Dopaminergic control of the globus pallidus and its impact

UNIVERSITÉ MOHAMMED V
FACULTÉ DES SCIENCES
Rabat
N° d’ordre 2759
THÈSE DE DOCTORAT
Présentée par
MAMAD Omar
Discipline : Biologie
Spécialité : NEUROSCIENCES
Dopaminergic control of the globus pallidus
and its impact on the subthalamic nucleus
and the pars reticulata of substantia nigra
CONTROLE DOPAMINERGIQUE DU GLOBUS PALLIDUS ET SON IMPACT SUR LE
NOYAU SOUS-THALAMIQUE ET LA SUBSTANCE NOIRE RETICULEE CHEZ LE RAT
Soutenue le 25 Février 2015
Devant le jury
Présidente:
Mme Nouria LAKHDAR-GHAZAL Professeur à la Faculté des Sciences de Rabat
Examinateurs :
Mr Mohammed ERRAMI
Professeur à la Faculté des Sciences de Tétouan
Mr Mohammed BENNIS
Professeur à la Faculté des Sciences de Marrakech
Mr Abdelhamid BENAZZOUZ
Directeur de Recherche INSERM, Bordeaux
Mr Wail BENJELLOUN
Professeur à la Faculté des Sciences de Rabat
Dédicace
A Mes parents,
Aucune dédicace ne saurait exprimer mon grand amour, mon estime et ma profonde affection.
Ce travail est le résultat de votre éducation et votre encouragement, je vous souhaite une
longue vie pleine de joie et de bonheur.
A mes chers frères, mes neveux et mes nièces
Mostapha, Hassan, Ibrahim, Abderazak, Mohamed, Fatima Zohra, Rachid, Soumia, Fahd,
Mohamed-Amine, Hafsa, Hajar et Sarah…
Pour le soutien que vous m‟avez toujours apporté.
Je vous souhaite tout le bonheur du monde
A Tous mes amis que j‟aime et que j'ai rencontré au cours de ces belles années à Bordeaux,
Soufienne, Amir, Karim, Nguen, Otmane, Jonathan , Yohann et Mohcine, pour votre aide à la
réalisation de ce projet, je vous souhaite une vie pleine de bonheur et de réussite.
2
Remerciements
Les travaux présentés dans la thèse ont été réalisés au sein de :
-
l‟Equipe « Rythmes biologiques, Neuroscience et Environnement » dirigée par le
Professeur Nouria Lakhdar-Ghazal, à la Faculté des Sciences de Rabat au Maroc
-
l‟Equipe « Monoamines, Stimulation Cérébrale Profonde et Maladie de Parkinson »
dirigée par le Docteur Abdelhamid Benazzouz à l‟Institut des Maladies
Neurodégénératives (CNRS UMR 5293) de l‟Université de Bordeaux en France
J‟ai eu la chance d‟être accueilli par ces deux laboratoires de localisations distinctes mais dont
les liens sont très forts.
Ce travail de thèse n‟aurait pas vu le jour sans mes deux directeurs de thèse à qui je tiens à
présenter mes vifs remerciements :
- le Pr. Wail Benjelloun, professeur à la Faculté des Sciences et Président de
l‟Université Mohammed V de Rabat, pour m‟avoir offert l‟opportunité d‟intégrer l‟école
doctorale de Neurosciences et de m‟avoir accueilli au sein de son laboratoire de recherche. Il
a accepté de m‟encadrer personnellement pendant la préparation de mon diplôme de Master et
puis pour mon diplôme de Doctorat. Son expérience, sa patience et sa disponibilité, malgré
ses nombreuses responsabilités, m‟ont été très bénéfiques durant toutes ces années de travail.
- le Dr. Abdelhamid Benazzouz, Directeur de Recherche INSERM, de m‟avoir
accueilli au sein de son équipe de recherche et de m‟avoir encadré pendant toute ma thèse. Je
le remercie pour son soutien moral et matériel dont j‟ai pu bénéficier. J‟étais et je reste très
touché par sa grande patience. Le Dr. Benazzouz m‟a permis de travailler sur un sujet
passionnant plein de challenges scientifiques. J‟ai beaucoup appris durant mon séjour à
Bordeaux sur les plans scientifique et humain. Vous êtes mon deuxième père et votre famille
est ma deuxième famille, je ne me suis jamais senti étranger ou loin de ma famille. Votre
simplicité et votre confiance en moi sont autant de preuves qui scellent le respect et l‟amitié
que j‟ai pour vous.
Je tiens à remercier tous les membres de mon jury d‟avoir accepté de juger ce travail.
Un grand merci au Pr. Nouria Lakhdar-Ghazal, Professeur à la Faculté des Sciences de
Rabat, d‟avoir aimablement accepté de présider le jury de cette thèse. Je la remercie de
m‟avoir fait découvrir ce fascinant domaine des Neurosciences et d‟avoir suivi
continuellement l‟état d‟avancement de mes travaux de recherche.
3
Un grand merci au Pr. Mohammed Errami, Professeur à la Faculté des Sciences de
Tétouan, d'avoir accepté d'être rapporteur de ma thèse et d‟effectuer un long déplacement afin
de siéger dans le jury.
Je souhaite exprimer ma gratitude au
Mr. Mohammed Bennis, Professeur à la
Faculté des Sciences de Marrakech, pour avoir accepté de participer a ce jury de thèse et
d‟être rapporteur de ce travail.
Mes remerciements vont aussi à : Claire de la ville, merci de m‟avoir formé et tout appris dès
mon arrivée au laboratoire. Un grand merci au Dr. Driss Boussaoud. Un immense merci au
Pr. Philippe De Deurwaerdère, a Mariam Sabbar, A Safa Bouabid, pour l‟amitié partagée,
aux bons moments passés au Village 3. A Emilie, tu m‟as surnommé : Monsieur Bonne
humeur, en fait c‟est aussi grâce à toi et aux bons moments passés au labo en ta présence. Un
grand merci à Mounia Rahmani, a Sarah Mounia Klouche, Anass. Mes remerciements
vont également à Fredo, pour son aide technique et la préparation des protocoles. Je souhaite
remercier: Melanie, Sylvia, Emilie S., Bérangère à Mark, Benoit, Brice, Aude, Camille,
Stephanie.E, Lea, Alexis,Coralie ,Youssra et Virane. Merci à Thomas, Du Zhuowei, Je
n‟oublie pas de remercier les membres de la Faculté des sciences de Rabat de m‟avoir donné
l‟opportunité de continuer mon parcours universitaire jusqu‟au bout. J‟adresse mes
remerciements au Pr. Saïd AMZAZI, Doyen de la Faculté et mon professeur durant mon
cycle de licence, j‟ai été ravi de travailler sous sa direction durant des activités universitaires.
Mes remerciements au Pr LFERDE Mohamed, directeur de l‟école doctorale pour sa
disponibilité et son aide précieuse dans les démarches administratives.
Je tiens à exprimer ma grande considération à tous mes chers Professeurs de la faculté
Pr. Soumaya Benomar, Pr Benabdelkhalek Mohammed, et Pr. Fouzia Bouzoubaa, Pr.
Khalid Taghzouti pour leur soutien moral et scientifique. A tous mes amis de thèse, Mounir,
à Nezha, Dounia, Mohcine, Ismail, Ala et Abedi. Je n‟oublierai jamais ma seconde mère,
Rabia Benazzouz, pour ses conseils précieux et pour son soutien moral durant mes années
passées à Bordeaux. A mes petites sœurs adorables Inès et Lina, je suis très chanceux d‟avoir
fait votre connaissance. Je remercie très chaleureusement Nadine.
Ce travail de recherche a été soutenu financièrement par l‟Université de Bordeaux
Segalen, le GDRI N198 (CNRS & INSERM France, et CNRST Maroc), Egide-Volubilis N°
20565ZM, la convention CNRS-CNRST Adivmar 22614 et le NEUROMED. Merci…
4
Le travail effectué pendant cette thèse a permis la publication de deux
articles dans des revues à comité de lecture :
Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Dopaminergic control of the
globus pallidus through activation of D2 receptors and its impact on the electrical activity of
subthalamic nucleus and substantia nigra reticulata neurons. PlosOne, 2015 In Press
Benazzouz A., Mamad O., Abedi P., Bouali-Benazzouz R. and Chetrit J. Involvement of
dopamine loss in extrastriatal basal ganglia nuclei in the pathophysiology of Parkinson‟s
disease. Frontiers in Aging Neuroscience, 2014, 13, 6:87. doi: 10.3389/fnagi.2014.00087.
eCollection 2014.
Communication affichées dans des Congrès et écoles internationaux
Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Dopamine control of the globus
pallidus and its impact on the subthalamic nucleus and the pars reticulata of substantia
nigra. 11th SONA international Conference, June 13-17, 2013, Rabat Morocco.
Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Control of the pallidosubthalamic and pallido-nigral pathways by dopamine D2 receptors in the rat. Final
meeting Neuromed Neuroscience, May 2-3, 2013, Marseille France.
Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Control of the pallidosubthalamic and pallido-nigral pathways by dopamine D2 receptors in the rat. Society For
Neuroscience SFN, October 13-17, 2012, New Orleans,USA.
Mamad O. Abedi.P, Delaville C., Benjelloun W. and Benazzouz A. Control of the pallidosubthalamic and pallido-nigral pathways by dopamine D2 receptors in the rat. 4th
Conference of Mediterranean Neuroscience Society (MNS), Septembre 30- October 03,
5
2012, Istanbul, Turquie.
Mamad O. Abedi.P, Delaville C., Benjelloun W. and Benazzouz A. Control of the pallidosubthalamic and pallido-nigral pathways by dopamine D2 receptors in the rat. 8ème forum
de Neuroscience FENS, Juillet 14-18, 2012, Barcelone, Espagne
Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Contrôle dopaminergique des
voies pallido-subthalamiques et pallido-nigrales par les récepteurs D2 chez le rat. Annual
conference of the International Research Group From Neuroscience, May-14-15,2012,
Marseille France.
Mamad O., Delaville C., Faggiani E., Benjelloun W. and Benazzouz A. Contrôle
dopaminergique du globus pallidus et conséquences comportementales. 4ème Ecole de
GDRI, Neurobiologie des adaptations à l'environnement. Octobre, 18 – 22, 2011.
Casablanca, Maroc.
Communication orales :
Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Contrôle dopaminergique des
voies pallido-subthalamiques et pallido-nigrales par les récepteurs D2 chez le rat. Annual
conference of the International Research Group From Neuroscience, May-14-15,2012,
Marseille France.
Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Control of the pallidosubthalamic and pallido-nigral pathways by dopamine D2 receptors in the rat. Final
meeting Neuromed Neuroscience, May 2-3,2013, Marseille France.
6
Résumé
Le travail de ma thèse porte sur l‟étude des contrôles exercés par la dopamine sur les
ganglions de la base (GB) chez le rat. Les GB sont un ensemble de structure sous-corticale
constitués principalement par le striatum, le globus pallidus (segment interne, GPi chez le
primate et noyau entopédonculaire, EP chez le rongeur ; et segment externe, GPe chez le
primate et GP chez le rongeur), le noyau sous-thalamique (NST), et la substance noire
(réticulata, SNr ; et compacta, SNc). Les GB sont impliqués dans le contrôle du mouvement et
leur dysfonctionnement conduisent à des troubles moteurs tels que ceux observés dans la
maladie de Parkinson. Le GPe occupe une position centrale au sein des circuits des GB en
jouant un rôle clé dans le contrôle du mouvement par son tonus GABAergique inhibiteur sur
les structures de sortie des GB. Comme pour le striatum, l‟activité des neurones du GP est
modulée par la dopamine. Le contrôle dopaminergique est médié par les récepteurs de type
D2 (RD2) qui modulent l‟activité neuronale de ce noyau qui reçoit une projection
dopaminergique directe de la SNc.
A
l‟aide
d‟outils
pharmacologiques
appropriés
(la
dopamine
ainsi
que
agoniste/antagoniste), nous avons étudié chez le rat anesthésie à l‟uréthane,
ses
l‟effet
modulateur de la dopamine sur l‟activité des neurones du GPe ainsi que son impact sur ses
deux structures efférentes qui sont le NST et la SNr en utilisant l‟électrophysiologie
extracellulaire in vivo.
La première partie du travail était consistait d‟abord à étudier l‟effet de l‟injection locale de la
dopamine sur l‟activité des neurones du GP. Ensuite montrer si la modulation dopaminergique
passe par les RD2 en utilisant un antagoniste sélectif des RD2, le sulpiride. Afin de confirmer
l‟implication des RD2, nous avons aussi utilisé le quinpirole (un agoniste sélective des RD2),
cette dernière a été réalisée après détermination de la concentration à laquelle les neurones du
GP ont présenté une réponse. L‟injection de l‟agent pharmacologique a été réalisée après 20
7
minutes d‟enregistrement de l‟activité basale des neurones et à condition que son activité de
décharge reste stable pendant toute cette durée.
Les données de notre étude ont montré que la dopamine, lorsqu'elle est injectée localement,
augmente la fréquence de décharge de la majorité des neurones du GP. Cette augmentation est
mimée par le quinpirole, et bloquée par le sulpiride. Cependant, l‟injection de la dopamine et
du quinpirole n‟a pas modifié le mode de décharge des neurones du GP. En parallèle,
l'injection de la dopamine, ainsi que le quinpirole, dans le GP réduit la fréquence de décharge
de la majorité des neurones du NST et de la SNr. Cependant, la dopamine et le quinpirole ne
changent pas le mode de décharge des neurones des deux structures.
Nos résultats sont les premiers à démontrer que la dopamine via les récepteurs D2 dans le
GPe joue un rôle important dans la modulation des voies GPe-NST et GPe-SNr et par
conséquent contrôle l‟activité des neurones du NST et de la SNr. De plus, nous démontrons
que la dopamine module la fréquence, mais pas le mode de décharge des neurones du GPe,
qui à son tour contrôle la fréquence, mais pas le mode de décharge des neurones du NST et de
la SNr.
L‟ensemble de ces travaux a permis d‟approfondir les connaissances sur l‟organisation
fonctionnelle des ganglions de la base et en particulier le rôle modulateur majeur de la
dopamine, via les récepteurs D2 au niveau du GP, et son impact sur la modulation des deux
voies pallido-subthalamique et pallido-nigral.
