collecting period for

each sample. B. S2/S1 ratio obtained either in the absence and in the presence of GDNF

as indicated below each bar; *p <0.05 as compared with the control (Student's t test).

Results are the mean SEM of 10 experiments performed in duplicate.


92

0

25

50

75

100

GDNF + + +(10 ng/ml, S2)

CGS 21680 - + -(10 nM, S1, S2)

SCH 58261 - - +(50 nM, S1, S2)

% in

cre

ase in

[3H

]do

pam

ine r

ele

ase b

y G

DN

F



(10 ng/ml) on [3H]dopamine release in striatal slices. Zero percent in the ordinates

represents the S2/S1 ratio obtained in the absence of GDNF for each experimental

condition indicated below the bars. Results are mean SEM from n experiments

performed in duplicate.*p<0.05 as compared with 0% (Student's t test); φp<0.05 as

compared with the effect of GDNF (10 ng/ml) in the absence of other drugs (left column)

(one-way ANOVA analysis followed by Bonferroni's test).

Results

93

3.1.6. Influence of A2A receptors and of GABA transmission

blockers upon dopamine release in striatal slices

GABAergic spiny neurons represent the majority of the cells in

the striatum (see e.g. Kawaguchi et al., 1995). A2A receptors are

expressed in those neurons and regulate GABAergic synaptic

transmission (see Discussion). Furthermore, inhibitory modulation of

dopamine release by glutamatergic or muscarinic cholinergic

transmission mostly depends on the activity of GABAergic neurons (Wu

et al., 2000; Zhang et al., 2002). Therefore, to evaluate if the

“paradoxical” CGS 21680- induced decrease of the effect of GDNF upon

dopamine release could be attributed to an influence of A2A receptors in

the striatal GABAergic circuits, we performed experiments where

GABAergic transmission was blocked and tested the effect of GDNF in

the absence and in the presence of the A2A receptor agonist. GABAergic

transmission was blocked with the GABAA and GABAB receptor

antagonists, bicuculline (10 μM) and CGP55845 (1 μM); under these

conditions, GDNF (10 ng/ml, added before S2) enhanced the evoked

release of tritium by 37±1.8% (n=4). When CGS 21680 (10 nM) was also

added together with bicuculline (10 μM) and CGP 55845 (1 μM), GDNF

(10 ng/ml) enhanced the evoked release of tritium by 74±5.6% (n=7,

p<0.05 as compared with absence of CGS 21680) clearly showing that

A2A receptor activation enhances the action of GDNF when GABAergic

transmission is blocked. In slices with GABAergic transmission blocked,

SCH 58261 (50 nM) prevented the effect of GDNF (10 ng/ml) on the

evoked release of dopamine (Figure 3.1.5). The presence of SCH 58261

(50 nM) or CGS 21680 (10 nM) or CGP 55845 (1 μM) plus bicuculline


94

(10 μM) during S1 and S2 did not significantly (p>0.05) influence the

S2/S1 ratio as compared with absence of any drug.

In the experiments described above, the fractional release of

tritium during the first stimulation period (S1) was not significantly

(p>0.05) modified by the presence of SCH 58261 (50 nM) or CGS 21680

(10 nM), but it was higher (p<0.05) in the slices where GABAergic

transmission was blocked with bicuculline (10 μM) and CGP 55845 (1

μM). This suggested that the evoked release of dopamine was not

influenced by the A2A receptor agonist or antagonist, but that blockade of

GABAergic transmission was enhancing dopamine release. To directly

evaluate the action of these drugs upon dopamine release, experiments

were designed where they were applied to the slices before S2 and the

S2/S1 ratios obtained in their presence compared with the S2/S1 ratios

obtained in the absence of drugs. As expected (see also Lupica et al.,

1990), neither CGS 21680 (10 nM) nor SCH 58261 (50 nM), when

applied before S2, influenced significantly (p>0.05) the evoked release of

tritium (%change by CGS: 0.31±4.6, n=4; % change by SCH: -6.8±4.4,

n=3). Inhibition of GABAergic transmission by the addition of bicuculline

(10 μM) and CGP 55845 (1 μM) before S2 increased the S2/S1 ratio by

60±3.7% (p<0.05, n=3), indicating that dopamine release in striatal

slices is under control of GABAergic transmission.

Results

95

0

25

50

75

100

GDNF + + +(10 ng/ml, S2)

Bicuculline + + +(10 M , S1, S2)

CGP 55845 + + +(1M , S1, S2)

CGS 21680 - + -(10 nM , S1, S2)

SCH 58261 - - +(50 nM , S1, S2)

% i

nc

rea

se

in

[3H

]do

pam

ine

rele

ase

by

G

DN

F



(10 ng/ml) on [3H]dopamine release in striatal slices in the presence of the GABAA

and GABAB receptor antagonists, bicuculline (10 μM) and CGP 55845 (1 μM). Zero

percent in the ordinates represents the S2/S1 ratio obtained in the absence of GDNF for

each experimental condition indicated below the bars. Results are mean SEM from n

experiments performed in duplicate.*p <0.05 as compared with 0% (Student's t test); φp

<0.05 as compared with the effect of GDNF (10 ng/ml) in the absence of other drugs (left

column) (one-way ANOVA analysis followed by Bonferroni's test).


96

3.1.7. Discussion

The present results first show that GDNF acutely increases

evoked dopamine release in rat striatal slices and synaptosomes and,

most importantly, that GDNF-induced enhancement of dopamine release

is modulated by adenosine A2A receptors, which provides a first

evidence for a crosstalk between adenosine A2A receptors and GDNF in

the striatum. Intranigral injections of GDNF (Hebert and Gerhardt, 1997)

or of an adenoviral vector encoding for GDNF (Smith et al., 2005)

enhance striatal dopamine levels and dopamine release even in 6-

hydroxydopamine lesioned (Smith et al., 2005) or old (Hebert and

Gerhardt, 1997) rats. Similar observations have been made in aged

Rhesus monkeys after repeated injections of GDNF in the lateral

ventricle (Grondin et al., 2003). GDNF has also been shown to increase

cell body excitability of cultured midbrain neurons (Yang et al., 2001).

Altogether, these evidences clearly show that GDNF enhances

dopaminergic function by promoting maturation and function of

nigrostriatal dopaminergic neurons, but do not demonstrate a direct

action of GDNF at striatal nerve endings. The isolated striatum does not

possess the cell bodies of dopaminergic neurons, which are located at

the substantia nigra. Therefore, the now reported enhancement of

dopamine release caused by GDNF acutely applied to striatal slices or

striatal synaptosomes shows that this neurotrophic factor acts on

dopaminergic nerve terminals, where it regulates dopamine release.

GDNF may then have a dual role in nigrostriatal dopaminergic neurons

to protect or even rescue dopaminergic nigrostriatal neuronal function:

one at the cell bodies in the nigra, promoting their maturation and

Results

97

sprouting, which secondarily leads to enhanced striatal dopamine levels

and dopamine release, and another localized at striatal nerve endings,

directly enhancing dopamine release. As is also shown, this direct

GDNF action is under control of adenosine A2A receptors, most probably

located presynaptically at dopaminergic nerve terminals, since the A2A

receptors/GDNF crosstalk could be observed in isolated synaptosomes,

where interactions at the circuit level are absent (see Raiteri and Raiteri,

2000). Blockade of GABAergic transmission enhanced the evoked

release of dopamine but reduced the effect of GDNF on dopamine

release (compare Figures 3.1.4 and 3.1.5) suggesting that part but not

all of the excitatory effect of GDNF on dopamine release depends upon

GABAergic transmission. Interestingly, the magnitude of the

enhancement of dopamine release caused by GDNF in synaptosomes,

where a crosstalk between different neuronal structures might be

virtually absent, was similar to that observed in slices upon inhibition of

GABAergic transmission. Further studies are required to investigate how

the functioning of GABAergic circuits in slices facilitates the effect of

GDNF and whether this also occurs in vivo. The striatal GABAergic

MSNs, which represent more than 90% of striatal neurons, receive

intrastriatal GABAergic inputs from axon collaterals of the medium spiny

neurons themselves and from GABAergic interneurons. Presynaptic

inhibitory A2A receptors in the GABAergic interneurons relieve the

inhibitory inputs to the spiny neurons, therefore enhancing its activity

(Mori et al., 1996). Postsynaptic A2A receptors in the GABAergic output

neurons inhibit D2 receptor functioning (see e.g. Ferré et al., 2003) and

inhibit NMDA receptor functioning (Wirkner et al., 2004). It is possible

that this multiplicity of A2A receptor mediated actions in striatal


98

GABAergic neurons contributes to the CGS 21680-induced inhibition of

the effect of GDNF on dopamine release, since this inhibition (1) was

observed in slices but not in synaptosomes; (2) was mimicked by

inhibitors of GABAergic transmission and (3) was fully absent when

GABAergic transmission was inhibited. The GABA-independent

enhancement of dopamine release caused by GDNF in slices was

facilitated by co-activation of adenosine A2A receptors, as it occurred in

isolated nerve endings. Both in synaptosomes and in slices, either in the

presence or in the absence of GABAergic transmission, the action of

GDNF was prevented by A2A receptor antagonists, which allowed us to

suggest that the facilitatory action of GDNF upon striatal dopamine

release requires co-activation of adenosine A2A receptors. In contrast

with what occurs in cholinergic and GABAergic striatal nerve endings

(Kirk and Richardson, 1994), dopamine release from striatal nerve

endings is not under control of adenosine A2A receptors (Jin et al., 1993;

Lupica et al., 1990 and present results), which led to the concept that

these receptors might be absent from striatal dopaminergic nerve

endings. However, we clearly show that A2A receptors in striatal

dopaminergic nerve terminals modulate the action of GDNF on

dopamine release. Therefore, the role of A2A receptors in the

dopaminergic nerve endings might be as modulators of the action of

other presynaptic receptors, rather than exerting a direct modulation of

dopamine release. This reinforces the idea (see Sebastião and Ribeiro,

2000) that a main role of A2A receptors in different brain areas is to

interact with receptors for other neuromodulators, a process probably

involved in the fine tuning of neuronal function.

Results

99

3.2. Adenosine A2A receptor modulation of the cortical-

induced protein phosphorylation in the striatum


Both enkephalinergic (D2- and A2A- regulated) and dynorphinergic

(D1- and A1- regulated) neurons receive corticostriatal afferents and

display excitatory postsynaptic responses to corticostriatal stimulation

(Kawaguchi et al., 1990), but corticostriatal stimulation selectively

activates MAPK and immediate early gene (IEG) expression in the

enkephalinergic neurons without activation in dynorphinergic neurons

(Berretta et al., 1997; Gerfen et al., 2002). A2A receptor-mediated PKA

activation can potentially phosphorylate different substrates (for instance

GluR1 subunit of the AMPA receptor at Ser845 and ERK1/2) involved in

synaptic plasticity (see Introduction, section 1.3.3). However, under

basal conditions, these PKA-dependent changes are impaired due to the

tonic activation operated by dopamine D2 receptors (for review, see

Ferré et al., 2004, 2005). Since stimulation of A2A (and mGlu5) receptors

inhibits dopamine binding to D2 receptors and activates MAPK (Popoli et

al., 2001; Ferré et al., 2002; Nishi et al., 2003), in the present work it was

assessed if a strong cortical glutamatergic input induced by electrical

stimulation would allow sufficient stimulation of A2A receptors (by

adenosine co-released with glutamate), overriding the tonic inhibition

operated by dopamine D2 receptors upon the phosphorylation of GluR1

and ERK1/2 proteins in the striatum.


100

3.2.2. Adenosine A2A receptor effect upon cortical stimulation-

induced protein phosphorylation

3.2.2.1 Adenosine A2A receptor effect upon cortical stimulation-

induced ERK1/2 phosphorylation

As reported previously (Sgambato et al., 1998; Gerfen et al.,

2002), cortical stimulation in the orofacial area of the lateral agranular

motor cortex (demonstrated by the selective elicitation of jaw

movements) induced a predominant phosphorylation of ERK1/2 in the

lateral caudate-putamen at the AP level of bregma 0.01.0 mm (Figures

3.2.1, 3.2.2A,B). Western blot assays were therefore performed from

punches of the lateral striatum corresponding to the area with maximal

ERK1/2 phosphorylation.

Cortical stimulation induced a significant increase in ERK1/2

phosphorylation in the ipsilateral striatum (14512.1%, n=10) compared

with electrode-implanted nonstimulated controls (1012.3%, n=10)

(Figure 3.2.2A,B). This effect was significantly counteracted by the

selective A2A receptor antagonist MSX-3 (3 mg/kg) (1068.1%, n=10)

(Figure 3.2.2A,C), and by the nonselective adenosine receptor

antagonist caffeine (10 mg/kg) (8317.2%, n=6) (Figure 3.2.2A,D), but

not by the A1 receptor antagonist CPT (4.8 mg/kg) (14317.9%, n=6)

(Figure 3.2.2A,E). None of the adenosine receptor ligands produced any

significant effect in sham non-stimulated animals. There were not

significant differences in the values of total ERK1/2 between the different

sham and stimulated groups (Figure 3.2.2A).

In these in vivo experiments the A2A receptor antagonist, MSX-3,

Results

101

was used instead of SCH 58261, which was used in vitro. This was

because SCH 58261 (but not MSX-3) was previously characterized in

the same in vitro conditions and MSX-3 (but not SCH 58261) in the

same in vivo conditions as those used in this study. Both compounds

have demonstrated their selectivity for A2A receptors at the concentration

(SCH 58261, 50 nM) and dose (MSX-3, 3 mg/kg i.p.) now used, as

shown before (Karcz-Kubicha et al., 2003; Ciruela et al., 2006).

3.2.2.2. Adenosine A2A receptor effect upon cortical stimulation-

induced AMPA receptor phosphorylation

Corticostriatal stimulation was also found to significantly increase

phosphorylation of the Ser845 residue of the GluR1 subunit of the AMPA

receptor in the ipsilateral striatum (18013.5%, n=11) as compared with

control, non-stimulated animals (1006.7%, n=13) (Figure 3.2.3A,B).

Striatal AMPA receptor phosphorylation was also completely

counteracted by MSX-3 (3 mg/kg) (1046.6%, n=9) or caffeine (10

mg/kg) (7310.3%, n= 5) (Figure 3.2.3A,C,D), whereas CPT (4.8 mg/kg)

(Figure 3.2.3A,E) did not significantly modify AMPA receptor

phosphorylation induced by cortical stimulation (1444.6%, n=7). Again,

none of the adenosine receptor ligands produced any significant effect in

sham non-stimulated animals. There were not significant differences in

the values of total GluR1 subunit of the AMPA receptors between the

sham and stimulated groups (Figure 3.2.3A).


102

Figure 3.2.1 - Immunohistochemical localization of striatal ERK1/2

phosphorylation induced by cortical electrical stimulation. A,D.

Immunohistochemical localization of phosphorylated ERK1/2 in coronal sections (AP

level of bregma 0.0) through the striatum ipsilateral to the implanted electrode from

stimulated (A) and sham nonstimulated (D) animals. B, C, E, F. Higher magnification of

the respective fields indicated in A and D, which shows cells displaying immunoreactivity

to phospho-ERK1/2 antibody in the lateral caudate-putamen of the stimulated animals.

Scale bars, 250m.

Results

103

0

50

100

150

200

Veh Veh + S

ER

K1/2

ph

osh

ory

lati

on

(%

of

co

ntr

ol)

0

50

100

150

200

Veh MSX-3

*

+ S

ER

K1

/2 p

ho

sh

ory

lati

on

(% o

f c

on

tro

l)

B.

C.

0

50

100

150

200

Veh CPT

+ S

ER

K1/2

ph

osh

ory

lati

on

(% o

f co

ntr

ol)

0

50

100

150

200

Veh Caffeine

*

+ S

ER

K1/2

ph

osh

ory

lati

on

(% o

f co

ntr

ol)

D. E.

A.

pERK1/2

Total ERK1/2 44 kDa

44 kDa

42 kDa

42 kDa

Ve

h

Ve

h+

S

MS

X-3

MS

X-3

+S

CP

T

CP

T+

S

Ca

ffe

ine

+S

Ca

ffe

ine

(1) (2) (3) (4)(0)(1)(0)

(2)(1) (4)(1) (3)(1)

0

50

100

150

200

Veh Veh + S

ER

K1/2

ph

osh

ory

lati

on

(%

of

co

ntr

ol)

0

50

100

150

200

Veh MSX-3

*

+ S

ER

K1

/2 p

ho

sh

ory

lati

on

(% o

f c

on

tro

l)

B.