Afin de compléter cette étude, il serait intéressant d‟étudier l'éventuelle implication des RD1
dans les réponses des neurones du GP ainsi que son impact sur le NST et la SNr, en utilisant
la même approche pharmacologique d'injection intrapallidale des agonistes et antagonistes des
RD1au niveau du GP. Comme la présente étude a été réalisée chez les animaux normaux,
nous proposons d'étudier l'effet modulateur de la dopamine dans le modèle de la maladie de
8
Parkinson chez le rat obtenu par l'injection stéréotaxique de 6-hydroxydopamine (6-OHDA)
dans le faisceau médial du télencéphale. Les résultats de ce projet permettront de comprendre
si les réponses des neurones du GP aux agents dopaminergiques sont semblables ou
différentes par rapport à celles obtenues chez des rats normaux. Afin d‟étudier les corrélats
comportementaux associés aux réponses électrophysiologiques, nous envisageons d„étudier
les effets des injections locales des agents dopaminergiques dans le GP sur le comportement
moteur des animaux normaux et d‟animaux dont le système dopaminergique est
préalablement lésé. Pour réaliser ce travail, l‟actimètrie utilisant l‟Open Field ainsi que le «
test de stepping» et le «Rotarod" seront utilisés.
9
Abstract
The work of my thesis is a part of integrative neurobiology and focuses on studying the
control exerted by dopamine on basal ganglia (BG), especially the "external part of globus
pallidus or GPe". GPe being a nucleus, which plays a key role in the control of movement by
exerting an inhibitory influence on the output structures of the BG circuitry. The action of
dopamine is mediated by D2 receptors that modulate neuronal activity of this nucleus that
receives direct dopaminergic projections from the substantia nigra compacta (SNc). Using
appropriate pharmacological tools (dopamine and its agonist/antagonist), we studied, in the
rat, the effects of dopamine on modulating the basal activity of GPe neurons and its impact on
the two major efferent structures, the subthalamic nucleus (STN) and the pars reticulata of
substantia nigra (SNr) using an extracellular electrophysiological approach combined with
local intracerebral microinjection of drugs in vivo.
Data of this thesis work showed that dopamine, when injected locally, increased the firing rate
of the majority of neurons in the GP. This increase of the firing rate was mimicked by
quinpirole, a D2R agonist, and prevented by sulpiride, a D2R antagonist. In parallel, the
injection of dopamine, as well as quinpirole, in the GP reduced the firing rate of majority of
STN and SNr neurons. However, neither dopamine nor quinpirole changed the tonic
discharge pattern of GP, STN and SNr neurons.
Our results are the first to demonstrate that dopamine through activation of D2Rs located in
the GP plays an important role in the modulation of GP-STN and GP-SNr neurotransmission
and consequently controls STN and SNr neuronal firing. Moreover, we provide evidence that
dopamine modulates the firing rate but not the pattern of GP neurons, which in turn control
the firing rate, but not the pattern of STN and SNr neurons.
10
Résumé
Le travail de ma thèse porte sur l‟étude des contrôles exercés par la dopamine sur les
ganglions de la base (GB) et plus particulièrement "le globus pallidus externe ou GPe". Le
GPe joue un rôle clé dans le contrôle du mouvement en exerçant un tonus inhibiteur sur les
structures de sortie des GB. L‟action de la dopamine est médiée par les récepteurs D2 qui
modulent l‟activité neuronale de ce noyau qui reçoit une projection dopaminergique directe de
la substance noire compacte (SNc). A l‟aide d‟outils pharmacologiques appropriés (la
dopamine ainsi que ses agoniste/antagoniste), nous avons étudié chez le rat l‟effet modulateur
de la dopamine sur l‟activité du GPe ainsi que son impact sur ses deux structures efférentes
qui sont le noyau sous-thalamique (NST) et la substance noire reticulée (SNr) en utilisant
l‟électrophysiologique extracellulaire in vivo.
Les données ont montré que la dopamine, lorsqu'elle est injectée localement, augmente la
fréquence de décharge de la majorité des neurones du GPe. Cette augmentation est mimée par
le quinpirole, un agoniste des récepteurs D2, et bloquée par le sulpiride, un antagoniste des
récepteurs D2. En parallèle, l'injection de la dopamine, ainsi que le quinpirole, dans le GP
réduit la fréquence de décharge de la majorité des neurones du NST et de la SNr. Cependant,
la dopamine et le quinpirole ne changent pas le mode de décharge des neurones du GPe, NST
et SNr.
Nos résultats sont les premiers à démontrer que la dopamine via les récepteurs D2 dans le
GPe joue un rôle important dans la modulation des voies GPe-NST et GPe-SNr et par
conséquent contrôle l‟activité des neurones du NST et de la SNr. De plus, nous démontrons
que la dopamine module la fréquence, mais pas le mode de décharge des neurones du GPe,
qui à son tour contrôle la fréquence, mais pas le mode de décharge des neurones du NST et de
la SNr.
11
Sommaire
List of figures: ........................................................................................................................................ 15
Tables .................................................................................................................................................... 16
I. Introduction ........................................................................................................................................ 17
I.1 The embryonic origin and the anatomy of GP ............................................................................. 22
I.2 Cytology and Morphological Characteristics of GPe Neurons..................................................... 26
I.3 Physiology and Classification of GPe neurons ............................................................................. 28
I.4 Functional Considerations ........................................................................................................... 35
I.4.1 GABAergic neurotransmission .............................................................................................. 38
I.4.2 GABA synthesis, enrichment and degradation ..................................................................... 38
I.4.3 GABA receptors .................................................................................................................... 40
I.5 Efferents of the GPe .................................................................................................................... 41
I.6 Afferents of the GPe .................................................................................................................... 45
I.6.1 GABAergic afferents of the Globus Pallidus ......................................................................... 45
I.6.2 Glutamatergic afferents of the GP ........................................................................................ 46
I.6.3 Dopaminergic afferents of the GP ......................................................................................... 49
I.7 Types of dopaminergic receptors in the GP ................................................................................. 52
I.8 Electrophysiological responses of GP neurons to dopamine drugs ............................................ 57
I.9 Behavioral study ........................................................................................................................... 59
General objectives ................................................................................................................................. 61
II. Materials and Methods ..................................................................................................................... 63
II.1 Study Model ................................................................................................................................ 63
II.2 Pharmacological substances........................................................................................................ 63
II.3 Electrophysiology in vivo in anesthetized rats ............................................................................ 65
II.3.1 Extracellular recording unit .................................................................................................. 65
II.3.2 Validation of the recording sites .......................................................................................... 74
II.3.3 Statistical analysis ................................................................................................................. 74
III. Results and Discussion...................................................................................................................... 76
PART 1: Effect of dopamine and its agonist (Quinpirole) on the electrical activity of GP neurons . 76
1.1 Effects of local injection of Dopamine in the globus pallidus on the firing rate of GP neurons
....................................................................................................................................................... 77
1.2 Effects of local injection of Quinpirole in the globus pallidus on the firing rate of GP neurons
....................................................................................................................................................... 82
Discussion part 1: The effect of dopamine, its agonist (Quinpirole) and antagonist (Sulpiride) D2
receptors on the activity of GP neurons ........................................................................................... 84
12
PART 2: The effect of local injection of dopamine and Quinpirole on the GP on the activity of the
STN and SNr neurons......................................................................................................................... 87
2.1 Effects of local injection of dopamine in the globus pallidus on the firing rate of STN neurons
....................................................................................................................................................... 88
2.2 Effects of local injection of quinpirole in the globus pallidus on the firing rate of STN neurons
....................................................................................................................................................... 91
2.3 Effects of local injection of dopamine in the globus pallidus on the firing rate of SNr neurons
....................................................................................................................................................... 93
2.4 Effects of local injection of dopamine in the globus pallidus on the firing rate of SNr neurons
....................................................................................................................................................... 95
Discussion part 2: The effect of local injection of dopamine and Quinpirole on the GP on the
activity of NST and SNr neurons ........................................................................................................ 97
IV. Conclusion and Perspectives .......................................................................................................... 100
References ........................................................................................................................................... 103
13
List of Abbreviations:
AOP: anterior preoptic area
A2A :adenosine receptors 2.
BG : Basal ganglia;
CB1:cannabinoid 1
CPu, caudoputamen nucleus;
DA: Dopamine;
DR1/2: Dopamine receptors 1/2.
EP : Entopeduncular nucleus;
H, Hippocampus;
GABA: γ-aminobutyric acid;
GPe : External segment of the globus pallidus;
GPi : Internal segment of the globus pallidus
LGE, lateral ganglionic eminence;
MGE, medial ganglionic eminence;
MSN: medium spiny neurons
POA, anterior preoptic area;
PCx, piriform cortex;
SNr : Substantia nigra reticulé;
SNc : Substantia nigra pars compacta;
STN: Subthalamic nucleus
SNr : Substantia nigra pars reticulata;
Str: Striatum
14
List of figures:
Figure 1: Representation of the cortico-subcortical loop motor circuit involving the basal
ganglia.
Figure 2: Schematic representation of the circuit of the basal ganglia
Figure 3: Anatomical Organization of the developing forebrain.
Figure 4: Neuronal diversity in the globus pallidus emerges from different and distant
progenitor pools.
Figure 5: Principal components of the basal ganglia showed the anatomical difference of the GP.
Figure 6 Example of rat GPe neurons and an axon.
Figure 7: Morphological reconstruction of biocytin-labelled neurons
Figure 8. Topography of GP cells.
Figure 9. Example of GP-TI Neurons in the structure of Globus Pallidus Neurons
Figure 10. Example of GP-TA Neurons in the structure of Globus Pallidus Neurons
Figure 11. Schematic drawing of transmitter release, transport, and synthesis at a GABAergic
synaptic terminal.
Figure. 12. Microcircuitry of the pallido-subthalamic projection
Figure 13: Simplified diagram of the main effrents projections of globus pallidus.
Figure 14: Human dopamine projections: representation of the four central dopaminergic
pathways.
Figure 15: Simplified diagram of the major affrents of GP neurons.
Figure 16: Example of accessories needed for surgery
Figure 17: Schematic of a triple and double glass micropipettes used for the recording in the
globus pallidus
Figure 18: Schematic of the injection electrode in the GP and the recording electrode used in the
STN and in the SNr.
Figure 19 : AlphaLab SnR: Multi-Channel workstation with complete acquisition
Figure 20: The three types of discharge mode of neurons of the subthalamic nucleus.
Figure 21: Location electrophysiological recording site in the (A) GP (B) STN, (C) SNr.
Figure 22: Intrapallidal microinjection of dopamine predominantly increased the firing rate
without changing the tonic firing pattern of GP neurons.
15
Figure 23 : Dopamine did not significantly change the the firing rate or the coefficient of
variation of the interspike intervals of GP neurons.
Figure 24: Intrapallidal microinjection of quinpirole predominantly increased the firing rate of
GP neurons in a dose-dependent manner without changing the tonic firing pattern.
Figure 25: Intrapallidal microinjection of dopamine predominantly decreased the firing rate
without changing the tonic firing pattern of STN neurons.
Figure 26: Intrapallidal microinjection of quinpirole predominantly decreased the firing rate
without changing the tonic firing pattern of STN neurons.
Figure 27: Intrapallidal microinjection of dopamine predominantly decreased the firing rate
without changing the tonic firing pattern of SNr neurons.
Figure 28: Intrapallidal microinjection of quinpirole predominantly decreased the firing rate
without changing the tonic firing pattern of SNr neurons.
Tables
Table 1: A summary of different technics used to classify the GP neurons
Table 2 Summary presentation of the distribution of dopamine receptors in the external globus
pallidus
Table 3: Functional effects of dopamine receptor agonists on the globus pallidus
Table 4: Pharmacological agents used for different experiments in this studies
Table 5: Overall assessment of the effect of dopamine and its agonist D2R (quinpirole) on the
firing rate of the GP, the STN and SNr neurons.
Table 6. Firing rates of GP, STN and SNr neurons before and after dopamine or quinpirole
injection into the GP.
Table 7: Table 2. Coefficient of variations of GP, STN and SNr neurons before and after
dopamine or quinpirole injection into the GP
Table 8 Overall assessment of the local injection of dopamine and quinpirole on the activity of
neurons in the STN.
Table 9 Overall assessment of the local injection of dopamine and quinpirole on the activity of
neurons in the SNr.
16
I. Introduction
The basal ganglia (BG) are a group of highly interconnected brain structures that are
intimately involved in a variety of processes including motor, cognitive and mnemonic
functions. One of their major roles is to integrate sensorimotor, associative and limbic
information in the production of context-dependent behaviors (DeLong, 1990, Prescott et al.,
2006, Chetrit et al., 2009, Acharya et al., 2011). Most findings about BG functions were
originally obtained from clinical observations and postmortem brain examination of patients
with major movement disorders, such as Parkinson's disease, Huntington's disease, and
hemiballismus (Bolam et al., 2000, Smith and Sidibe, 2003, Bolam et al., 2009).
Interest in BG research has been kindled by the striking motor symptoms encountered
in these pathological conditions. Despite improvements in diagnostic tools and the wealth of
information derived from experimental and clinical studies, the exact contribution of the BG
to the functioning of the brain is not precisely known. This uncertainty is exemplified by the
current controversy on the implication of BG in motor versus cognitive functions (Albin et al.,
1989, Pelayo et al., 2003).
Voluntary motor is essentially a phenomenon of cortical origin. It involves the primary
motor cortex, the premotor area, the supplementary motor area and the prefrontal and parietal
cortices (see Figure 1). The neuronal activity of these cortical areas is regulated by a set of
cortico-subcortical loops where the BG are involved (Gerfen et al., 1990).
The BG comprised the striatum (caudate nucleus and putamen), the external globus
pallidus (GPe in primate, equivalent of GP in rodents), the internal globus pallidus (GPi in
primate, equivalent of the entopeduncular nucleus, EP, in rodents), the subthalamic nucleus
(STN), and the substantia nigra pars compacta and reticulata (SNc and SNr, respectively)
(DeLong, 1990, Bai et al., 2007).
17
Figure 1: Representation of the cortico-subcortical loop motor circuit involving the basal
ganglia. According to (Graybiel, 1990).
As the primary input of the basal ganglia, the striatum and STN receive glutamatergic
inputs from the cortex and thalamus. In the striatum, inputs from the cortex and thalamus both
form excitatory synaptic connections on medium spiny neurons (MSN) in which cortical
afferents are from the sensory, motor, and associative cortices (Bolam et al., 2000), and
thalamic afferents originate from the intralaminar thalamic nuclei (Doig et al., 2010).