C.

0

50

100

150

200

Veh CPT

+ S

ER

K1/2

ph

osh

ory

lati

on

(% o

f co

ntr

ol)

0

50

100

150

200

Veh Caffeine

*

+ S

ER

K1/2

ph

osh

ory

lati

on

(% o

f co

ntr

ol)

D. E.

A.

pERK1/2

Total ERK1/2 44 kDa

44 kDa

42 kDa

42 kDa

Ve

h

Ve

h+

S

MS

X-3

MS

X-3

+S

CP

T

CP

T+

S

Ca

ffe

ine

+S

Ca

ffe

ine

(1) (2) (3) (4)(0)(1)(0)

(2)(1) (4)(1) (3)(1)

Figure 3.2.2 - Striatal ERK1/2 phosphorylation induced by cortical electrical

stimulation. A. Representative Western blots. B. ERK1/2 phosphorylation induced by

cortical electrical stimulation. C,D. A2A receptor antagonists, MSX-3 (C) and caffeine (D)

counteracted ERK1/2 phosphorylation induced by cortical stimulation. E. A1 receptor

antagonist, CPT, does not counteract ERK1/2 phosphorylation induced by cortical

stimulation. Results are shown in meansSEM (n = 6-10 per group) of representative

Western blots. p<0.05, significantly different compared with vehicle-treated non

stimulated group (ANOVA with Tukey-Kramer tests). *p<0.05, significantly different

compared with vehicle-treated stimulated group (ANOVA with Tukey-Kramer tests).


104

0

50

100

150

200

Veh Veh+S

Glu

R1 p

ho

sh

ory

lati

on

(%

of

co

ntr

ol)

B.

C. D. E.

A.

pGluR1

Total GluR1 100 kDa

100 kDa

Veh

Veh

+S

MS

X-3

MS

X-3

+S

CP

T

CP

T+

S

Caff

ein

e+

S

Caff

ein

e

(1) (2) (3) (4)(0)(1)(0)

(2)(1) (4)(1) (3)(1)

0

100

200

Veh MSX-3

+ S

*

Glu

R1 p

ho

sh

ory

lati

on

(% o

f c

on

tro

l)

0

50

100

150

200

Veh Caffeine

*

+ S

Glu

R1 p

ho

sh

ory

lati

on

(% o

f co

ntr

ol)

0

50

100

150

200

Veh CPT

Glu

R1 p

ho

sh

ory

lati

on

(% o

f c

on

tro

l)

+ S

0

50

100

150

200

Veh Veh+S

Glu

R1 p

ho

sh

ory

lati

on

(%

of

co

ntr

ol)

B.

C. D. E.

A.

pGluR1

Total GluR1 100 kDa

100 kDa

Veh

Veh

+S

MS

X-3

MS

X-3

+S

CP

T

CP

T+

S

Caff

ein

e+

S

Caff

ein

e

(1) (2) (3) (4)(0)(1)(0)

(2)(1) (4)(1) (3)(1)

0

100

200

Veh MSX-3

+ S

*

Glu

R1 p

ho

sh

ory

lati

on

(% o

f c

on

tro

l)

0

50

100

150

200

Veh Caffeine

*

+ S

Glu

R1 p

ho

sh

ory

lati

on

(% o

f co

ntr

ol)

0

50

100

150

200

Veh CPT

Glu

R1 p

ho

sh

ory

lati

on

(% o

f c

on

tro

l)

+ S

Figure 3.2.3 - Striatal GluR1 phosphorylation induced by cortical electrical

stimulation. A. Representative Western blots. B. GluR1 phosphorylation induced by

cortical electrical stimulation. C,D. A2A receptor antagonists, MSX-3 (C) and caffeine (D)

counteracted GluR1 phosphorylation induced by cortical stimulation. E. A1 receptor

antagonist CPT does not counteract GluR1 phosphorylation induced by cortical

stimulation. Results are shown in meansSEM (n = 5-13 per group) of representative

Western blots. p<0.05, significantly different compared with vehicle-treated non

stimulated group (ANOVA with Tukey–Kramer tests). *p<0.05, significantly different

compared with vehicle-treated stimulated group (ANOVA with Tukey–Kramer tests).

Results

105

3.2.3. Discussion

In the striatum, there are two major types of glutamatergic

synapses, the D1 receptor-regulated and the D2 (and A2A) receptor-

regulated glutamatergic synapses, mostly localized at the heads of the

dendritic spines of the GABAergic dynorphinergic and enkephalinergic

neurons, respectively (Ferré et al., 2005). In the GABAergic

dynorphinergic neuron, D1 receptor stimulation modulates neuronal

excitability and glutamatergic neurotransmission by inducing PKA-

mediated phosphorylation of different substrates. These substrates

include ERK1/2 and Ser845 of the GluR1 subunit of the AMPA receptor

(Wolf et al., 2003), both critical steps in the establishment of plastic

changes in excitatory synapses, involving recruitment of AMPA

receptors to the postsynaptic density (Malinow and Malenka, 2002; Lee

et al., 2003; Thomas and Huganir, 2004). As mentioned above (see

rationale and 1.3.3), D1 receptor-mediated mechanisms by which

dopamine can potentiate the function and plasticity of glutamatergic

synapses in the GABAergic dynorphinergic neuron is operated by

adenosine activating A2A receptors. At the postsynaptic site, A2A

receptors form heteromeric complexes with D2 and metabotropic

glutamate mGlu5 receptors (Ferré et al., 2005) and costimulation of A2A

and mGlu5 receptors exerts a synergistic effect on their ability to inhibit

dopamine binding to the D2 receptor and on MAPK activation (Popoli et

al., 2001; Ferré et al., 2002; Nishi et al., 2003). In the present work it

was postulated that a strong cortico-limbic-thalamic glutamatergic input

would produce a sufficient amount of intrasynaptic glutamate and

adenosine (most probably derived from ATP co-released with


106

glutamate), with a significant stimulation of postsynaptic A2A receptors.

Then, under conditions of strong glutamatergic input, adenosine and A2A

receptors can provide a mechanism of facilitation of plastic changes in

the excitatory synapses of the GABAergic enkephalinergic neurons

(Ferré et al., 2005). The present results strongly support this hypothesis,

because phosphorylation of ERK1/2 and GluR1 at Ser845 induced by

stimulation of corticostriatal afferents was completely dependent on A2A

receptor function.

Results

107

3.3. Adenosine A2A receptor modulation of GDNF effects on

the glutamatergic corticostriatal pathway


In accordance with its ability to bolster nigrostriatal innervation,

GDNF affords neuroprotection in Parkinson‟s disease. As mentioned in

3.1, GDNF facilitates dopamine release in a manner dependent on

adenosine A2A receptor activation. GDNF also affords beneficial effects

in animal models of Huntington‟s disease (reviewed in Alberch et al.,

2004), where the control of the viability of dopaminergic neurons is not

considered a prominent etiological factor. Dysfunction of the

glutamatergic system may be a general feature of neurodegenerative

diseases (Lipton and Rosenberg, 1994) and, accordingly, is present in

Parkinson‟s disease (Day et al., 2006) as well as Huntington´s disease

(Cepeda et al., 2007). GDNF protects several types of neurons against

glutamate-induced toxicity in different brain areas (Yoo et al., 2006;

Wong et al., 2005) and it was observed an up-regulation of GDNF and

its receptors after excitotoxic insults to the striatum (Alberch et al.,

2002). Thus, the trophic factor has been suggested as a potential

therapeutic approach in neurodegenerative conditions secondary to

excitotoxic-induced damage (Araújo et al., 1997; Perez-Navarro et al.,

1996; 1999).

These observations lead to the hypothesis that GDNF may act

through broader mechanism of action to preserve striatal circuits from

injury, which might also involve the control of glutamatergic inputs, a

hypothesis that has not yet been experimentally tested. Thus, in the


108

present work, it was tested if GDNF controlled the corticostriatal

glutamatergic pathway in an A2A receptor-dependent manner, as it

occurred with the dopaminergic influx to the striatum.