The transmission of cortical information through the basal ganglia occurs through 2
routes: the direct and indirect pathways. Striatal MSN neurons involved in the direct pathway
express high level of D1 dopamine receptors and project directly onto the two principal basal
ganglia output structures, the GPi and SNr. MSN neurons involved in the indirect pathway
highly express D2 dopamine receptors and project to the GPe (Gerfen et al., 1990).
In the direct pathway corticostriatal information is transmitted directly from the
striatum to the output nuclei through an inhibitory GABAergic projection. In the indirect
18
pathway corticostriatal information is transmitted indirectly to the output nuclei via the
complex network interconnecting inhibitory projections from the striatum to GPe and GPe to
STN and an excitatory projection from the STN to the GPi and SNr (Shink et al., 1996). The
direct and indirect pathways act in opposition one to another to control movement, which
indicates segregated information processing (Albin et al., 1989, DeLong, 1990, Doig et al.,
2010, Do et al., 2012) (Figure 2).
Figure 2: Schematic representation of the cortico-basal ganglia-thalamo-cortical circuit.
GPe GPi (External and internal segment of the globus pallidus) STN (subthalamic nucleus)
SNc and SNr (Substantia nigra pars compacta and reticulata). Blue arrows: GABAergic
inhibition, red arrows glutamatergic excitations, green arrow: dopaminergic projections. D1
and D2: D1 and D2 dopaminergic receptors. Adapted from (Albin et al., 1989).
19
In the current model of the functional organization of the BG, the GPe in primate (or
GP in rodents) is considered as a relay linking the striatum to the output structures of the BG,
the GPi (or the entopeduncular nucleus, EP in rodents) and the SNr. The projections from GPe
to these structures, through the STN, use γ-aminobutyric acid (GABA) as neurotransmitter
(Shink et al., 1996, Hauber and Lutz, 1999).
Major pallidal afferents using GABA as
neurotransmitter originate in the striatum, while glutamatergic afferents arise from the STN
(Pelayo et al., 2003). Besides these afferents, GPe neurons also receive dopaminergic
projections from the SNc (Fallon and Moore, 1978). A major role of dopamine in the GPe has
been suggested by findings that intrapallidal dopamine receptor blockade produced massive
akinesia in the rat (Hauber and Lutz, 1999) and in contrast, intrapallidal microinjection of
dopamine partially restored the motor deficits induced by 6-hydroxydopamine (6-OHDA) in
the rat model of Parkinson‟s disease (PD) (Alexander et al., 1990, Galvan et al., 2001).
Recent studies from our team have shown that dopamine depletion in the GPe induced
a significant decrease of the firing rate of GPe neurons and motor deficits on the rat. This
indicates that DA exerts an excitatory effect on GPe neurons (Bouali-Benazzouz et al., 2009,
Abedi et al., 2013). Dopamine acts by binding to specific membrane receptors (Gingrich and
Caron, 1993) that belong to the G protein-coupled receptors, otherwise known as the seventransmembrane domain receptors. Five distinct dopamine receptors have been isolated,
characterized and subdivided into two subfamilies, D1- and D2-like, on the basis of their
biochemical and pharmacological properties. The D1-like subfamily comprises D1 and
D5 receptors, while the D2-like subfamily includes D2, D3 and D4 receptors (Vallone et al.,
2000). Much evidences indicated that both dopamine D1 and D2 receptors are expressed in
the GPe (Alexander et al., 1990) (for more details see table 1). Dopamine receptors are found
at pre-and postsynaptic localization in GPe. Most of the presynaptic dopamine receptors are
20
thought to be D2R, and are located on terminals of the GABAergic striatopallidal projection
(Campo et al., 2003, Feresin et al., 2003, Pelayo et al., 2003). Recently, in vitro patch clamp
recordings showed that activation of D1 receptors increased the frequency but not the
amplitude of the spontaneous excitatory postsynaptic currents, suggesting a presynaptic
facilitation of glutamate transmission in the globus pallidus (Hernández et al., 2007).
Together, these evidences demonstrate that dopamine in the GPe may play a key role
in the modulation of the neuronal activity in the motor circuits, confirming that the GPe is a
key structure of basal ganglia network playing an important role in the motor control.
In this thesis, we will first describe relevant features of the general anatomy of the GPe
followed by an overview of the current state of knowledge about the functional modulatory
role of dopamine in the GPe and its impact on its efferent structures.
21
I.1 The embryonic origin and the anatomy of GP
The term “GP” comes from the pale appearance of GP in Nissl stains. This is due to
the low density of neurons in this structure, which are surrounded by a massive volume of
axons (white matter) (Parent and Hazrati, 1995) .
The GP is a subcortical structure that belongs to the basal ganglia. As the other nuclei
of the system, it is involved in a wide variety of motor and affective behaviors and in
sensorimotor integration as well as in cognitive functions (DeLong, 1990, Hauber and Lutz,
1999, Bolam et al., 2000, Prescott et al., 2006, Chetrit et al., 2009, Acharya et al., 2011, Abedi
et al., 2013).
The forebrain is considered as one of the most complex structures of the mammals. In
this region, cell migration plays an essential role in the development,
each neuron is
generated by a proliferative area and then migrates to its final destination (Marin and
Rubenstein, 2003). The embryonic origin of the GP and its anatomical differentiation has
been previously reported.
The embryonic origin of the GP
The brain has a stereotypical architecture that implement progressively during
embryonic development. It initially formed from neural tube subdivisions: three primary
vesicles, the forebrain, midbrain and hindbrain, which then form five structures, the
telencephalon, diencephalon, midbrain, metencephalon and myelencephalon (see Figure 3)
(Marin et al., 2002, Marin and Rubenstein, 2003). The telencephalon has two major regions:
the pallium (roof), which gives rise to the cerebral cortex and hippocampus and the
subpallium (base), which give rise to the structures of BG (Rubenstein et al., 1998, Cobos et
al., 2001, Marin and Rubenstein, 2003). The region of subpallium is formed by reliefs called
lateral ganglionic eminences (LGE) and medial ganglionic eminences (MGE), more of
22
these two structures, it is also formed by the anterior preoptic area (AOP), which located
more ventrally (Marin and Rubenstein, 2003) (see Figure 3). It has typically also been
assumed that neurons in the GP derive from the MGE (see figure 3) (Nobrega-Pereira et al.,
2010). This important node basal ganglia nucleus contains several distinct classes of
projection neurons but few interneurons (Kita and Kitai, 1994, Cooper and Stanford, 2000).
Figure 3: Anatomical Organization of the developing forebrain.
A : Schema of a sagittal section through the brain mouse showing the main subdivisions of
the forebrain, the diencephalon and the telencephalon. In the telencephalon, the pallium is
depicted in lighter gray than the subpallium. (B) Schema of a transversal section through the
telencephalon, indicating some of its main subdivisions. LGE, lateral ganglionic eminence;
MGE, medial ganglionic eminence; POA, anterior preoptic area (Marin and Rubenstein,
2003).
Recently, the molecular profile of GP neuronal types, their lineage and their
proportion have been determined (Nobrega-Pereira et al., 2010). Indeed, majority of GP
neurons are from two embryonic sources: the MGE (70/ %) and LGE (25%). The remaining
5 % are from the POA (Nobrega-Pereira et al., 2010). Several studies have suggested the
existence of five neuronal types within the GP classified according to their molecular
specificity (Ferland et al., 2003, Takahashi et al., 2003, Kaoru et al., 2010) (see Figure 4 B).
23
Figure 4: Neuronal diversity in the globus pallidus emerges from different and distant
progenitor pools. A: Schematic representation of a transversal hemisection depicting the
putative routes of migration of GP neurons. B: Schematic representation and table of neuronal
diversity in the GP, based on the molecular profile of its constituents and their differential
origin. H, Hippocampus; CPu, caudoputamen nucleus; PCx, piriform cortex; Str, striatum
(Nobrega-Pereira et al., 2010).
The anatomical differentiation of the pallidal complex
The anatomical studies on rodents and on primates have identified a difference in the
organization of the pallidal complex. In rodents and carnivores, the pallidal complex is
composed of the globus pallidus (GP) and the entopeduncular nucleus (EPN). The GP is
located medially to either the caudate putamen complex (rodents) or the putamen (carnivores),
and the EPN lies within the internal capsule. Thus, in these species the two parts of the
pallidal complex are widely separated (Smith and Sidibe, 2003, Jaeger and Kita, 2011a). In
human and non-human primates, the pallidal complex lies medial to the putamen, and located
laterally to the internal capsule. In these species, the pallidal complex is further subdivided
into lateral or external (GPe, equivalent of GP in rodents) and medial or internal (GPi,
equivalent of EPN in rodents) segments by a dorsoventral sheet of white matter called the
medial or internal medullary lamina. The GPe is also separated from the putamen by another
24
sheet of white matter, the lateral or external medullary lamina (Smith and Sidibe, 2003, Kita,
2010, Jaeger and Kita, 2011a) (Figure 5 A and B).
A
B
Figure 5: Principal components of the basal ganglia showed the anatomical difference of
the GP. (A) The pallidal complex in rodents (GP and EPN); in this species the GP is located medially
to either the caudate putamen complex. (B) Shows the pallidal complex (GPe and GPi) in primates,
which lies medial to the putamen, and lateral to the internal capsule.
25
I.2 Cytology and Morphological Characteristics of GPe Neurons
In human, GPe constitutes approximately ¾ of the total volume of the pallidal
complex with a cell density greater than that of the GPi. The neurons in both GPe and GPi use
GABA as a neurotransmitter (Shink et al., 1996, Hauber and Lutz, 1999). The majority of
GPe neurons are enriched with peptides such as the parvalbumin (PV), and a few of them
also express the calretinin (CR) (Shink et al., 1996, Hauber and Lutz, 1999). These neurons
have large aspiny firstly then varicose and finally dendrites (Figure 6A). Dendrites of GPe
neurons form a disk-like dendritic field with the plane of the disc parallel to the lateral
medullary lamina (Yelnik et al., 1984, Kita, 1996).
Hoover and Marshall (2002) demonstrated that a substantial population (42%) of
globus pallidus neurons contains preproenkephalin mRNA, and that globus pallidus neurons
retrogradely labeled after FluoroGold injections into the striatum are more frequently
preproenkephalinergic, compared to the population of pallidosubthalamic neurons. (Hoover
and Marshall, 2002).
Several studies have shown that in rodents and monkeys GPe projection neurons send
out local collateral axons (Kita, 1994, Sato et al., 2000a, Sato et al., 2000b, Sadek et al.,
2007). These local projections of axons end on soma and proximal dendrites and can generate
powerful inhibition to GPe neurons (Figure 6). GPe and GPi are primarily made up of
relatively large cells with triangular or polygonal cell bodies that give rise to thick, sparsely
spined, poorly branching dendrites. These morphological characteristics were found in nonhuman primate (Fox et al., 1974, Difiglia et al., 1982, Francois et al., 1984, Percheron et al.,
1984, Yelnik et al., 1984) and in rodents and other species (Iwahori and Mizuno, 1981, Kita
and Kitai, 1994, Nambu and Llinas, 1997).
26
Figure 6: Example of rat GPe neurons and an axon. A: A neuron with large aspiny firstly,
then varicose, and finally dendrites with occasional complex endings (arrow heads) with
appendages. The scale in B also applies to A. B: A neuron with sparsely spineous dendrites.
C: An axon of a GPe neuron that has extensive local axon collaterals in GPe and multiple
small terminal fields in GPi (Kita, 2010).
27
I.3 Physiology and Classification of GPe neurons
In addition to their morphological characteristics, GP neurons have been classified
according to their electrophysiological and neurobiochemical properties. In this part I will try
to describe in a chronological order and by the technique used for the classification of pallidal
neurons (Table 1). The majority of these studies have been conducted in rodents but also a
part in primates.
Electrophysiological
properties in
Neuro-biochemical properties
Electrophysiological
vivo and in vitro recording
*Waveform
of
extracellular
and
Neuro-
biochemical properties
Two groups of neuronal
The visual inspection of
recording (Bergstrom et al., 1982,
population
current
1984).
PV+:60%)
&
40%)(Kita,
1994,
* The sensitive to the amplitude of
injected current (Nambu and Llinas,
1997).
* Increases or decrease in activity
(DeLong, 1971, Gardiner and Kitai,
(around
(PPE:
Hoover
morphology
clamp
and
(Cooper and
Stanford, 2001b), (Kita &
and Marshall, 1999, 2002).
Kitai, 1991).
3 calcium binding proteins:
Firing pattern (Mallet et al.,
PV,CB & CR) (Cooper and
2008)
Stanford, 2002)
1992, Goldberg and Bergman, 2011,
Qi and Chen, 2011).
Table 1: Summary of different techniques used to classify pallidal neurons
28
Electrophysiological and neurobiochemical properties:
Early studies revealed that there are at least two different subpopulations of pallidal
neurons on the basis of the waveform of extracellular recordings, Type I (negative/positive
waveform) and Type II (positive/negative waveform). In this study no significant differences
were observed in the firing pattern or number of cells per track between these cell types,
although the Type II neurons had a faster mean firing rate in the locally anesthetized animals.
one part of both cell types could be antidromically activated from the subthalamic nucleus,
although Type II neurons had significantly slower conduction velocities. Type I neurons are
inhibited by systemic apomorphine, while type II neurons are excited by systemic
apomorphine (Bergstrom et al., 1982, 1984, Kelland et al., 1995). Other studies showed that
type I neurons were silent at the resting membrane level and generated a burst of spikes with
strong accommodation to depolarizing current injection. Type II neurons fired at the resting
membrane level or with small membrane depolarization, and their repetitive firing was very
sensitive to the amplitude of injected current and showed weak accommodation (Nambu and
Llinas, 1997). In vivo unit recordings from monkey GPe distinguish two types of pallidal
neurons on the basis of their baseline activity patterns. The most numerous type of neuron
exhibits high-frequency firing interspersed with spontaneous pauses, while the other type
exhibits low-frequency firing and bursts. Both types of neurons change their activity in
relation to limb movement, and in most cases these changes consist of increases in the firing
activity (DeLong, 1971, Gardiner and Kitai, 1992, Goldberg and Bergman, 2011).