3.3.2. GDNF control of the glutamatergic input to the striatum

3.3.2.1. GDNF facilitates K+-evoked glutamate release from striatal

synaptosomes

In striatal synaptosomes incubated with [3H]glutamate, the initial

(first three collected samples) basal release of tritium was 2.72±0.05% of

the total tritium incorporated in the synaptosomes (fractional release)

(n=6) (Figure 3.3.1). The first stimulation period (S1) caused a near 1.5

fold increase in the amount of tritium release; the second stimulation

period (S2) caused a slightly smaller increase in tritium release than the

first one, so the S2/S1 ratio obtained was 0.91±0.05 (n=6). To evaluate

the action of GDNF upon glutamate release from synaptosomes, we

used a concentration of GDNF (10 ng/ml) known to facilitate dopamine

release from the same preparation (see 3.1.2 and also Gomes et al.,

2006) as well as to increase dopamine levels and number of

dopaminergic neurons in culture (Schatz et al., 1999).

When GDNF (10 ng/ml) was present during S2, the S2/S1 ratio

was increased to 1.1±0.08 (p<0.05 as compared with absence of GDNF

in the same synaptosomal batch), corresponding to a 13.1±2.8% (n=5,

p<0.05) enhancement of [3H]glutamate release (Figure 3.3.2).

Results

109

3.3.2.2. GDNF-mediated enhancement of K+-evoked glutamate

release depends on A2A receptor activation

As illustrated in Figure 3.3.2, the GDNF-induced enhancement of

glutamate release was abolished (p<0.05, n=3) when the A2A receptor

antagonist was present. In two of these experiments the effect of GDNF

in the absence of SCH 58261 was also tested in the same synaptosomal

batch and the usual excitatory effect of GDNF (10 ng/ml) was observed.

The abolishment of the effect of GDNF by the A2A receptor antagonist

suggests that the effect of GDNF in striatal glutamatergic nerve endings

requires tonic activation of adenosine A2A receptors. In the presence of

CGS 21680, GDNF caused a marked increase in the S2/S1 ratio (Figure

3.3.2), corresponding to a facilitatory effect of 29±6.9% (n=3). This effect

of GDNF was significantly (p<0.05) greater than that observed in the

absence of CGS 21680; the potentiation of the GDNF effect by CGS

21680 should be attributed to A2A receptor activation, since it was

prevented by the selective (Zocchi et al., 1996) A2A receptor antagonist

SCH 58261 (50 nM; Figure 3.3.2). The presence of CGS 21680 (10 nM)

or SCH 58261 (50 nM) during S1 and S2 did not significantly modify the

S2/S1 ratio (p>0.05). The amount of tritium released during S1 in the

absence of any drug or in the presence of CGS 21680 (10 nM) or SCH

58261 (50 nM) was also not significantly different (p>0.05).


110

0 3 6 9 12 15 18

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

GDNF, 10 ng/mlS2

S1 S2

CGS 21680 (10 nM)S1, S2

Time (min)

Fra

cti

on

al

Re

lea

se

(%

)

Figure 3.3.1 - GDNF enhances electrically evoked [3H]glutamate release from

striatal synaptosomes. Averaged time course of [3H]glutamate release experiments.

Striatal synaptosomes were labelled with [3H]glutamate as described in Experimental

Procedures. Release was evoked twice (S1 and S2) by chemical stimulation (20 mM KCl,

30 sec), as indicated by the bars above the abscissa. CGS 21680 (10 nM) was added 15

min before S1 onwards, and was present throughout all the experiment in both control

and test conditions (indicated by the upper horizontal bar). GDNF was added to the test

chambers 2 min before S2, as indicated by the upper horizontal bar (filled circles),

whereas it was not added to the parallel control chambers (open circles). Each point

represents the mean±SEM of the results obtained in 4-5 experiments performed in

duplicate.

Results

111

-5

0

5

10

15

20

25

30

35

'

GDNF + + + +(10 ng/ml, S2)

CGS 21680 - - + -(10 nM, S1, S2)

SCH 58261 - + - +(50 nM, S1, S2)

% i

nc

rea

se

in

[3H

]glu

tam

ate

re

lea

se

by

G

DN

F

Figure 3.3.2 - Blockade of the effect of GDNF by the A2A receptor antagonist, SCH

58261, and its potentiation by the A2A receptor agonist, CGS 21680. The effect of

GDNF on the evoked release of glutamate was estimated by changes in the amount of

[3H]glutamate release in S2 as referred to S1 (S2/S1 ratio). 0% represents the S2/S1 ratio

in the absence of GDNF. Results are mean±SEM from 3-5 experiments performed in

duplicate. *p<0.05 as compared with the effect of GDNF in the absence of other drugs

(left column) (one-way ANOVA analysis followed by Bonferroni's test); 'p<0.05 as

compared with the effect of GDNF in the presence of CGS 21680 (third column from left)

(one-way ANOVA analysis followed by Bonferroni's test). The amount of tritium released

during S1 under the different conditions was not significantly different (p>0.05, one-way

ANOVA analysis followed by Bonferroni's test); the presence of CGS 21680 or SCH

58268 also did not significantly affect the S2/S1 ratio (p>0.05, one-way ANOVA analysis

followed by Bonferroni's test).


112

3.3.3. In vivo A2A receptor modulation of the activation state of

GDNF receptor upon cortical stimulation

3.3.3.1. Cortical stimulation induces GDNF receptor

phosphorylation in the striatum

Cortical stimulation in the orofacial area of the lateral agranular

motor cortex induced an increase (1367.3%, n=8) in Ret

phosphorylation in the ipsilateral striatum compared to electrode-

implanted non-stimulated controls (1007.8%, n=6) (Figure 3.3.5A,B).

Total Ret levels were not significantly affected by cortical stimulation in

any group of animals (Figure 3.3.5A).

3.3.3.2. Cortical stimulation-induced phosphorylation of GDNF

receptor in the striatum requires A2A receptor tonic activation

The dependency on A2A receptor activation was evaluated in a

parallel group of animals where cortical stimulation was performed after

administration of a selective A2A receptor antagonist, MSX-3 (3 mg/kg,

i.p.) or after the administration of a selective A1 receptor antagonist, CPT

(4.8 mg/kg, i.p.). The dose of the adenosine antagonists was previously

shown to provide motor activation by selectively antagonizing A1

receptors (CPT) or A2A receptors (MSX-3) (Karcz-Kubicha et al., 2003).

In animals under MSX-3, cortical stimulation failed to induce Ret

phosphorylation (1216.1%, n=8) (Figure 3.3.4A,C). In contrast,

previous systemic administration of the A1 receptor antagonist CPT did

not counteract the influence of cortical stimulation upon Ret

Results

113

phosphorylation (13710.2%, n=7) (Figure 3.3.5A,D). As shown in

Figures 3.3.3A, total Ret levels were not significantly affected by cortical

stimulation in any group of animals.

0

50

100

150

Veh CPT

Ret

ph

osh

ory

lati

on

(%

of

co

ntr

ol)

+ S

0

50

100

150

Veh Veh+S

Ret

ph

osh

ory

lati

on

(%

of

co

ntr

ol)

B. C. D.

A.

pRet

Total Ret 170 kDa

170 kDa

Veh

Veh

+S

MS

X-3

MS

X-3

+S

CP

T

CP

T+

S

(1) (2) (3)(0)

(1)(0)(2)(1) (3)(1)

0

50

100

150

Veh

MSX-3

*

Ret

ph

osh

ory

lati

on

(% o

f c

on

tro

l)

+ S

0

50

100

150

Veh CPT

Ret

ph

osh

ory

lati

on

(%

of

co

ntr

ol)

+ S

0

50

100

150

Veh Veh+S

Ret

ph

osh

ory

lati

on

(%

of

co

ntr

ol)

B. C. D.

A.

pRet

Total Ret 170 kDa

170 kDa

Veh

Veh

+S

MS

X-3

MS

X-3

+S

CP

T

CP

T+

S

(1) (2) (3)(0)

(1)(0)(2)(1) (3)(1)

0

50

100

150

Veh

MSX-3

*

Ret

ph

osh

ory

lati

on

(% o

f c

on

tro

l)

+ S

Figure 3.3.3 - Striatal phosphorylation of the GDNF receptor component Ret is

induced by cortical electrical stimulation and depends on A2A receptor activation.

A. Representative Western blots. B. Ret phosphorylation induced by cortical electrical

stimulation. C. A2A receptor antagonist, MSX-3 counteracted Ret phosphorylation

induced by cortical stimulation. D. A1 receptor antagonist CPT does not counteract Ret

phosphorylation induced by cortical stimulation. Results are shown in meansSEM (n =

6-8 per group) of representative Western blots. p<0.05, significantly different compared

with vehicle-treated non stimulated group (ANOVA with Tukey–Kramer tests). *p<0.05,

significantly different compared with vehicle-treated stimulated group (ANOVA with

Tukey–Kramer tests).