The GP is almost exclusively composed of GABAergic projection neurons. Early
studies suggest that the neuronal population in the GP can be neuro-biochemically subdivided
into two groups: the parvalbumin positive cells (60%) and the neuropeptide precursor
preproenkephalin (PPE) mRNA containing neurons (40%) (Kita, 1994, Hoover and Marshall,
29
1999, 2002). Calcium-binding proteins are known to have unique buffering characteristics
that may confer specific functional properties upon pallidal neurons. Indeed, differential
calcium binding protein expression may underlie the electrophysiological heterogeneity
observed in the rat globus pallidus (Cooper and Stanford, 2002). Later studies reported that
the GP neurons can be differentiated by three calcium binding proteins: PV, calbindin D-28k
(CB) and calretinin (CR) (Cooper and Stanford, 2002). The PV positive neurons take about
60% of total pallidal neuronal population and distribute throughout the GP with the highest
present in the lateral part as mentioned before (Kita and Kitai, 1991, Cooper and Stanford,
2002). The CB containing neurons constitute around 2% of total GP neurons and can be
observed throughout the GP in a complementary pattern to PV cells (Cooper and Stanford,
2002). The CR neurons are very sparse (less than 1%) and are not labelled by colloidal gold
particles, thus they may represent a subpopulation of pallidal interneurons. No co-expression
of calcium binding proteins is observed in the GP. Due to the fact that approximately 30% of
GP neurons are not labelled by any of the three calcium binding proteins, it turns that every
GP neuron appears to express either one single type of calcium binding protein or none at all
(Cooper and Stanford, 2002).
Other classification of types of GP neurons based on the visual inspection of current
clamp electrophysiological properties and morphology of biocytin-filled neurons, have been
classified into three subgroups. Firstly type A, their somata were variable in shape while their
dendrites were highly varicose. Then type B their cells were the smallest encountered, oval in
shape with restricted varicose dendritic arborisations. Finally type C with extensive dendritic
branching (see Figure 7 and 8) (Cooper and Stanford, 2001b). These results confirm the
neuronal heterogeneity in the GP. The driven activity and population percentage of the three
subtypes correlates well with the in vivo studies (Kita & Kitai, 1991). For example type A
30
cells seem to correspond to type II neurons of Nambu and Llinas (1994, 1997) that described
as fired spontaneously at the resting membrane level. While the small diameter type B cells
display morphological similarities with those described by Millhouse (1986). The rarely
encountered type C cells may well be large cholinergic neurons. These findings provide a
cellular basis for the study of intercellular communication and network interactions in the
adult rat in vitro slices (Cooper and Stanford, 2001b).
Figure 7: Morphological reconstruction of biocytin-labelled neurons: A, examples of
large multipolar type A neurones with extensive dendritic branching, which were mainly
varicose. B, representative examples of small oval type B GP cells whose dendrites were
predominantly varicose. C, examples of large pyramidal type C cells with extensive dendritic
trees (Cooper and Stanford, 2000).
31
Figure 8: Topography of GP cells: The location of each biocytin-filled GP neurone was
plotted onto stylised drawings of coronal (A) slices (obtained from Paxinos & Watson 1986).
There appears to be a homogenous distribution of neuronal types A, B and C throughout the
GP (Cooper and Stanford, 2000).
Recently, new electrophysiological studies support that the GP neurons can be
grouped into two subpopulations according to their neuronal firing patterns: a major group of
GP neurons (about 75%) preferentially discharge during the inactive component of the
cortical slow oscillation when most cortical, striatal and STN neurons are quiescent, thus
named as GP-TI neurons; another population of GP neurons (more than 20%) are likely
discharge during the active component and called GP-TA neurons (Mallet et al., 2008)
(Figure 9 and 10). The GP-TI neurons are considered as the prototypic GP neurons, which
target the downstream BG nuclei including the STN, EP and SNr. Most GP-TI neurons
express PV while none of them express PPE. Besides the long-range axon collaterals that
project to the BG downstream nuclei, the GP-TI neurons give rise to extensive local
collaterals and some of them even have collaterals modestly innervate the striatal GABAergic
interneurons. The GP-TA neurons are exclusively PPE positive and only innervate the
striatum with the special GABAergic/enkephalinergic projections. They also emit local axon
collaterals although those collaterals are relatively restricted and the number of boutons is
much smaller than GP-TI cells (Mallet et al., 2012). The same study has defined the axonal
32
and dendritic architecture of GP-TI neurons and GP-TA neurons based on the long-range and
local axonal projections of some well-labeled cells (Mallet et al., 2012). They also
reconstructed the local axon collaterals and proximal extrinsic projections of three more GPTI neurons.
The GP-TA neuronal projections may be an important source of striatal enkephalin,
thus play a role in the regulation of MSNs firing (Blomeley and Bracci, 2011).
Figure 9: Example of GP-TI Neurons in the structure of GP neurons
The full reconstructions of GP-TI neurons. In red show the Somata, in blue axons, and in
green axonal boutons. As the picture show each neuron was prototypic in its long-range
axonal projections descending to the STN and other BG. And also each neuron gave rise to
extensive local axon collaterals in GPe, some cells additionally innervated the Str. (Mallet et
al., 2012).
33
Figure 10: Example of GP-TA Neurons in the structure of Globus Pallidus Neurons
The full reconstructions of GP-TA neurons. Red color shows the Somata, in blue axons, and
in green axonal boutons. (Mallet et al., 2012).
The GP is controlled by multiple projections, different circuits and various
neurotransmitters, thus its function is under integrated regulations. As what has been
discussed above, there are mainly two types of GABAergic inhibitory inputs in the GP, which
are from the striatum (striato-pallidal input) and from local collaterals of neighbouring
pallidal neurons (pallido-pallidal input). The striatopallidal synapses are usually distributed at
distal dentritic compartments and express relatively higher GABAA α2 subunits;
comparatively, the pallido-pallidal synapses are more somatic and proximal, containing more
GABAA α3 subunits (Gross et al., 2011).
The next part will be devoted to the different afferent and efferent projections of
the GP, and also to the role of the GP in the basal ganglia.
34
I.4 Functional Considerations
In the current accepted model of the functional organization of the basal ganglia, the
GPe is considered as a simple "relay station" in the indirect pathway connecting the striatum
to the GPi/SNr, directly or indirectly through the STN (DeLong et al., 1985, Albin et al.,
1989, Alexander et al., 1990). However, recent studies highlighted the GPe more than just a
relay playing a central role in the integration and processing of information in the circuit
(Parent et al., 2000). Anatomical studies using anterograde double labeling have indeed
shown that projections from the striatum and STN neurons converge to the GPe. Knowing
that these two structures receive cortical projections, it seems more likely that cortical
information is integrated at the level of GPe neurons (Smith et al., 1998). Although the
precise functional role of this interaction between the striatum and the STN at GPe level is not
well characterized, it is not inappropriate to suggest that the activation of the inhibitory
(GABA) and/or excitatory (glutamatergic) pathways influence the discharge firing of GPe
neurons. Indeed, studies in non-human primate have shown that the firing activity of GPe
neurons varied in relation to the movement. They showed that GPe neurons exhibit a unique
spectrum of properties different from those of cortical neurons in retrieval of behavioral goals
from visual signals and the specification of actions, which are two crucial processes in goaldirected behavior. This indicates that the GP may play an important role in detecting
individual behavioral events (Arimura et al., 2013).
To explore the influence of subthalamic and striatal projections on the neuronal
activity of GPe, Hatanaka and Colleagues (2007) have recorded the electrical activity of GPe
neurons during the execution of a motor task. They have concluded that GPe neuronal activity
was modulated by its GABAergic and glutamatergic afferents (Hatanaka et al., 2007).
Furthermore, while STN lesions did not change the firing rate and patterns of GP neurons in
35
normal rats, it normalized the firing pattern in 6-hydroxydopamine rat model of PD, by
changing the abnormal bursts to a tonic regular firing characteristic of the normal situation
(Ni et al., 2000). Other studies in non-human primate (Nambu et al., 2000, Kita et al., 2004)
have shown that the GPe and GPi receive their main excitatory inputs from the STN. The
STN recorded neurons, in quietly resting awake animals are spontaneously active. Thus the
activity of GPe and GPi neurons, which are capable of generating autonomous firing, is
modified by background glutamatergic inputs from the STN. The contribution of STN inputs
to the basal firing activity of GPe and GPi neurons were examined by chemical blockade of
STN with local injection of the potent and long acting GABAA receptor agonist, muscimol.
Muscimol injection into the STN in awake monkeys resulted in a dramatic decrease of the
firing rate of GPe neurons, to complete silence in some neurons. This finding indicates that a
tonic level of excitatory input plays an important role in the basal firing rate of GPe neurons
in vivo (Nambu et al., 2000, Kita et al., 2004).
The existence of direct projections from the SNr to GPe / GPi, considered strategic for
controlling the activity of these output structures (Albin et al., 1989, Hazrati et al., 1990, FinkJensen and Mikkelsen, 1991), seems to put the GPe in a central position in the treatment of
cortical information within the circuit of the basal ganglia. Then the GPe modulates the
excitability of output structures of the basal ganglia (Obeso et al., 2006). Indeed, the activity
of these structures governs and predicts the engine since reduction or abnormal increase in the
frequency of neuronal discharge status is associated respectively with dyskinesia or
parkinsonism. In addition, studies in monkeys have shown that the firing rate of GPe neurons
varies inversely with that of SNr/GPi neurons (Filion and Tremblay, 1991). Thus in the
parkinsonian state, the electrical activity of GPe neurons is characterized by an abnormally
low firing rate while the frequency of discharge of SNr/GPi neurons is abnormally increased.
36
The pallido-subthalamic projections are a key element in the indirect pathway that
conveys striatal information to the output structures of the basal ganglia. Via its GABAergic
projections, the GP has a strong tonic inhibition of neurons in the STN. Indeed, extracellular
recordings were early shown that electrical stimulation of the GP suppressed the spontaneous
activity of subthalamic neurons (Kita and Kitai, 1987). In addition, the experiments conducted
by Fujimoto and Kita in 1993 revealed that the GP modulated the STN response to other
afferents, particularly those from the cerebral cortex (Fujimoto and Kita, 1993). Thus,
suppression of the action of GP by injury increased the response of STN neurons to cortical
stimulation. Indeed, experimental studies have shown that lesions of the GP resulted in
significant changes in the spontaneous activity and the pattern of discharge of neurons in the
STN (Ryan and Clark, 1992), so that the GP is considered a key structure for controlling the
activity of the STN, making its neurons below a certain threshold of activity (Campo et al.,
2003).
It was also suggested that, when animal is in motion, the STN and the striatum are
stimulated simultaneously by excitatory signals from the cortex. This stimulation of striatal
neurons leads to inhibition of GABAergic neurons of the GP, resulting in a disinhibition of
STN neurons, which then are free to respond to cortical stimulation. Also, Fujimoto and Kita
(1993) established a relationship between the "pause" in the discharge of GP neurons in
animals during movements and the increased discharge activity of STN neurons and their
ability to discharge in burst in response to excitations from the cortex and elsewhere
(Fujimoto and Kita, 1993). However, the pallido-subthalamic interaction plays a crucial role
in the mechanism of inhibition-disinhibition in this closed circuit pallido-subthalamo-pallidal,
since the state of inhibition of neurons in the STN will be restored, thanks to the excitatory
afferents from STN neurons to the GP, which will reactivate the pallido-subthalamic
37
inhibitory pathway. A summary of different actions of dopamine and it‟s agonist on the GP
neurons are shown on the table 3.
I.4.1 GABAergic neurotransmission
γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammalian brains,
together with glycine, which is mainly distributed in the spinal cord, they compose the
inhibitory neurotransmission system in the mammalian CNS. In mammalian brains,
GABAergic inhibition is essential for controlling excitatory signal transmission, maintaining
the excitatory/inhibitory balance of neuronal circuits and filtering input/output information
(Smith and Kittler, 2010). Once GABAergic neurons are activated, GABA is released to the
inhibitory synaptic cleft from presynaptic compartment and binds to specific transmembrane
receptors on the plasma membrane of pre-, post- and extra-synaptic regions. The major effect
of GABA is inhibitory in adult brain, but during embryonic or early postnatal development
stage it also can be excitatory (Li and Xu, 2008). In mature mammalian brains, the binding of
GABA to its receptors results in the influx of Cl- and hyperpolarization of the neuron
therefore inhibits the generation of action potentials (Goetz et al., 2007). Deficits in the
GABAergic neurotransmission is involved in various psychiatric and psychological diseases,
including epilepsy, Down syndrome, anxiety disorders, depression, schizophrenia, and autism
(Fritschy, 2008, Rudolph and Mohler, 2013). In some neurodegenerative diseases such as
Huntington
disease
and
Parkinson‟s
disease,
the
dysfunction
of
GABAergic
neurotransmission also contributes to the motor symptoms.
I.4.2 GABA synthesis, enrichment and degradation
In GABAergic neurons, GABA is synthesized from the excitatory neurotransmitter glutamate
using the enzyme glutamate decarboxylase (GAD). There are two isoforms of GAD, GAD65
(also named GAD2) and GAD 67 (also named GAD1), which are named by their molecular
38
weight. The GAD65 is reported to directly interact with the vesicular GABA transporter
VGAT (or VIAAT, vesicular inhibitory amino acid transporter), indicating that when
glutamate is present at the presynaptic cytosol of GABAergic neurons, it is rapidly converted
into GABA and enriches the presynaptic vesicles (Jin et al., 2003). There are membranebound glutamate transporters EAAT3 (Excitatory amino-acid transporter 3) at presynaptic
terminals of inhibitory neurons, which are responsible for taking up glutamate to presynaptic
cytosol and serve as GABA synthesis source (Conti et al., 1998a, He et al., 2000) (Figure. 11).
Recent studies show that glutamine may also serve as an important source for GABA
synthesis in immature tissue or during periods of increased synaptic activity (Liang et al.,
2006, Brown and Mathews, 2010).
Figure 11: Schematic drawing of transmitter release, transport, and synthesis at a
GABAergic synaptic terminal. The axonal ending of an inhibitory interneuron (PRE) is
drawn on the left, a glial cell (GLIA) on the right. Bottom structure indicates postsynaptic
membrane of a target cell (POST), for example, a pyramidal neuron. Transporters are marked
by flanking arrows, and synthesizing or degrading enzymes are marked by a centred arrow.
Transporters are colour matched to substrates: GABA is shown as blue particles, glutamate in
red, and glutamine in green. GS: glutamine synthetase, Mit: mitochondrion, PAG: phosphateactivated glutaminase, SV: synaptic vesicle, and VATPase: vacuolar-type H+-ATPase. For
other abbreviations, see the main text. From (Roth and Draguhn, 2012).