114

3.3.4. Discussion

The present findings provide the first demonstration of the ability

of GDNF to directly enhance glutamate release dependently on the co-

activation of A2A receptors. It was recently shown by using hippocampal

preparations (Koeberle et al., 2008) that GDNF exerts an anti-apoptotic

effect by up-regulating glutamate transporters in the CNS and it acutely

modulates synaptic transmission in the midbrain dopaminergic neurons,

which co-release dopamine and glutamate (Bourque and Trudeau, 2000;

Wang et al., 2003). However, a direct evaluation of the actions of GDNF

upon striatal glutamate release has not been performed so far.

It is usually conceived that the beneficial effects of GDNF in

Parkinson´s disease result from its protective and/or restorative effects

acting as a growth factor on nigrostriatal dopaminergic neurons

(reviewed in Hurelbrink and Barker, 2004), but GDNF also exerts

beneficial effects in animal models of Huntington‟s disease (reviewed in

Alberch et al., 2004), where the control of the viability of dopaminergic

neurons is not considered a prominent etiological factor. Furthermore,

there is increasing evidence that neurodegeneration begins with a

synaptic dysfunction which later evolves into an overt damage of

neurons through a dying back mechanism (Coleman and Perry, 2002;

Wishart et al., 2006). This has been documented in either Parkinson‟s

(reviewed in Dauer and Przedborski, 2003) or Huntington‟s disease (Li

et al., 2001; 2003) and suggests that the beneficial role of GDNF in

different neurodegenerative diseases affecting striatal circuits can also

be adequately explained by considering its direct effects within the

striatum and, according to the present data, in glutamatergic terminals.

Results

115

Another important conclusion from this study is the re-inforcement of the

importance of adenosine A2A receptors in the control of the effects

operated by GDNF. In fact, it was now observed that GDNF causes a

minor effect on the release of glutamate, which was enabled upon

activation of A2A receptors. Presynaptic A2A receptors localized in

glutamatergic nerve terminals have been recently demonstrated to exert

an important modulatory role of striatal glutamate release (Ciruela et al.,

2006). The main source of adenosine that activates these presynaptic

A2A receptors seems to be ATP which is co-released with glutamate

(Ferré et al., 2005). As pointed out in the Introduction (see 1.3.3.), A2A

receptors localized in glutamatergic nerve terminals form heteromers

with A1 receptors and function as a concentration-dependent switch, by

which low concentrations of adenosine (by acting at A1 receptors) inhibit

glutamate release and high concentrations of adenosine (by acting at

A2A receptors, which shuts down A1 receptor signalling), facilitate

glutamate release (Ferré et al., 2007). The present results indicate that

GDNF may provide an additional key component for the operation of the

A1-A2A receptor heteromer.

The in vitro approaches were also extended to in vivo studies

further supporting the pivotal role of A2A receptors to enable the

functioning of the modulation system operated by GDNF. It was

observed that cortical stimulation of the orofacial area of the lateral

agranular motor cortex increases Ret phosphorylation in the ipsilateral

striatum, which indicates that stimulation of cortical afferents promotes

activation of the effector of the GDNF receptor in the striatum.

Accordingly, GDNF protects several types of neurons against glutamate-

induced toxicity in different brain areas (Yoo et al., 2006; Wong et al.,


116

2005) and it was observed an up-regulation of GDNF and its receptors

after excitotoxic insults to the striatum (Alberch et al., 2002). The cortical

stimulation-induced of Ret phosphorylation requires co-activation of A2A

receptors, further suggesting that GDNF actions in the striatum are

seriously impaired when A2A receptors are not free to be activated by its

endogenous ligand. As described in the previous section (see 3.2 and

also Quiroz-Molina et al., 2006), under conditions of a strong cortico-

limbic-thalamic input, the electrical stimulation-induced phosphorylation

of different substrates implicated in plasticity (such as ERK1/2) is

dependent upon A2A receptor activation and it is important to notice that

GDNF also has the ability to enhance the phosphorylation of ERK1/2

(Kobori et al., 2004; Salvatore et al., 2004; Yang et al., 2001).

Altogether, these observations suggest that GDNF, dependently on A2AR

activation, controls glutamatergic inputs to the striatum and, on the other

hand, may be recruited as a defensive response to glutamate

excitotoxicity and/or possibly involved in plastic changes in the striatum.

Results

117

3.4. GDNF receptor components distribution in the rat striatum,

cellular and subsynaptic localization and co-localization with A2A

receptor


The striatum is a complex network of neuronal circuits, which are

triggered by glutamatergic afferents, are mainly GABAergic in nature

and are modulated by a main external dopaminergic nigrostriatal input

(Gerfen, 2004). There are also cholinergic interneurons and GABAergic

neurons (Tepper and Bolam, 2004). To understand the role of the GDNF

presynaptic modulation system, it is important to determine which

type(s) of nerve terminals are equipped with the GDNF receptor system.

In spite of some studies on the regional and cellular distribution of GDNF

receptor components at the mRNA expression level (Wang et al., 2008;

Cho et al., 2004; Sarabi et al., 2003; Widenfalk et al., 1997; Nosrat et al.,

1997; Trupp et al., 1997), a systematic analysis of GDNF receptor

distribution among different striatal nerve terminals at the protein level

has not yet been reported so far. The distribution of GDNF receptors in

several neuronal populations in the striatum was therefore analysed in

order to further support a physiological role of GDNF upon

neurotransmitter release.

Presynaptic modulation systems can either directly control

release of neurotransmitters or indirectly modify it through the control of

the metabolism and/or viability of synaptic contacts. The former are

expected to be located in the active zone (i.e. where neurotransmitter

release takes place), where the later are not required to display such a


118

confined localization. To gauge the possible relevance of the GDNF

receptor system as a modulator of neurotransmitter release, I next

investigated the cellular and sub-synaptic localization of Ret and GFR1,

in particular their localization in the active zone. Finally, it seemed of

particular importance to evaluate if GDNF receptor components co-

localize with adenosine A2A receptors at the cellular level, particularly at

glutamatergic and dopaminergic terminals, where a functional interplay

between GDNF and A2A receptors was observed.

3.4.2. GDNF receptor proteins are located in striatal nerve terminals

As illustrated in Figure 3.4.1, both Ret (Figures 3.4.1A,B) and

GFR1 (Figures 3.4.1A,C) immunoreactivity were present in

synaptosomal membranes with a density similar to that found in total

membranes from the whole striatum.

Both proteins were present in the active zone (Figure 3.4.2A).

Thus, when quantifying the relative density of Ret and GFR1

immunoreactivity in the three sub-synaptic fractions (presynaptic active

zone, postsynaptic density and extra-synaptic fractions) in three different

assays from different groups of rats, it was found that Ret and GFR1

immunoreactivity is mainly located in the presynaptic active zone fraction

(34±5.5% and 35±7.3% of total immunoreactivity, n=5) and in the

postsynaptic density (50±3.0% and 52±8.9% of total immunoreactivity,

n=5) and has a significantly (p<0.05) lower relative abundance in the

extrasynaptic fraction of nerve terminals (17±3.4% and 12±3.1% of total

immunoreactivity, n=5) (Figures 3.4.2B,C).

The next step was to estimate if the proteins constituting the

Results

119

GDNF receptor were confined to a reduced number of nerve terminals or

if most nerve terminals in the striatum were equipped with this

modulation system. For that purpose, a double immunocytochemical

labelling of Ret or GFR1 was carried out with a marker of nerve

terminals, synaptophysin, a protein located in synaptic vesicles. As

illustrated in Figure 3.4.3A, there was a co-localization of synaptophysin

with either Ret or GFR1. Thus, 56±2.3% (n=3) of the general population

of striatal nerve terminals, identified as synaptophysin immunopositive

elements were also endowed with Ret receptor component and 61±1.2%

(n=4) of the referred elements are equipped with GFR1 component of

the receptor (Figure 3.4.3B). Therefore, a high proportion of the nerve

endings at the striatum possess GDNF receptor complex.


120

A.

40 60 80

0

25

50

75

100

125Synaptosomal membranes

Total membranes

Amount of protein ( g)

Ret

imm

un

ore

acti

vit

y

(% o

f m

axim

um

)

B.