39
GABA is enriched in presynaptic vesicles of GABAergic neurons by VGAT, which is
embedded in the vesicular membrane and uses the electrochemical gradient for H+ to absorb
GABA into small synaptic vesicles (Hsu et al., 1999, Ahnert-Hilger and Jahn, 2011, Roth and
Draguhn, 2012). Additionally, chloride gradients between vesicle lumen and presynaptic
cytosol may contribute to the vesicular loading of GABA (Ahnert-Hilger and Jahn, 2011,
Riazanski et al., 2011, Roth and Draguhn, 2012). It is estimated that the concentration of
GABA within vesicles could be as high as 1000 folds comparing to presynaptic cytosol
(Edwards, 2007). After release, GABA is cleared and taken by membrane-bound GABA
transporters (GATs) into neurons or glial cells. GAT-1 is expressed mainly on neurons while
GAT-3 is predominantly observed on glial cells, although the different GAT isoforms are
partially overlapping (Minelli et al., 1996, Ribak et al., 1996, Conti et al., 1998b). This uptake
also contributes to the modulation of GABAergic neurotransmission (Figure 11).
GABA is finally degraded by GABA transaminase (GABA-T) in the mitochondria of neurons
and glial cells. GABA-T induces transamination of GABA and α-ketoglutarate, producing
succinic semialdehyde and glutamate (Kugler, 1993). It is estimated that more than 90% of all
GABA in the mammalian CNS is degraded in this way and contributes to energy metabolism
in the TCA cycle (Roth and Draguhn, 2012). Taking together, the GABA concentration in
synaptic vesicles, cytosol and extracellular space is the result of the balance among synthesis,
enrichment and degradation. The equilibrium of these mechanisms is important to maintain
the physiological role of GABAergic neurotransmission.
I.4.3 GABA receptors
There are mainly two types of GABA receptors, which are chloride permeable ligand-gated
ion channels (GABAA receptors) and metabotropic G-protein-coupled GABAB receptors
(GABABRs). GABAA receptors (GABAARs) facilitate fast inhibition and play the major role
40
of inhibitory receptors; GABABRs mediate a “slow” and “late” inhibition, or function as autoreceptors that control the typical negative feedback loop of synapses when expressed
presynaptically (Isaacson et al., 1993, Misgeld et al., 1995, Scanziani, 2000). Evidence shows
that that synaptically released neurotransmitters saturate their receptors (Clements, 1996).
Therefore, the strength of GABAergic synapses highly depends on the number of postsynaptic
GABAARs (Otis et al., 1994, Nusser et al., 1997).
I.5 Efferents of the GPe
Individual neurons of the GPe innervate basal ganglia output nuclei (GPi and SNr) as
well as the STN and SNc (Figure 12). About one quarter of them also innervate the striatum
and are in a position to control the output of the striatum powerfully as they preferentially
contact GABA interneurons. A small number of GPe neurons also innervate the dorsal
thalamus, inferior colliculus, the pedunculopontine tegmentum and the thalamic reticular
nucleus.(Shink et al., 1996, Bolam et al., 2000). GPe axons form large symmetric boutons that
contain small round or elongated vesicles and multiple mitochondria, and form symmetric
synapses on the somata and proximal dendrites of GPi and STN neurons, similar to the local
collateral axons (Chang et al., 1983, Shink and Smith, 1995). In the Str, GPe axons terminate
on aspiny GABAergic interneurons and the dendritic shafts of spiny projection neurons. The
topographic arrangements of the GPe-STN and GPe-striatal projections are in register with
that of the STN-GPe and striatal-GPe projections, suggesting the existence of precise
reciprocal loops (Smith et al., 1998) .
Experiments with sensitive anterograde tracers, such as PHA-L or biocytin, show that
the primate GPe projects to most of the core structures of the basal ganglia including the
striatum, EP/GPi, SNr, STN as well as the reticular nucleus of the thalamus and the
41
pedunculopontine tegmental nucleus (Albin et al., 1989, Amirkhosravi et al., 2003b, Tong and
Melara, 2007) (Figure 13).
The pallido-striatal projection has been considered minor, however, several studies
have suggested that this projection is heavier than previously thought and that it might play a
significant role in controlling the activity of the striatum (Kita and Kita, 2001).
From a physiological point of view, the GABA mediated inhibitory effect of the GPe
is considered essential for the control of the subthalamic nucleus, so that the latter structure
could adequately exert its powerful glutamate-mediated driving effect on the basal ganglia
(Kita and Kitai, 1987).
As previously described the projection from GP to the STN uses GABA as a
transmitter (Shink et al., 1996, Hauber and Lutz, 1999), and exerts a strong inhibitory action
on STN neurons. Morphological studies have revealed that GP neurons may be subdivided
into two main groups according to their main projection target and chemical features. Neurons
projecting to the STN (~60%) contain calcium-binding protein parvalbumin whereas those
projecting to striatum (~40%) predominantly express preproenkephalin and make synapsis
with striatal interneurons (Aldana et al., 2003) (Franquet et al., 2003). Neurons in the lateral
portion of GP target specifically the lateral two-thirds of the STN (Albin et al., 1989).
Other studies showed that neurons projecting to the STN were localized in the rostral part of
the GP .
The projection arises from the subpopulations of pallidal neurons belonging to the
categories of aspiny and spiny neurons located mainly in the lateral part of the GP (Totterdell
et al., 1984, Smith and Bolam, 1989, Arenas et al., 2003).
42
Figure 12: Microcircuitry of the pallido-subthalamic projection. Individual pallidal (GP)
neurons that project to the STN possess local axon collaterals and innervate other structure of
BG. This part is reproduced from Bevan et al (Bevan et al., 1998).
Pallido-nigral terminals display specific ultrastructural features. They have a large
size, contain pleomorphic vesicles, numerous mitochondria and form symmetric synaptic
contacts preferentially with the perikarya and proximal dendrites of the SNr projection
neurons. These features contrast those of the striato-nigral terminals that are of a smaller size,
possess few mitochondria and contact predominantly more distal portions of the dendrites of
SNr cells (Totterdell et al., 1984, Smith and Bolam, 1989, von Krosigk et al., 1992). The GP
sends massive inhibitory projections to the output nuclei that terminate as groups of large
varicosities that are closely positioned around the soma and proximal dendrites of GPi and
SNr (Natarajan and Yamamoto, 2011).
43
Figure 13: Simplified diagram of the main efferent projections of GP neurons. The main
projection sites to the GPe are the GPi, STN, and Striatum as mentioned above. A small
number of GPe neurons also innervate the dorsal thalamus, SNr and pedunculopontine
nucleus (NPP). According to (Tepper et al., 2007).
44
I.6 Afferents of the GPe
The globus pallidus does not receive massive projections from the cerebral cortex or
the thalamus, which is a major difference compared to the striatum regarding the connections.
The most important afferent connections of the GP arise from the striatum (GABAergic
fibers), and from the STN (glutamatergic afferent fibers). GP receives also other connections
from the GPi, substantia nigra pars compacta (SNc), raphe, and pedunclopontine tegmentum
(Albin et al., 1989, Hazrati et al., 1990, Deschenes et al., 1996, Amirkhosravi et al., 2003a,
Yasukawa et al., 2004) (Figure 15).
In both rats and monkeys, striatal projection neurons project to the GP/GPe, with
approximately half of the total number only and the other half emitting collaterals to the
GP/GPe on the way to the GPi and the SNr (Albin et al., 1989, Wu et al., 2000).
I.6.1 GABAergic afferents of the Globus Pallidus
The major GABAergic afferent fibers input to the GP/GPe is derived from the
striatum. The striatum contains two groups of projection neurons; one project to GP/GPe only
and the other to GP/GPe, GPi, and SNr. The Striatum-GP/GPe neurons contain GABA,
dynorphin, and enkephalin and express D2-like dopamine receptors (D2Rs). The StriatumGPe/GPi/SNr neurons contain GABA, substance-P and express D1-like dopamine receptors
(D1Rs). The striatal input to GPe/GPi is GABAergic, and inhibits GPe/GPi neurons (Kita,
1992) .
All GPe projection neurons that were examined with single cell staining had local
axon collaterals, some having dense axonal arborizations with numerous boutons and others
having short axons with less numerous boutons (Kita and Kitai, 1994). Most of the terminal
fields of collateral axons could be found in the same medio-lateral zone of the GPe, where the
parent cells were located.
45
The GPe also receives sparse afferents from the GPi and SNr (Fink-Jensen and
Mikkelsen, 1991) this study were examined by use of the anterogradely transported
lectin Phaseolus vulgaris-leucoagglutinin (PHA-L). The use of PHA-L into different parts of
the GPi resulted in a moderate number of labeled nerve fibers in the ipsilateral part of GP.
The fibers showed a heterogeneous morphology: some were of small caliber with few delicate
varicosities, others were of medium caliber with several more bulbous nerve terminals.
Restricted injections in the dorsal and ventral parts of the GPi, respectively, displayed that the
dorsal part of the GPi projects to the dorsal and rostral parts of the dorsal pallidum and the
ventral part to the ventral and caudal parts (Fink-Jensen and Mikkelsen, 1991).
The SNr and the GPi provide major output nuclei of the BG where the final stage of
information processing of this system takes place. These cell groups are mainly composed of
GABAergic neurons, integrate inputs from all other component nuclei of the BG (Deniau et
al., 2007).
Another study on non-human primates suggested that the biotinylated dextran amine
injected in the GPi was transported retrogradely to perikarya in the GP/GPe and the STN and
then anterogradely, via axon collaterals, to the STN and the GP/GPe respectively (Shink et al.,
1996). This suggestion was supported by injections of biotinylated dextran amine
or Phaseolus vulgaris-leucoagglutinin in regions of the GP/GPe that corresponded to those
containing retrogradely labelled cells following injections in the GPi (Shink et al., 1996).
I.6.2 Glutamatergic afferents of the GP
The GP/GPe receives glutamatergic projections from the STN. Studies with the
anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in the rat indicate that the
pallidal complex and the SNr are the main targets of the subthalamic nucleus (Kita and Kitai,
1987, Albin et al., 1989). The STN is the only glutamatergic component of the BG and
46
provides the main source of glutamatergic inputs to BG output nuclei. In rodent, the STN
projection to the SNr originates from the whole nuclei, with most STN neurons sending
branched axons to GP/GPe, EP/GPi and SNr (Deniau et al., 1978a, Deniau et al., 1978b, Van
Der Kooy and Hattori, 1980).
Extracellular recording experiments revealed that electrical stimulations of the
GP/GPe suppressed the spontaneous firing of STN neurons. Recent studies reported that the
firing rate and patterns of STN neurons is regulated by the reciprocally connected GABAergic
from GP and glutamaergic afferents from the cortex (Marin et al., 2003). Activation of striatal
neurons lead to the inhibition of GP neurons, which disinhibits STN neurons leaving them
free to respond to cortical influence. The inhibited state at the level of the STN is restored by
the subthalamic excitatory input to GP neurons and the reactivation of the pallido-subthalamic
pathway. (
In the light of these results, the GP/GPe can no longer be regarded only as part of a
closed intrinsic loop with the STN. Instead, it must be viewed as a structure that can influence
most components of the basal ganglia and thus occupies a crucial position in the indirect
pathway of the basal ganglia circuitry (Albin et al., 1989).
The GP/GPe receives glutamatergic inputs from the STN to modulate the local
GABAergic neurotransmission. Evidence shows that both striatal-pallidal and local axon
collateral terminals have group III mGluRs (Bradley et al., 1999). Activation of these
receptors can presynaptically reduce both GABAergic and glutamatergic transmission in the
GP/GPe (Matsui and Kita, 2003). The group I mGluRs are also observed in the GP but their
function is not clearly revealed (Paquet and Smith, 2003, Mitrano and Smith, 2007). As a part
of the indirect pathway and the target of striatal projections, the GP/GPe neurons express
adenosine A2A receptors, dopamine D2 receptors as well as the cannabinoid CB1 receptors at
the striato-pallidal terminals. Those receptors control the GABAergic and glutamatergic tune
47
in the GP and are involved in motor dysfunctions like Parkinson‟s disease (Kita, 2007, Jaeger
and Kita, 2011b). In addition, the GP receives abundant serotoninergic (5-HT) innervation
from the dorsal raphe (Charara and Parent, 1994). The main effect of the 5-HT projections is
to suppress the glutamatergic excitation (through 5-HT1A-R) and GABAergic inhibition
(through 5-HT1B-R) (Kita et al., 2007, Hashimoto and Kita, 2008, Rav-Acha et al., 2008).
48
I.6.3 Dopaminergic afferents of the GP
In addition to the GABAergic and glutamatergic afferents, anatomical studies have
shown that GP/GPe also receives dopamine fiber terminals. Dopamine is one of the major
neurotransmitter in the basal ganglia and in the central nervous system in general, although
the amount of DA producing neurons does not represent more than 0.3% of brain cells.
Anatomical studies have distinguished eight major dopaminergic pathways in the brain, with
the three main originate in the midbrain (see Figure 14).
Figure 14: Representation of the three major central dopaminergic pathways.
49
The mesolimbic pathway links the DA-producing cells in the ventral tegmental area
(VTA) in the midbrain to the nucleus accumbens (NA), which is located in the ventral
striatum and is a part of the limbic system within the amygdala. The mesolimbic pathway is
thought to be involved in social emotional behavior and motivation, and is often associated
with feelings of reward and desire, particularly because of the connection to the nucleus
accumbens, which is also associated with these states.
The mesocortical pathway also involves neurons in the ventral tegmental area that
innervate the frontal cortex and surrounding structures. It is essential to the normal cognitive
functions of the dorsolateral prefrontal cortex, and is thought to be involved in motivation and
emotional response, attention, initiative, planning, decision making, working memory, and
other higher cognitive functions.
Finally, the nigrostriatal pathway projects from the SNc to the striatum (caudate and
putamen), but also to other structures of the basal ganglia, involved in motor control. Loss of
dopamine neurons in the SNc results in a marked reduction in dopamine function, which is
one of the main pathological features of Parkinson's disease. The symptoms of the disease
typically do not show themselves until 80-90% of dopamine function has been lost (Smith and
Kieval, 2000, Smith and Villalba, 2008).
The existence of a dopaminergic innervation of the GP/GPe has been demonstrated in
rats, monkeys and humans (Jan et al., 2000, Debeir et al., 2005). The GP/GPe neurons are
innervated by nigral dopamine fibers, by separate fiber system and also by collaterals of
nigro-striatal fibers. This has been shown in rodents (Lindvall and Bjorklund, 1979; Debeir et
al., 2005; Anaya-Martinez et al., 2006), in non-human primate (Nobin and Bjorklund, 1973;
Parent and Smith, 1987; Lavoie et al., 1989; Parent et al., 1989; Francois et al., 1999;
Hedreen, 1999; Jan et al., 2000) and in human brains (Bjorklund and Nobin, 1973, Cossette et
50
al., 1999) (Nobin and Bjorklund, 1973; Cossette et al., 1999; Francois et al., 1999; Jan et al.,
2000). There are two types of morphologically different nigrostriatal projections to the
GP/GPe. One type of axons innervates massively the striatum and sparsely extrastriatal
structures including the GP, and the other type of axons are the inverse (Cossette et al., 1999).