40 60 80

0

25

50

75

100


Total membranes

Amount of protein (g)

GF

R

1im

mu

no

rea

cti

vit

y

(% o

f m

ax

imu

m)

C.

A.

40 60 80

0

25

50

75

100


Total membranes

Amount of protein ( g)

Ret

imm

un

ore

acti

vit

y

(% o

f m

axim

um

)

B.

40 60 80

0

25

50

75

100


Total membranes

Amount of protein (g)

GF

R

1im

mu

no

rea

cti

vit

y

(% o

f m

ax

imu

m)

C.

Figure 3.4.1 - GDNF receptor proteins (Ret and GFRα1) are present in

synaptosomal membranes with a similar density to that found in total membranes

from the whole striatum. A. Representative Western blots showing that both Ret and

GFRα1 immunoreactivity is located in membranes from purified nerve terminals with a

density similar to that found in total striatal membranes. B. Quantification of the

immunoreactivity of Ret (panel A). C. Quantification of the immunoreactivity of GFR1

(panel A). Results are shown in mean±SEM of 4 experiments.

Results

121

0

10

20

30

40

50

60

70

Pre Post Extra

Re

t im

mu

no

rea

cti

vit

y

(% o

f co

ntr

ol)

0

10

20

30

40

50

60

70

Pre Post Extra

GF

R

1 i

mm

un

ore

ac

tiv

ity

(%

of

co

ntr

ol)

B.

C.

A.

Ret

GFRα1

SNAP25

PSD95

Synaptophysin

Pre Post Extra SF

0

10

20

30

40

50

60

70

Pre Post Extra

Re

t im

mu

no

rea

cti

vit

y

(% o

f co

ntr

ol)

0

10

20

30

40

50

60

70

Pre Post Extra

GF

R

1 i

mm

un

ore

ac

tiv

ity

(%

of

co

ntr

ol)

B.

C.

A.

Ret

GFRα1

SNAP25

PSD95

Synaptophysin

Pre Post Extra SF

Figure 3.4.2 - GDNF receptor proteins (cRet and GFRα1) are located in synapses,

namely in the presynaptic active zone of striatal nerve terminals. A. Representative

Western blots showing that Ret and GFRα1 immunoreactivity is mainly located in the

presynaptic active zone and in the postsynaptic density, as evaluated upon subsynaptic

fractionation of purified striatal nerve terminals into the presynaptic active zone (enriched

in SNAP25), postsynaptic density (enriched in PSD95) and extra-synaptic fraction

(enriched in synaptophysin); the last lane (SF) shows immunoreactivity in the

synaptosomal fraction before subsynaptic fractionation. B. Quantification of the

immunoreactivity of Ret (panel A). C. Quantification of the immunoreactivity of GFR1

(panel A). Results are shown in mean±SEM of 5 experiments.


122

0

25

50

75

Ret GFR1

% s

yn

ap

top

hysin

im

mu

no

po

sit

ive n

erv

e t

erm

inals

A.

B.

GFR1

Ret

MergedGDNF

Receptor Syn

0

25

50

75

Ret GFR1

% s

yn

ap

top

hysin

im

mu

no

po

sit

ive n

erv

e t

erm

inals

A.

B.

GFR1

Ret

MergedGDNF

Receptor Syn

Figure 3.4.3 - Ret and GFRα1 are located in more than half of striatal nerve

terminals. A. Representative double labelling immunocytochemical co-localization of the

Ret and GFRα1 immunoreactivity with synaptophysin in striatal nerve terminals. As

shown in B., more than half of striatal nerve terminals (synaptophysin-immunopositive)

were endowed with Ret and GFRα1 immunoreactivity (mean±SEM of 3-4 experiments).

Results

123

3.4.3. Mapping GDNF receptor components in different types of

striatal nerve terminals

Double immunocytochemical studies were carried out looking at

the co-localization of Ret or of GFR1 immunoreactivity with markers of

the different types of nerve terminals. As shown in Figures 3.4.4A and

3.4.5A, only few GABAergic terminals (immunopositive for vesicular

GABA transporters) were endowed with Ret (18±2.5%, Figure 3.4.4B) or

GFR1 (21±1.2%, Figure 3.4.5B) immunoreactivity (n=4). The

percentage of cholinergic terminals (immunopositive for vesicular

acetylcholine transporters) equipped with at least one of the GDNF

receptor components is even lower (Ret: 4.7±2.7%; GFR1: 5.3±3.1%,

n=4, Figures 3.4.4B and 3.4.5B). In contrast, numerous dopaminergic

terminals (immunopositive for tyrosine hydroxylase) were

immunopositive for GDNF receptor proteins (Ret: 61±1.6%; GFR1:

56±0.3%, n=3-4, Figures 3.4.4B and 3.4.5B). Likewise, a large

percentage of striatal glutamatergic terminals (immunopositive for

vesicular glutamate transporters 1 and 2) were immunopositive for

GDNF receptor components (Ret: 45±2.4%; GFR1: 53±0.9%, n=3-4,

Figures 3.4.4B and 3.4.5B).


124

vGLU

T1/2

0

10

20

30

40

50

60

70

TH

vGAT

vA

ChT

% c

o-l

oca

lizati

on

Re

t im

mu

no

rea

cti

vit

y

A.

B.

Ret

vGluT1/2

vGAT

vAChT

TH

MergedNerve terminal

vGLU

T1/2

0

10

20

30

40

50

60

70

TH

vGAT

vA

ChT

% c

o-l

oca

lizati

on

Re

t im

mu

no

rea

cti

vit

y

A.

B.

Ret

vGluT1/2

vGAT

vAChT

TH


Figure 3.4.4 - Ret protein is mainly located in dopaminergic and glutamatergic,

rather than GABAergic and cholinergic nerve terminals in the rat striatum. A.

Representative double labelling immunocytochemical co-localization of Ret and different

markers of different types of nerve terminals, namely tyrosine hydroxylase (TH, marker

of dopaminergic neurons, first row), vesicular glutamate transporters types 1 and 2

(vGluT1/2, markers of glutamatergic terminals, second row), vesicular GABA transporter

(vGAT, marker of GABAergic terminals, third row) and vesicular acetylcholine transporter

(vAChT, marker of cholinergic terminals, last row). B. Average number of each type of

terminals endowed with Ret, showing that this protein constituting the GDNF receptor is

more abundantly located in dopaminergic and glutamatergic terminals of the rat striatum.

The results are mean±SEM of 3-4 experiments.

Results

125

A.

B.

GFR1

GFR1

TH

vGLUT1/2

vGAT

vAchT


vGAT

0

10

20

30

40

50

60

70

TH

vGLU

T1/2

vAChT

% c

o-l

oca

lizati

on

GF

R

1im

mu

no

rea

cti

vit

y

A.

B.

GFR1

GFR1

TH

vGLUT1/2

vGAT

vAchT


vGAT

0

10

20

30

40

50

60

70

TH

vGLU

T1/2

vAChT

% c

o-l

oca

lizati

on

GF

R

1im

mu

no

rea

cti

vit

y

Figure 3.4.5 - GFR1 protein is mainly located in dopaminergic and glutamatergic,

rather than GABAergic and cholinergic nerve terminals in the rat striatum. A.

Representative double labelling immunocytochemical co-localization of GFR1 and

different markers of different types of nerve terminals, namely tyrosine hydroxylase (TH,

marker of dopaminergic neurons, first row), vesicular glutamate transporters types 1 and

2 (vGluT1/2, markers of glutamatergic terminals, second row), vesicular GABA

transporter (vGAT, marker of GABAergic terminals, third row) and vesicular acetylcholine

transporter (vAChT, marker of cholinergic terminals, last row). B. Average number of

each type of terminals endowed with GFR1, showing that this protein constituting the

GDNF receptor is more abundantly located in dopaminergic and glutamatergic terminals

of the rat striatum. The results are mean±SEM of 3-4 experiments.


126


glutamatergic and dopaminergic nerve terminals

The tight control by A2A receptors of the effects of GDNF in both

glutamatergic and dopaminergic terminals should require that both

receptor systems should be located in a subset of both glutamatergic

and dopaminergic terminals. Triple immunocytochemical analysis of the

GDNF receptor proteins (Ret and GFR1), A2A receptors and markers of

either glutamatergic (vGluT1/2) or dopaminergic (TH) terminals were

therefore performed.