In the striatum, it is well known that dopamine plays a key role in the control of the overall
balance of the activity between direct and indirect pathways (Gerfen et al., 1990, Chen et al.,
2011). Dysfunctions in the dopamine system, including in the GP, are involved in the
pathophysiology of several central nervous system diseases, including Parkinson‟s disease
(Chen et al., 2011). Several studies using immunohistochemical analysis of tyrosine
hydroxylase and biochemistry have shown a degeneration of the nigro-palidal pathway in
animal models (Bergman et al., 1994; Jan et al., 2000; Anaya-Martinez et al., 2006) and
parkinsonian patients (Jan et al., 2000; Rajput et al., 2008).
In addition, the PV positive and negative (PPE positive) GP neurons positively and
negatively modulated, respectively, by dopaminergic transmission (Hoover and Marshall,
2002). This may implicate a novel dopaminergic regulation in the BG macrocircuit and a
dichotomous role of GP in dopaminergic dysfunction such as PD.
51
I.7 Types of dopaminergic receptors in the GP
Dopamine acts via five receptor subtypes subdivided into two receptor families: D1
(D1 and D5 subtypes) and D2 (D2, D3 and D4 subtypes). All are prototypic of G-proteincoupled receptors with dopamine D1 receptors being positively linked to adenylate cyclase
and D2 receptors had negative coupling to the enzyme (Kebabian and Calne, 1979, Richfield
et al., 1987, Camps et al., 1990). Experimental studies reported that functional dopamine
receptors are expressed in the striatum, GPe, GPi and STN and that dopamine modulates their
neuronal activity through a variety of mechanisms via pre- and post-synaptic sites (Smith and
Villalba, 2008, Rommelfanger and Wichmann, 2010)( Benazzouz et al., 2014) (see table 2).
In the striatum, which is the main target structure of nigral dopamine projections, D1
and D2 receptors are present in medium-sized spiny projection neurons throughout the dorsal
and ventral striatum, whereas D3 receptors are confined to the limbic-related ventral striatum.
D1 and D2 receptors are largely segregated into the two major populations of striatofugal
neurons: striatonigral substance-P-containing neurons, which possess a high level of D1
receptors, and striatopallidal enkephalin-immunoreactive neurons, which are enriched with
D2-receptor mRNA (Campo et al., 2003). This segregation, however, is not exclusive: striatopallidal neurons also contain low levels of D1 receptors and striatonigral neurons possess low
levels of D2 receptors (Surmeier et al., 1996, Aizman et al., 2000). In the ventral striatum, D3
receptors are partly co-localized with D1 and D2 receptors on striatofugal neurons (Le Moine
and Bloch, 1996). Intrinsic cholinergic interneurons also contain high levels of D2 and D5
receptors. Low levels of D4 and D5 receptors have been reported in striatofugal neurons but
the exact projection sites of these neurons remain to be established (Le Moine et al., 1990,
Bergson et al., 1995, Defagot et al., 1997). Presynaptic D2 autoreceptors and heteroreceptors
52
on cortical glutamatergic terminals have also been reported in pharmacological studies
(Chesselet, 1984).
In the GP/GPe, D2Rs are found in approximately 40-50% of all pallidal neurons
(Floran et al., 1997). Most of the presynaptic dopamine receptors in the GP/GPe are thought
to be D2Rs and are located on terminals of the GABAergic striatopallidal projection (Campo
et al., 2003, Feresin et al., 2003, Pelayo et al., 2003). More controversially, it appears that a
small number of D1 receptors are present on the terminals of GABAergic symmetrical
synapses in the rodent GP (Levey et al., 1993, Yung et al., 1995). Other studies have shown
the presence of D3, D4 receptors in the rat (Khan et al., 1998, Rivera et al., 2003) and D5 in
rats and monkeys (Khan et al., 2000) (for more details see Table 2).
Table 2: Summary presentation of the distribution of dopamine receptors in the external
globus pallidus: M, monkey; R, rodent. (Adapted from, (Rommelfanger and Wichmann,
2010)
Structure
Synaptic
location
Pre
GPe
D1
D1
D2
D5
D2
Axons,
GABA
terminals R
GABA terminals
R (Levey et al., 1993)
(Levey et al.,
1993, Yung et
al., 1995)
Glutamate
terminals M
D3
D4
Axons,
terminals
R
(Rivera et al.,
2003)
(unpublished observations)
Dendrites R
(Yung et al., 1995)
Perikarya R
Post
(Ciliax et al.,
1999, Khan et al.,
2000), M (Ciliax
et al., 1999)
Perikarya R
(Khan et al., 1998)
Perikarya
Perikarya R (Mrzljak
R
et al., 1996,
(Khan et al.,
1998)
Khan et al.,
1998)
A previous study using electron microscopy confirmed the presence of presynaptic
D2-receptors in the monkey GPe on putative GABAergic axons and terminals, with sparse
labeling of putative glutamatergic terminals (DeLong, 1990). Another study showed that
53
dopamine also inhibits GABA transmission from the globus pallidus to the thalamic reticular
nucleus via presynaptic D4 receptors (Shink et al., 1996).
In the striatum, and to a lesser degree in the GP/GPe, dopamine appears to be essential
for balancing the activity of the two pathways producing opposite effects in the striatal cells
that project to the direct and indirect pathways through D1 and D2 receptors (Fallon and
Moore, 1978, Gerfen et al., 1990, Keefe and Gerfen, 1995, Ryczko et al., 2013).
Given the predominance of D2Rs in GP/GPe, it is likely that most functional actions
of dopamine in GPe are mediated via D2Rs. Activation of pallidal D2Rs has been shown to
increase the electrical activity of GP/GPe neurons. For instance, activation of D2Rs in the rat
GP increases the expression of the immediate early gene c-fos (Billings and Marshall, 2003),
and infusions of the non-specific dopamine receptor agonist apomorphine into the rat GP
increases pallidal neuronal activity (Napier et al., 1991). Other studies in primates have shown
that the neuronal activity in GPe increased after infusions of the quinpirole in the globus
pallidus, and that infusions of the D2R antagonist sulpiride lowered pallidal firing rates,
suggesting that the pallidal D2Rs are occupied by endogenous dopamine under normal
conditions (DeLong, 1990).
One key to understand the effects of DA in the GP is characterizing the affected
neuron populations. Approximately two-thirds of GP neurons contain the calcium binding
protein parvalbumin (PV) and these cells project predominantly to down-stream structures of
the indirect pathway, namely the STN and output nuclei (Amaya et al., 2003, Amirkhosravi et
al., 2003a, Matsuda et al., 2003).
In addition to dopamine D2Rs, a lower level of D1Rs has been detected in axons and
terminal boutons forming symmetric, putatively GABAergic synapses in the rodent GP
(Levey et al., 1993, Yung et al., 1995) (Ciliax et al., 1999, Raevskii et al., 2003, Porritt et al.,
2005).
54
Figure 15: Simplified diagram of the main afferents of GP neurons. The external segment
of the pallidum (GPe) receives major inputs from two input nuclei of the basal ganglia, the
neostriatum (Striatum) and the subthalamic nucleus (STN). The internal segment of the
pallidum (GPi) and the substantia nigra pars reticulata (SNr) are the main output nuclei of the
basal ganglia. Other projection cholinergic from pedunculopontine nucleus (NPP) and
dopaminergic from substantia nigra pars compacta (SNc). According to (Tepper et al., 2007).
55
Table 3: Functional effects of dopamine receptor agonists in the globus pallidus.
(Rommelfanger and Wichmann, 2010)
Structure
Effects of dopamine or nonspecific D1LR/D2LR agonists
Increases firing rate
(Napier et al., 1991)
GPe
Decreases GABA transmission
(Bergstrom and Walters, 1984)
D1 agonist
effects
D2 agonist effects
Increases glutamate
release (Hernández et
al., 2007)
Increases glutamate
release
(Floran et al., 1990)
Increases firing rate
(unpublished observations)
Increased c-fos
(Billings and Marshall,
2003)
Increases GABA release (Floran et al.,
1990)
Contralateral dystonic posture (Chen et
al., 2011)
Increases the number and duration of
Increase the activity
of GP (Chen et al.,
2011)
Decreases GABA release
(Floran et al., 1997)
Decreases GABA-A
currents (Cooper and
Stanford, 2001a, Shin et al.,
2003)
Decreases glutamate release
(Hernández et al., 2007)
High-Voltage Spindle (Yang et al., 2013)
56
I.8 Electrophysiological responses of GP neurons to dopamine drugs
Although dopamine innervation of the GP/GPe is considered to be scarce, it plays an
important role in the modulation of GP/GPe neuronal activity (Lindvall and Bjorklund, 1979,
Lavoie et al., 1989). Indeed, local microinjection of dopamine and dopamine agonists into the
GP reduced the effectiveness of the response of GP/GPe neurons to GABA application
(Bergstrom and Walters, 1984). Moreover, microinjection of dopamine increased the firing
rate of GP neurons by the attenuation of iontophoretically applied GABA or striatal
stimulation in vitro (Nakanishi et al., 1985). Systemic dopamine administration also
mimicked direct dopamine GP/GPe application by increasing the activity of GP/GPe neurons
(Bergstrom et al., 1982, Carlson et al., 1990), although in these studies, actions of dopamine
in other nuclei cannot be discounted.
Several studies have shown that the nigro-pallidal dopaminergic pathway may directly
modulate the electrical activity of GP/GPe neurons (Bernal et al., 2003). Fuchs showed that
local administration of cocaine or amphetamine in the rat induced dopamine increases in the
GP (Johansen et al., 2003, Fuchs et al., 2005). Other studies showed that local administration
of dopamine produced changes in GP/GPe neuronal firing rate by activating dopamine
receptors (Chen et al., 2011, Qi and Chen, 2011, Hadipour-Niktarash et al., 2012).
In addition to the predominant action of dopamine on D2Rs, several studies demonstrated
that activation of dopamine D1Rs produced different effects on the firing rate of GP/GPe
neurons in the rat (Ruskin et al., 1998, Chen et al., 2011, Qi and Chen, 2011).
Recently, in vitro patch clamp studies in rodent brain slices suggested that activation of
presynaptic D1Rs facilitates glutamate release in the globus pallidus (Hernández et al., 2007)
while activations of D2LRs reduces it.
57
In animal models of PD with dopamine cell lesions, the electrical activity of GP neurons
was profoundly impaired. Early work in the MPTP monkey allowed the identification of a
reduction in the firing rate of neurons in the GPe (Filion and Tremblay, 1991). The regular
activity interspersed with pauses in the normal monkeys was replaced by a bursty activity of
action potentials, which is an electrophysiological pathological signature of Parkinson's
disease (Filion and Tremblay, 1991, Nini et al., 1995). Another characteristic of Parkinson's
disease is the emergence of a synchronization between the neurons of the GP/GPe (Nini et al.,
1995). The mechanisms responsible for this neuronal synchronization between neurons in the
GP/GPe are not known, but some authors believe that this synchronization can appear with
changes in intrinsic neuronal properties (Deister et al., 2013). Immunohistochemical analysis
of tyrosine hydroxylase in the GP/GPe of animal models and parkinsonian patients has shown
a degeneration of the nigro-pallidal pathway suggesting that the absence of direct
dopaminergic modulation of GP/GPe could contribute to the disruption of the neuronal
activities in this nucleus in Parkinson's disease.
58
I.9 Behavioral study
The GP/GPe has long been associated with the control of motor behavior. In primates,
firing rates of many pallidal neurons changed during limb movements (Sekita-Krzak et al.,
2003). Both clinically and experimentally, lesions of the GP can affect voluntary movements
(Bochynska et al., 2003, Shin et al., 2003), as can direct intrapallidal infusion of drugs,
particularly those binding to GABA receptors.
This dopaminergic projection to GP attracted scant attention in studies of the basal
ganglia, yet it appears to contribute importantly to the function of GP/GPe neurons and their
involvement in motor behaviors. Intrapallidal dopaminergic manipulations can affect GP
neuronal firing rate as well as motor behavior (Hauber and Lutz, 1999, Joel et al., 2003,
Marino et al., 2003, Qi and Chen, 2011, Hadipour-Niktarash et al., 2012). In the last study of
our teams, it has been shown that 6-OHDA-injection into the GP resulted in a reduction of
locomotor activity as measured in the open field test. This locomotor impairment has also
been demonstrated by our stepping test results showing that the same DA depletion induced a
marked and long-lasting impairment in the forelimb contralateral to the lesion (Abedi et al.,
2013). These results fit with a previous study (Bouali-Benazzouz et al., 2009), in which it has
been shown that 6-OHDA injection into the GP produced deficits of dopaminergic
transmission that caused asymmetrical motor impairment (see figure 15). Indeed,
subcutaneous injection of apomorphine, a non-selective dopamine agonist, induced a
rotational behavior. The observed motor effects can be due to the depletion of DA into the GP
or into the striatum or a combined depletion into the two structures (Hauber and Lutz, 1999,
Fuchs et al., 2005).
In general, activation of D1Rs or D2Rs in the rodent GP appeared to facilitate
movement. In support of this notion, local intrapallidal infusions of D1R or D2R antagonists
59
were found to induce akinesia (Hauber and Lutz, 1999) and catalepsy (Costall et al., 1972) in
rats, likely by blocking the effects of endogenous dopamine on these receptors. Similarly,
intrapallidal infusion of D1R agonists (Sanudo-Pena and Walker, 1998) increased general
movement. Other studies have demonstrated that infusion of D1R agonists and D2R agonists
(Koshikawa et al., 1990) induces stereotypic jaw movements. Taking into account these
findings, we thus provide additional arguments that dopamine transmission within the GP is
necessary to achieve motor control and that its lack plays a role in generating the motor
symptoms.
60
General objectives
Despite the large amount of clinical and experimental works that has been devoted to
understanding the functional role of basal ganglia, the functional role of dopamine in the
modulation of extrastriatal nuclei of the system is still poorly investigated.
The overall objective of the present work is to provide a better understanding of the
mechanisms by which dopamine modulates the electrical activity of globus pallidus neurons
and also to determine the impact of this modulation on the control of the neuronal activity of
the most efferent structures of the GP, which are the STN and SNr.
To address these issues, we conducted an electrophysiological work in the rat by using
single and multi-channel extracellular microrecordings to investigate the responses of GP
neurons and also of STN and SNr neurons to local pharmacological manipulation using
dopaminergic agents locally injected into the GP.
- The first part of the study focused on studying the effects of local microinjection of
dopamine on the electrical activity of GP neurons.
- Based on the assumption that the action of dopamine in the GP is predominantly
mediated by dopamine D2Rs, we sought to verify this hypothesis by studying the effects of a
selective agonist of D2Rs (quinpirole) on the electrical activity of GP neurons. Blockade of
the responses of GP neurons to quinpirole will be carried out by a selective D2R antagonist
(sulpiride).
- Finally, in order to study the impact of dopamine in the GP on the control of the
neuronal activity of its two main efferent structures, we investigated the effect of
61
microinjections of dopamine and quinpirole in the GP on the electrical activity of STN and
SNr neurons.
In summary, data of this thesis demonstrates that the neuronal activity of GP can be
modulated by dopamine and its agonist (Quinpirole) through D2 receptors, and the activation
of these receptors was involved in major excitation (activation) of GP neurons. Furthermore,
demonstration of the modulation of the pallido-subthalamic and pallido-nigral pathways are
made, which are considered as an important part of the output of the basal ganglia.
62
II. Materials and Methods
II.1 Study Model
Adult male Sprague Dawley rats, weighing 280–380g, were used for in vivo
electrophysiological experiments. Animals were provided by the “Centre d‟Elevage Depré”
(Saint Doulchard, France) and arrived at least 1 week before use. They were housed four per
cage under artificial conditions of light (light/dark cycle; lights on at 7:00 A.M.), temperature
(24°C), and humidity (45%) with food and water available ad libitum. All efforts were made
to minimize the number of animals used and their suffering. All animal experiments were
carried out in accordance with European Communities Council Directive 2010/63/UE. The
study received approval from the local Ethics Committee under the number 50120136-A
(Bordeaux, France).
II.2 Pharmacological substances
Drugs were chosen on the basis of their different affinity for their preferential
receptors. Dopamine was used at dose of the 2µg/20nl. Quinpirole was chosen as D2R
agonist. The drug was purchased from Sigma (Saint-Quentin Fallavier, France) and was
dissolved in sterile saline (0.9% NaCl). A dose of 8µg/20nl was chosen after the realization of
doses response to see witch concentration had an effect on the activity of the firing rate of the
globus pallidus neurons. The first D2R antagonist used was Raclopride at dose of 4µg/20 nl
then 8µg/20 nl and 12 µg/20nl, all this dose does not had an effect on the activity of globus
pallidus neurons. Then we used Sulpiride as the second D2R antagonist: S (−)-sulpiride ((−)5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrodinyl) methyl]-2-methoxy-benzamide). Sulpiride was
dissolved in distilled water added with a few drops of HCl and the final pH of 6.5–7.2 was
63
titrated with NaOH. A dose of 6µg/20nl was chosen after realization of dose response (4µg,
6µg and 10µg); this dose had an effect on the activity of neuron of globus pallidus.
Before the start of the experiments, the microelectrodes were filled with saline solution
or drug effect experiments, respectively. To test the integrity of the recording-injection
system, the system was flushed (at 4 nl/sec) for 5 sec. The pump was then switched off, and
the microelectrode was inserted into the brain. Once a neuron was isolated with sufficient
recording quality and the recording was stable for at least 120 sec, we began the data
collection with a recording of the neuron's baseline activity for 20 min, followed by recording
during the injection of drug and for at least 60 min thereafter.
In this study, different dopaminergic pharmacological substances were used. Their
common name, their property, the concentration at which they were used intraperitoneal
injection, or at the local GP and supplier of each are shown in Table 4.
Table 4: Pharmacological agents used for different experiments in this studies. DCAA:
aromatic amino acid decarboxylase. DAergique: dopamine.
Usuel Name
Property
Concentrations
Volume injected
utilisées
Electrophysiology
Fournisseur
SKF38393
Agoniste D1-like
1µg
20nl
Sigma
SCH23390
Antagoniste D1-like
2µg
20nl
Sigma
Raclopride
Antagoniste D2-like
4µg,8µg, 12µg
20nl
Sigma
Quinpirole
Agoniste D2-like
2µg,4µg ; 8µg
20nl
Sigma
Supiride
Antagoniste D2-like
12.5mg,25mg ; 40mg
2 ml ip
Sigma
2µg
20nl
Sigma
12.5 mg/kg
5µl
Sigma
5 ml / kg
2ml
Sigma
Dopamine
hydrochloride
6-OHDA
Neurotoxine
Desipramine
Inhibits
he
reuptake of
norepinephrine
64
II.3 Electrophysiology in vivo in anesthetized rats
II.3.1 Extracellular recording unit
II.3.1.1 Principle of stereotactic surgery
Brain stereotaxis is used to locate and reach, with the stereotactic frame (Figure 16B), a
brain structure accurately given in the three dimensions of space. It defines the position or the
exact location of the structure using a coordinate system in space and to reach it.
The coordinates of the brain structures are grouped in stereotactic atlas specific to each
species (rat, cat, rabbit, dog, monkey, man). The coordinates we used for locating structures in
rats are those given by the Atlas stereotactic coordinates of Paxinos and Watson (1998)(
Figure 16A) . Determining these coordinates is based on a bony landmark bregma, which is
the point between the coronal suture and sagittal suture on the top of the skull (Figure 16 C).
A
B
C
Figure 16: Example of accessories needed for surgery: A ,Atlas stereotactic coordinates
B, David Kopf stereotaxic frame for rats, and related accessories and C, the use of
coordinates is based on a bony landmark bregma .
65
The animals were previously anesthetized with urethane 20% dissolved in 0.9% NaCl (1.3
g / kg). The rats were immobilized on the stereotactic frame, itself placed in a Faraday cage.
A midsagittal incision is made in the scalp to expose the skull, which is then dried to
distinguish anatomical landmarks bregma and lambda. Using stereotactic atlas of Paxinos and
Watson (1996), the theoretical position of the target structures is determined by the following
coordinates:
- Globus Pallidus : AP : -1mm ; L : -3 mm ; P : 5,0 – 7,5 mm
- Subthalamic nucleus: AP : -3,8mm ; L : -2,5 mm ; P : 6,8 – 8,5 mm
- Substantia nigra pars reticulata: AP : -5,3 mm ; L : -2,5 mm ; P : 7,2 – 8,6 mm
A craniotomy is performed to place the recording electrode in the three values of the
stereotactic coordinates. The dura is then disengaged before lowering the electrode into the
desired structure.
66
II.3.1.2 Characteristic of recording electrodes:
Recording in the globus pallidus, Subthalamic nucleus and Substantia nigra reticulata:
Extracellular single-unit recordings were made in rats anesthetized with urethane (1.2
g/kg, i.p.). For microrecording and simultaneous microinjection of drugs in the GP, a doublebarreled pipette assembly, similar to that described previously (Akaoka and Aston-Jones,
1991, Delaville et al., 2012, Chetrit et al., 2013), was used. The tips of recording and injection
micropipettes were separated by 150 to 200
m. For microrecording in the STN and
simultaneous microinjection of drugs in the GP, the microinjection pipette was placed into the
GP and the recording electrode was placed into the STN. The injection pipette was filled with
a dopaminergic drug and the recording micropipette with an impedance of 8 to 12 MΩ, was
filled with 4% pontamine sky blue in 0.09% NaCl. The micropipettes were placed into the
targeted nucleus according to the stereotactic coordinates given in the brain atlas of Paxinos
and Watson (1996) for the GP (AP: 0.9 mm posterior to bregma, L: 3 mm from the midline,
DV: 5.5 - 7.5 mm from the dura), STN (AP: -3.8 mm posterior to bregma, L: 2.5 mm from the
midline, DV: 7.5-8.5 mm from the dura) and SNr (AP: -5.3 mm posterior to bregma, L: 2.5
mm from the midline, DV: 7.5-8.5 mm from the dura).. Extracellular neuronal activity was
amplified, band pass-filtered (300–3000 Hz) using the Neurolog system (Digitimer,
Hertfordshire, UK), displayed on an oscilloscope and transferred via a Powerlab interface to a
computer equipped with Chart 5 software (AD Instruments, Charlotte, USA). Only neuronal
activity with a signal-to-noise ratio of 3:1 was recorded and used for additional investigation.
Basal firing of GP and STN neurons was recorded for 20 min before drug injection to
ascertain the stability of the discharge activity, then a dopaminergic drug or the saline vehicle
was injected into the GP at a volume of 20 nl, using brief pulses (200 ms) of pneumatic
pressure (Picospritzer III, Royston Herts, UK). This small volume was used to avoid the risk
67
of losing the recorded cell due to pressure effects. In all rats, the central part of the nucleus
was targeted. At the end of each session, the recording site was marked by electrophoretic
injection (Iso DAM 80; WPI, Hertfordshire, UK) of Pontamine sky blue through the
micropipette at a negative current of 20 μA for 8 min. GP and STN neuronal activity was
analyzed off line with a spike discriminator using a spike histogram program (AD
Instruments, Charlotte, USA), and firing parameters were determined using Neuroexplorer
(AlphaOmega, Nazareth, Israel). The firing patterns of GP ,STN and SNr neurons were
analyzed using the coefficient of variation of the interspike intervals and also the density
histograms according to the method developed by (Kaneoke and Vitek, 1996), as previously
described (Belujon et al., 2007, Chetrit et al., 2013). An algorithm using Matlab computer
software programming was used. Tonic regular and irregular versus burst firing were
distinguished by analyzing the distribution of the interspike intervals. Tonic firing had a
Gaussian distribution whereas burst firing had a Poisson distribution. Figure 17 and 18 shows
schematically a "triple glass micropipettes" ,”double glass micropipettes” and the technic used
in the recording in the STN and SNr".
Figure 17: Schematic of a triple and double glass micropipettes used for the recording in
the globus pallidus. The recording pipettes and injections are stretched, broken respectively
unless 1μm and 20 microns, meetings, spacing peaks of about 200 microns, and under a
dissecting microscope. Of glass rods are added to solidify the pipette.
68
Figure 18: Schematic of the injection electrode in the GP and the recording electrode
used in the STN and in the SNr. Recording pipettes are placed at the STN / SNr and the
target injection it is placed at the GP were used for the same coordinates GP.
II.3.1.3 Signal acquisition
To characterize the neuronal activity of the structures of the basal ganglia (GPe, STN and
SNr) recorded following local injections of dopamine and quinpirole, unit extracellular
recordings of neurons were performed in rats anesthetized with urethane 20%. The
spontaneous activity of neurons is filtered (bandwidth of 300 Hz to 3 kHz) and amplified
using the "Neurolog" system (Digitimer, USA).
The signal acquisition is performed using the "PowerLab" system (AD Instruments,
USA). After checking the stability of the activity of the neuron, the spontaneous activity of
the neuron was recorded continuously for ten minutes. The first 20 minutes are used to ensure
the stability of the activity of the neuron. Then, the drug is injected locally at the site (globus
pallidus) by air pressure (Picospritzer, Intracel LTD, USA). Neuronal activity was recorded
until an eventual return to baseline.
69
II.3.1.4 Analysis of the recorded neural activity:
The recordings are analyzed "off-line" using three software signal processing. The Chart
software "Spike histogram" (ADInstruments, USA) provides a first step to discriminate action
potentials from background noise.
The
"Neuroexplorer"
software
(AlphaIOmega,
Germany)
to
determine
the
electrophysiological parameters such as average firing rate corresponding to the number of
action potentials per second, the average interval between action potentials (Interspike
interval or ISI) or the coefficient of variation of ISI, defined as the ratio of the standard
deviation of the average ISI, used here as an index of uniformity of the discharge mode. Then,
we used an algorithm "pattern", developed in the laboratory allows us to determine how the
firing of neurons in the method Kaneoke and Vitek (1996). This method is based on the
concept of density distribution and a rigorous statistical definition of discharge mode. Thus,
three modes are identified: regular, irregular, and burst (see Figure 19 and 20).
We only included recordings from cells that were confirmed to be in the target structures
(GPe, STN, and SNr) based on the probe coordinates during recording sessions, by the firing
properties of recorded neurons, and through histological analyses.
70
Figure 19: AlphaLab SnR: Multi-Channel workstation with complete acquisition.
The AlphaLab SnR revolutionizes the way neuroscience research is performed in the
laboratory, making it the most advanced data acquisition system currently available on the
market. The AlphaLab SnR is a comprehensive, fully integrated, high-throughput system that
allows users to record and stimulate from a virtually endless number of channels, with
unprecedented ease and flexibility. The system brings microelectrode recording to a new
level–from passive observation to active control and sophisticated interaction with neural
circuits.
71
Figure 20: The three types of discharge mode of neurons of the subthalamic nucleus.
A. regular, B. irregular, C. in burst. On the left, a crude sample record. In the middle of the
histogram inter-spikes interval (ISI). On the right, the density histogram (Chetrit et al., 2009).
72
At the end of each electrophysiological recording session, and to locate the recording
site, iontophoretic injection (DAM 80i, WPI, UK) Sky Blue of Pontamine is performed via
the recording pipette. The animal was sacrificed and the brain removed, frozen in isopentane
at - 45 ° C and stored at -80 ° C.
To locate the electrophysiological recording site or in situ injection site, histochemical
staining with acetylcholinesterase is performed on histological sections containing the target
structure (Figure 21). In fact, this color has allowed us to distinguish the different structures of
the cuts, and contrast it produces easier to identify the point of blue Pontamine, materializing
the electrophysiological recording site. Only rats with a blue dot in the structures of interest
were selected for statistical analysis of electrophysiological data.
The sections were rinsed with 0.2 M acetate buffer (27.2 g of sodium acetate in a liter
of distilled water, pH 5.9) before being incubated for 4 hours with stirring in the incubation
solution (180 ml of 0.2 M acetate buffer, 0.75 g of glycine, 0.5 g of copper sulfate, 0.2 g of
acetylcholine iodide). After rinsing with 0.2 M acetate buffer, the slides were found in
ammonium sulphide (diluted 1:100 in 0.2 M acetate buffer), then washed three times before
being mounted between slide and cover slip.
73
Figure 21: Location electrophysiological recording site in the (A) GP (B) STN, (C) SNr.