As illustrated in Figure 3.4.6A,B, 13±0.9% (n=3) of the vGluT1/2-

positive striatal nerve terminals are simultaneously equipped with both

A2A and Ret receptor and 9.4±1.6% (n=3) of the vGluT1/2 positive nerve

terminals are equipped with both A2A and GFR1 receptor. Likewise, we

also found that 12±1.0% (n=3) of the TH-positive nerve terminals are

equipped with both A2A and Ret receptor and that 11±0.4% (n=3) of the

TH-positive nerve terminals are equipped with both A2A and GFR1

receptor (Figure 3.4.7A,B).

Since the localization of A2A receptors in dopaminergic terminals

is frequently questioned (e.g. Svenningsson et al., 1999), a confirmatory

double immunocytochemical study was carried out using striatal

synaptosomes to assess the presence of A2A receptor in striatal

dopaminergic nerve terminals [labelled with TH or DAT (dopamine

transporter) immunoreactivity]. We found that 21±4.3% (n=6) of the TH-

immunopositive striatal nerve terminals were endowed with A2A receptor

immunoreactivity and 22±1.1% (n=5) of the DAT-immunopositive striatal

nerve terminals were endowed with A2A receptor immunoreactivity

Results

127

(Figure 3.4.8). This constitutes the first direct demonstration for the

presence of A2A receptors in dopaminergic terminals in the striatum,

reinforcing previous functional data showing that A2A receptors enable

GDNF actions in striatal dopaminergic nerve endings (see also Gomes

et al., 2006).

A.

Ret

GFR1

0

5

10

15

cRet GFR1

A2AR (+)

% v

GL

UT

1/2

im

mu

no

po

sit

ive

te

rmin

als

en

do

wed

wit

h A

2A

an

d R

et

or

GF

R

1

B.

A2AR

GDNF

ReceptorvGluT1/2 Merged

A.

Ret

GFR1

0

5

10

15

cRet GFR1

A2AR (+)

% v

GL

UT

1/2

im

mu

no

po

sit

ive

te

rmin

als

en

do

wed

wit

h A

2A

an

d R

et

or

GF

R

1

B.

A2AR

GDNF


Ret

GFR1

0

5

10

15

cRet GFR1

A2AR (+)

% v

GL

UT

1/2

im

mu

no

po

sit

ive

te

rmin

als

en

do

wed

wit

h A

2A

an

d R

et

or

GF

R

1

B.

A2AR

GDNF


Figure 3.4.6 - GDNF receptor proteins (Ret and GFRα1) are co-located with

adenosine A2A receptors in glutamatergic nerve terminals of the rat striatum. A.

Representative triple labelling immunocytochemical co-localization of adenosine A2A

receptors and either Ret (upper row) or GFRα1 (lower row) in glutamatergic nerve

terminals (immunopositive for vesicular glutamate transporters types 1 and 2, vGluT1/2),

which is quantified in panel B. Average number of glutamatergic terminals endowed with

either A2AR and Ret/GFR1 (mean±SEM of 3 experiments).


128

0

5

10

15

cRet GFR1

A2AR (+)

% T

H i

mm

un

op

os

itiv

e t

erm

ina

ls

en

do

wed

wit

h A

2A

an

d R

et

or

GF

R

1

Ret

GFR1

A.

B.

A2AR

GDNF

ReceptorTH Merged

0

5

10

15

cRet GFR1

A2AR (+)

% T

H i

mm

un

op

os

itiv

e t

erm

ina

ls

en

do

wed

wit

h A

2A

an

d R

et

or

GF

R

1

Ret

GFR1

A.

B.

A2AR

GDNF

ReceptorTH Merged


adenosine A2A receptors in dopaminergic nerve terminals of the rat striatum. A.

Representative triple labelling immunocytochemical co-localization of adenosine A2A

receptors and either Ret (upper row) or GFRα1 (lower row) in dopaminergic nerve

terminals (immunopositive for tyrosine hydroxylase, TH), which is quantified in panel B.

Average number of dopainergic terminals endowed with either A2AR and Ret/GFR1

(mean±SEM of 3 experiments).

Results

129

0

10

20

30

TH DAT

% T

H o

r D

AT

im

mu

no

po

sit

ive

for

A2A

re

ce

pto

rs

A.

B.

A2AR MergedNerve

terminal

TH

DAT

0

10

20

30

TH DAT

% T

H o

r D

AT

im

mu

no

po

sit

ive

for

A2A

re

ce

pto

rs

A.

B.

A2AR MergedNerve

terminal

TH

DAT

A2AR MergedNerve

terminal

TH

DAT

Figure 3.4.8 - Adenosine A2A receptors are localized at striatal dopaminergic nerve

terminals. A. Representative double labelling immunocytochemical co-localization of

adenosine A2A receptors in dopaminergic terminals, labelled either with tyrosine

hydroxylase (TH, upper row) or dopamine transporters (DAT, lower row). B. Average

number of each type of TH- or DAT-immunopositive terminals endowed with A2A

receptors (mean±SEM of 5-6 experiments).


130

3.4.5. Discussion

The present results provide direct morphological evidence

indicating that adenosine A2A receptors co-locate with Ret and GFR1,

GDNF receptor components, in a subset of dopaminergic and

glutamatergic terminals of the rat striatum. Indeed, there was a co-

localization of A2A receptors and Ret/GFR1 immunoractivity in striatal

nerve terminals and, in particular, in nerve terminals endowed with the

tyrosine hydroxylase (TH) and with the vesicular glutamate transporters

type1 and type 2 (vGlut 1/2), that is, dopaminergic and glutamatergic

nerve terminals. These observations are consistent with the ability of

GDNF to presynaptically enhance the release of dopamine (see 3.2. and

also Gomes et al., 2006) and glutamate (see 3.3. and also Gomes et al.,

2009) from striatal nerve endings, upon the control of adenosine A2A

receptors.

The main question arising from these observations relates to the

subcellular distribution of A2A receptors in the striatum. Indeed, the

majority of these receptors is located outside nerve terminals in striatal

tissue, namely in the postsynaptic density (Hettinger et al., 2001), where

they exert their main striatal function, the control of the responsiveness

of MSNs. It should be emphasised that these results are not in

opposition to the well-defined postsynaptic role for adenosine in this

brain area. Instead, they suggest a complementary role for A2A receptors

at the presynaptic site, where they interact with receptors for

neurotransmitters or trophic factors, in a strategic position to modulate

synaptic functioning. Previous studies by others (Rodrigues et al., 2005;

Ciruela et al., 2006) have documented the presence of adenosine A2A

Results

131

receptors at the striatal glutamatergic terminal. At these terminals, the

authors report the functional interaction between A2A receptors and

glutamate metabotropic group 5 receptors (Rodrigues et al., 2005) or A1

receptors (Ciruela et al., 2006) in the control of neurotransmitter release,

which is consistent with the existence of receptor heteromers (Ferré et

al., 2005; Ciruela et al., 2006). If evidence exists documenting the

presence of A2A receptors at the glutamatergic terminal, their presence

at the dopaminergic terminals of the striatum was still a matter of debate

before the present work. As stated above (see 3.1.7), adenosine does

not control striatal dopamine release via A2A receptors receptors (Jin et

al., 1993; Lupica et al., 1990 and present results, see 3.1.2) and this

observation led to the idea that these receptors should be absent from

this locus. However, we clearly demonstrate the presence of the

receptor at the dopaminergic nerve ending by performing morphologic

studies, which further confirm the presynaptic modulatory role of A2A

receptors upon dopamine release (see 3.1.2).

Regarding GDNF, it was necessary to determine which type(s) of

nerve terminals are equipped with its receptor proteins, since a

systematic study at the protein level has not been reported so far.

Double immunocytochemical labelling of Ret or GFR1 with a marker of

nerve terminals, synaptophysin, a protein located in synaptic vesicles,

showed that a high proportion of the nerve endings at the striatum

possess GDNF receptor complex. However, only few GABAergic and

cholinergic terminals were endowed with Ret or GFR1 immunoreactivity,

in contrast to the numerous dopaminergic and glutamatergic terminals

immunopositive for GDNF receptor proteins, which may suggest a major

role for GDNF in the control of dopamine and glutamate release. Studies


132

on the subsynaptic localization of Ret and GFR1 revealed that the

proteins are mainly located in the presynaptic active zone fraction

(where neurotransmitter release takes place, in agreement with the

presynaptic modulatory role proposed for GDNF) and in the postsynaptic

density and have a significantly lower relative abundance in the

extrasynaptic fraction of nerve terminals.

133

CO

NC

LU

SIO

N

Conclusion

135

4. CONCLUSION

The main goal of the present work was to investigate the possible

modulation role of adenosine A2A receptors of the GDNF-mediated

effects at the neurotransmitter release level in the striatum. The main

conclusions are that: (a) GDNF facilitates dopamine and glutamate

release in a manner dependent on adenosine A2A receptor activation; (b)

in vivo stimulation of the cortical (glutamatergic) afferents triggers

phosphorylation of the GDNF receptor protein, Ret, in the striatum, an

effect that requires the tonic activation of A2A receptors; (c) GDNF

receptor proteins are located in striatal nerve terminals, mainly

glutamatergic and dopaminergic, and are co-located with adenosine A2A

receptors.

After the publication of the first results included in this thesis,

namely on the functional interaction between GDNF and adenosine

(Gomes et al., 2006), Yamagata et al. (2007) found that adenosine

regulates GDNF expression and production in primary cultures of rat

astrocytes. To date, these are the only reports firmly establishing a

relation between adenosine and GDNF. Thus, the observations detailed

in the present thesis provide a first evidence for a functional crosstalk

between adenosine A2A receptors and GDNF and allow ascribing a novel

role for GDNF in the striatum, acting on a short time scale as a

presynaptic neuromodulator, apart from acting in a larger time scale

as a trophic factor. In fact, we now report that GDNF presynaptically

controls the release of dopamine and glutamate, in accordance with the

localization of the GDNF receptor proteins in dopaminergic and

glutamatergic terminals. This ability of GDNF to modulate


136

neurotransmitter release seems restricted to the control of glutamate

and dopamine since only few GABAergic and cholinergic terminals were

endowed with Ret/GFR1 immunoreactivity.

In contrast with what occurs in cholinergic and GABAergic striatal

nerve endings (Kirk and Richardson, 1994), dopamine release from

striatal nerve endings is not under control of adenosine A2A receptors

(Jin et al., 1993; Lupica et al., 1990 and present results), which led to the

concept that these receptors might be absent from striatal dopaminergic

nerve endings. However, we clearly show that A2A receptors in striatal

dopaminergic nerve terminals modulate the action of GDNF on

dopamine release. Therefore, the role of A2A receptors in the

dopaminergic nerve endings might be as modulators of the action of

other presynaptic receptors, rather than exerting a direct modulation of

dopamine release. A2A receptors localized in glutamatergic nerve

terminals form heteromers with A1 receptors and function as a

concentration-dependent switch, by which fluctuations in adenosine

concentrations inhibit (by acting at A1 receptors) or facilitate glutamate

release (Ciruela et al., 2006; Ferré et al., 2007). The present results

indicate that GDNF can provide an additional key component for the

operation of the A1-A2A receptor heteromer, reinforcing the idea (see

Sebastião and Ribeiro, 2000) that a main role of A2A receptors in

different brain areas is to interact with receptors for other

neuromodulators, a process probably involved in the fine tuning of

neuronal function.

The observation that GDNF controls the two key transmitter

systems involved in defining the firing pattern of striatal circuits has

profound implications for the conception of the role of GDNF in striatal

Conclusion

137

circuitry. This simultaneous ability of GDNF to presynaptically control the

release of glutamate (the trigger of striatal circuits) as well as the release

of dopamine (the main modulator defining the ON state of the targeted

medium spiny neurons) predicts a powerful effect of GDNF on striatal

plasticity, which is considered a key event both in striatal physiology

as well as major adapting and contributing factor in neurodegenerative

diseases affecting striatal circuits (Calabresi et al., 2007). Whereas it is

known that A2A receptors control synaptic plasticity in the basal ganglia

(D‟Alcantara et al., 2001), it remains to be defined if GDNF participates

in synaptic plasticity changes at corticostriatal synapses in an adenosine

A2A receptor dependent manner. Likewise, the functional interaction

between A2A receptors and GDNF to control the demise of

neurodegenerative processes affecting striatal circuits should also be

considered. The beneficial role of GDNF in different neurodegenerative

diseases affecting striatal circuits can be adequately explained by

considering the direct effects of GDNF within the striatum and, according

to the present data, in glutamatergic and dopaminergic terminals.

However, the present findings that the tonic activation of A2A receptors

determines the activation and function of the GDNF modulation system,

clearly point towards the need of further studies on the consequences of

long-term therapy with A2A receptor blockers in neurodegenerative

diseases where GDNF may have a beneficial role.

These in vitro observations were extended with in vivo studies

further supporting the pivotal role of A2A receptors to enable the

functioning of the modulation system operated by GDNF. Thus, I

observed that cortical stimulation increased Ret phosphorylation in the

striatum compared to electrode-implanted non-stimulated controls, an


138

effect counteracted by A2A receptor antagonism. These results indicate

that stimulation of cortical afferents promotes activation of the effector of

the GDNF receptor in the striatum, a process that requires co-activation

of A2A receptors. The ability of the GNDF system to facilitate

neurotransmitter release and to become activated upon recruitment of

the corticostriatal pathway suggests that GDNF might be part of a

positive feedback loop potentiating corticostriatal transmission. This loop

is under tight control by adenosine A2A receptor, so that GDNF facilitates

the release of transmitters, which in turn may result in enhanced

synaptic activity and further facilitates activation of the GDNF receptor

complex (Figure 4.1).

Finally, I obtained direct morphological evidence to support the

tight interaction between A2A and GDNF receptors, showing for the first

time the co-location of GDNF and A2A receptors at both dopaminergic

and glutamatergic terminals in the striatum.

In summary, I now provide the first direct morphological evidence

indicating that GDNF receptor proteins are located in striatal

glutamatergic and dopaminergic nerve terminals and that these

receptors are co-located with A2A receptors in a subset of these

terminals. This supports the ability of GDNF to facilitate dopamine and

glutamate release in a manner dependent on the activation of A2A

receptors in both dopaminergic and glutamatergic nerve terminals.

Phosphorylation of Ret, the first step of the transducing signalling

triggered by GDNF after binding to its receptor, also requires co-

activation of A2A receptors, further suggesting that GDNF actions in the

striatum are seriously impaired when A2A receptors are not free to be

activated by its endogenous ligand. Altogether, these findings prompt a

Conclusion

139

novel view on the role of GDNF in striatal circuits, where it fulfils a

synaptic short term neuromodulation role apart from its long term role as

a neurotrophic factor. Furthermore, the synaptic localization of the

GDNF modulation system controlling glutamatergic synapses in a

manner tightly controlled by A2A receptors opens a novel working

hypothesis to understand the control of synaptic plasticity and

neurodegeneration by these two receptor systems.

Glutamate

Dopamine

1

2

A2AR

GDNF

Ret

P

P

Striatal A2AR-mediated

Ret phosphorylation

SNc

Cerebral cortex

Dorsal striatum

Output

structu

re

MSN

1

2

Corticostriatal efferents

stimulation

A. B.

Glutamate

Dopamine

1

2

A2AR

GDNF

Ret

P

P


Ret phosphorylation

SNc

Cerebral cortex

Dorsal striatum

Output

structu

re

MSN

1

2


stimulation

Glutamate

Dopamine

1

2

A2AR

GDNF

Ret

P

P

GlutamateGlutamate

DopamineDopamine

1

2

A2AR

GDNF

Ret

P

P


Ret phosphorylation

SNc

Cerebral cortex

Dorsal striatum

Output

structu

re

MSN

1

2


stimulation

SNc

Cerebral cortex

Dorsal striatum

Output

structu

re

MSN

1

2


stimulation

A. B.

Figure 4.1 - Hypothetical model for a new role of adenosine A2A receptor in striatal

physiology - focus on the functional interaction and receptor co-localization with

GDNF. A. Neuronal inputs (glutamatergic and dopaminergic from cortex and substantia

nigra pars compacta) to the striatum and stimulation of the corticostriatal glutamatergic

efferents. B. A magnified scheme of presynaptic glutamatergic (1) and dopaminergic (2)

terminals, showing A2AR-mediated Ret phosphorylation, subsequent to the corticostriatal

stimulation and the overall result, neurotransmitter release.


140

141

RE

FE

RE

NC

ES

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143

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