Contrast the product of the acetylcholine esterase staining on sections facilitates locating the
point of Pontamine Blue (sky blue) (arrows).
II.3.2 Validation of the recording sites
After completion of the experiments, animals were sacrificed by an overdose of urethane,
the brains removed, frozen in isopentane at –45 °C and stored at –80 °C. Fresh-frozen brains
were cryostat-cut into 20 μm coronal sections and acetylcholine esterase staining was used as
previously described (Chetrit et al., 2009) to determine the location of the Pontamine sky blue
dots marking the recording site in the recorded structure. Only brains in which both the
recording and drug injection were shown to be in the GP were used for data analysis.
II.3.3 Statistical analysis
Statistical analyses were performed using Sigmaplot (Systat Software, San Jose, USA). For
electrophysiological data recording of the globus pallidus, subthalamic nucleus and substantia
nigra reticulata normalized firing rates, before and after drug injection, were compared using a
t-test, (p< 0.05). In addition, the coefficient of variation of the interspike intervals was
74
analyzed to determine the changes in the firing pattern during the time after drug injections
using a using a t-test, (p< 0.05).
75
III. Results and Discussion
PART 1: Effect of dopamine and its agonist (Quinpirole) on the electrical
activity of GP neurons
The objective of the first part of the study is to characterize the effects of dopamine on
GP neurons and the subtype of dopamine receptors involved in this modulation. To achieve
this part, we studied the responses of GP neurons to the local injection of dopamine and D2
receptor agonist and antagonist, quinpirole and sulpiride respectively.
In addition to GABAergic and glutamatergic afferents, the GP receives a
dopaminergic innervation from the SNc as collateral fiber endings of the nigro-striatal
pathway and also own endings of the nigro-pallidal projections (Francis et al, 1999. Kieval
and Smith, 2000, Bouali-Benazzouz et al, 2009;. Rommelfanger and Wichmann, 2010). On
the other hand, the GP sends inhibitory GABAergic projections to almost all the other nuclei
of the basal ganglia, including the STN and SNr (Chang et al, 1981. Kincaid et al, 1991.
Moriizumi et al. , 1992, Parent and Hazrati, 1995b), which puts it in a key position within the
BG network playing an important role in the motor control.
At first, we studied the electrophysiological activity of the GP by locally injecting
dopamine at a concentration of 2μg/200nl (volume injected is 20nl). This manipulation has
been performed using a dual electrode. Then, to determine if the DA modulates the neuronal
activity of GP via D2R receptors we used the same protocol of recording and injected
intraperitoneally an antagonist of D2 receptors, sulpiride, at the dose of 6 mg/kg to block the
effect of DA. To confirme the involvement of D2 receptors, quinpirole, a D2 receptor agonist,
was infused into the GP. For that we used a dose response (at 2 or 4 or 8 µg/200nl saline
(NaCl 0.9%) to verify witch concentration (dose) had an effect on the GP neurons.
76
The table 5 shows a general assessment of the injection of dopamine and quinpirole in
the GP and its effect on the activity of GP neurons:
Structures/ Effects
Increase
No effect
Decrease
Totals
Dopamine GP-GP
34
14
2
50 neurons
Quinpirole GP-GP
19
5
5
29 neurons
Table 5: Overall assessment of the responses of GP neurons to intrapallidal injection of
dopamine and its D2R agonist (quinpirole). Note that majority of GP neurons increased
their firing rate in response to dopamine or its D2R agonist.
1.1 Effects of local injection of Dopamine in the globus pallidus on the firing rate of
GP neurons
The effect of local injection of dopamine and D2R agonist/antagonist on the
spontaneous discharge of GP neurons was investigated in 40 animals under urethane
anesthesia. In control conditions (before drug injection), the mean firing rate of GP neurons
was 19.32±1.05 spikes/sec (n=84) and most of the cells exhibited a regular discharge pattern
as shown by the coefficient of variation of the interspike intervals and density histograms
(Figure 22.A).
Control microinjection of saline (0.9% NaCl) into the GP revealed no significant
effects on the firing rate and pattern of neuronal activity in the GP (data not shown).
However, intrapallidal injection of dopamine predominantly induced an excitatory effect on
GP neurons (Figure 22. BC). It significantly increased the firing rate of 34 out of 50 GP
neurons (68%, Figure 22.D) with a percentage increase of 45% (p<0.001, paired t-test, Figure
22.B-D, Table 6). This effect occurred within 2-3 minutes after the injection and lasted 30-40
minutes. In only 2 GP tested cells (4%), dopamine decreased the firing rate with a percentage
decrease of 28% and in 14 GP neurons (28%) dopamine did not alter the firing rate (p>0.05,
77
Figure 22.D and Figure 23.A,B,C, Table 6). In all GP tested neurons, local dopamine injection
did not change the firing pattern as the coefficient of variation of the interspike intervals did
not significantly change compared to before injection (p>0.05, Table 7). Analysis of the
density histograms (Figure 22.AC) (Kaneoke and Vitek, 1996) showed similar results, e.g. the
absence of significant changes of the firing pattern (p>0.05, Chi2 test).
To test the hypothesis that the increased firing rate observed in GP cells after local
microinjection of dopamine results from the activation of dopamine D2Rs, these receptors
were blocked by the injection of sulpiride, a selective D2R antagonist. In all GP tested cells,
sulpiride blocked the excitatory effect induced by dopamine (Figure 22.E).
Table 6: Firing rates of GP, STN and SNr neurons before and after dopamine or
quinpirole injection into the GP
FR increase cells
Before
After
FR decrease cells
Before
After
Non responsive cells
Before
After
18.64±1.62
21.15±8.95
15.20±9.43*
22.63±2.07
22.63±2.12
ns
Quinpirole 14.31±2.14 19.44±2.72**
STN
neurons
Dopamine 4.72±0.60 6.66±0.96*
20.22±3.31
10.87±1.56**
24.68±3.96
25.61±3.99
ns
5.12±0.79
3.43±0.56*
6.39±0.72
6.22±0.60
Quinpirole 11.92±6.69 18.86±4.10*
SNr
neurons
Dopamine 9.03±2.90 13.10±3.78*
Quinpirole 21.82±6.00 32.12±7.40*
11.36±2.51
7.23±1.43*
12.91±2.48
13.33±2.67
ns
9.41±1.24
5.73±1.03*
12.30±4.16
12.54±4.64
ns
16.10±2.11
8.99±1.60*
12.91±2.48
13.33±2.67
ns
GP
neurons
Dopamine
27.03±2.44***
ns
FR: firing rate in spikes/sec; values are presented as the mean ± SEM. Statistical analysis
using paired t-test was performed; *: p<0.05, **: p<0.01, ***: p<0.001 in comparison with
before drug injection.
78
Figure 22: Intrapallidal microinjection of dopamine predominantly increased the firing
rate without changing the tonic firing pattern of GP neurons.
(A-C) A representative example of GP neuron before (AB) and after (BC) microinjection of
dopamine (DA) into the GP showing an increase of its firing rate with spike train (A1C1),
firing rate histogram (B), raster display of random segments of recording (A2C2), insterspike
interval histogram (A3C3) and density histogram (A4C4) of the same GP neuron.
(D) Circular plot representing the percentage of GP neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
(E) Representative firing rate histogram with the response of a GP neuron showing an
increase of its firing rate after dopamine injection corresponding to the effect observed in the
majority of GP neurons.
(E) The excitatory effect of dopamine (DA) was prevented by the selective D2R antagonist,
sulpiride (Sulp). Note that DA increased the firing rate (E1E2) without changing the
coefficient of variation (E3) of GP neurons and that after the injection of sulpiride
(DA+Sulp), DA had no effect on the firing rate (E1E2). *p<0.05
79
Table 7: Coefficient of variations of GP, STN and SNr neurons before and after
dopamine or quinpirole injection into the GP
GP neurons
STN neurons
SNrneurons
Before
After
Before
After
Before
After
Dopamine
0.32±0.06
0.27xx±0.04ns
1.12±0.02
1.10±0.02ns
1.16±0.08
1.15±0.07ns
Quinpirole
0.34±0.06
0.36±0.06ns
1.12±0.03
1.12±0.03ns
1.18±0.06
1.16±0.05ns
Values are presented as the mean ± SEM. Statistical analysis using paired t-test was performed; ns: non
significant difference in comparison with before drug injection.
80
Figure 23: Dopamine did not significantly change the firing rate or the coefficient of
variation of the interspike intervals of GP neurons. (A) is a representative example of the
firing rate histogram showing no effect on the firing rate of the GP neurons. The gray vertical
line represents the time of injection.
81
1.2 Effects of local injection of Quinpirole in the globus pallidus on the firing rate
of GP neurons
To confirm the importance of the D2Rs in the effects induced by dopamine, we tested
the effect of intrapallidal injection of quinpirole, a selective D2R agonist. First, in a set of
experiments, we investigated the dose response of quinpirole at the doses of 0.2, 0.4, and 0.8
g. Local microinjection of quinpirole significantly affected the firing rate of GP neurons in a
dose-dependent manner (F=26.42, p<0.001, Figure. 24A). In contrast to the doses of 0.2 and
0.4 g, which did not affect the firing rate (p>0.05 for the two doses), 0.8 g significantly
increased the firing rate of the majority of GP neurons (19 out of 29, 66%) with a percentage
increase of 36% (p<0.01, paired t-test, Figure 24.A-E, Table 6). In 5 out of 29 GP tested cells
(17%), quinpirole significantly decreased the firing rate (-46.24%, p<0.01, Figure 24.F-H,
Table 6) and in 5 out of 29 GP tested neurons (17%) dopamine did not significantly alter the
firing rate (p>0.05, Table 6). In all GP tested neurons, quinpirole did not change the firing
pattern as the coefficient of variation of the interspike intervals did not significantly change
after the injection of quinpirole compared to control conditions (p>0.05, Figure. 24, Table 7).
Analysis of the density histograms showed similar results, e.g. the absence of significant
changes in the firing pattern (p>0.05, Chi2 test).
82
Figure 24: Intrapallidal microinjection of quinpirole predominantly increased the firing
rate of GP neurons in a dose-dependent manner without changing the tonic firing
pattern.
(A) Histograms showing the dose response effects of quinpirole (Quin 0.2, 0.4 and 0.8 g) on
the firing rate (A1) and the coefficient of variation of the interspike intervals (A2) of GP
neurons. ***p<0.001.
(B-D) A representative example of GP neuron before (BC) and after (CD) microinjection of
quinpirole (Quin) into the GP showing an increase of its firing rate with spike train (B1D1),
firing rate histogram (C), raster display of random segments of recording (B2D2), insterspike
interval histogram (B3D3) and density histogram (B4D4) of the same GP neuron.
(E) Circular plot representing the percentage of GP neurons showing an increase, a decrease
or no change of their firing rate after the local injection of quinpirole.
(F-H) A representative example of GP neuron before (FG) and after (GH) microinjection of
quinpirole (Quin) into the GP showing a decrease of its firing rate with spike train (F1H1),
firing rate histogram (G), raster display of random segments of recording (F2H2), insterspike
interval histogram (F3H3) and density histogram (F4H4) of the same GP neuron.
83
Discussion part 1: The effect of dopamine, its agonist (Quinpirole) and
antagonist (Sulpiride) D2 receptors on the activity of GP neurons
In the present study, we provide evidence that dopamine, through activation of D2Rs,
exerts an excitatory effect on the majority of GP neurons in vivo. Thus, dopamine-induced
firing rate increase was mimicked by the selective D2R agonist, quinpirole, and prevented by
the selective D2R antagonist, sulpiride. However, neither dopamine nor quinpirole changed
the discharge pattern, demonstrating that dopamine, through activation of D2Rs, modulates
the firing rate but not the pattern of GP neurons under physiological conditions. The
possibility that the absence of change of the firing pattern may be influenced by the use of
urethane anesthesia cannot be completely rule out. However, this possibility may be
minimized, as dopamine depletion in the rat, under the same conditions of anesthesia, has
been shown to induce burst activity in GP neurons (Ni et al., 2000) and also in STN neurons
(Hassani et al., 1996, Ni et al., 2001, Belujon et al., 2007, Chetrit et al., 2013). Furthermore,
similar burst activity has been reported in non-anesthetized MPTP-treated monkeys (Bergman
et al., 1994) and in patients with Parkinson‟s disease (Hutchison et al., 1998, Benazzouz et al.,
2002). Our results are consistent with previous studies showing that quinpirole increased the
firing rate of GP neurons in the rat (Querejeta et al., 2001) and of GPe neurons in non-human
primate (Hadipour-Niktarash et al., 2012). Quinpirole also increased the expression of the
immediate early gene c-fos, which is a marker of neuronal activity (Billings and Marshall,
2003). The firing rate increase, which represents the major effect of dopamine on GP neurons,
may be explained by the action of this neurotransmitter on D2Rs located pre-synaptically on
GABA striato-pallidal fibers (Smith and Villalba, 2008). Thus, dopamine, like quinpirole,
reduces GABA release by activating D2Rs, resulting in a disinhibition of GP neurons. This is
consistent with an early study, which showed that iontophoretic injection of dopamine or
amphetamine reduced GABA transmission in the GP (Bergstrom and Walters, 1984).
84
Accordingly, in vitro data demonstrated that dopamine, through activation of D2Rs on striatopallidal terminals, exerts an inhibitory effect on GABA release in the rat GP (Floran et al.,
1997, Cooper and Stanford, 2001a). These studies, together with ours, suggest that the striatopallidal GABAergic inhibition is under the control of presynaptic D2Rs and that local
depletion of dopamine may contribute to the changes in GP neuronal activity observed in
animal models of Parkinson's disease. Thus, intrapallidal injection of 6-hydroxydopamineinduced dopamine depletion in GP resulted in a decrease of the firing rate of GP neurons
(Bouali-Benazzouz et al., 2009). This supports the key role played by dopamine at
extrastriatal sites, suggesting that dopaminergic drugs may play their anti-parkinsonian effects
through activation of D2Rs located in GP in addition to their action in the striatum.
In addition to their localization on GABA fibers, D2Rs are also located
presynaptically on glutamatergic afferents originating from the STN and the parafascicular
nucleus of the thalamus (Smith and Villalba, 2008). Their activation has been suggested to
reduce glutamatergic release in GP of in vitro slices (Hernandez et al., 2006). This may
explain why in some of our GP tested cells dopamine, like quinpirole, reduced their firing
rate. The fact that this effect was observed in only a minority of GP neurons compared to
those showing an increase in their firing rate, is consistent with the reduced number of D2Rs
located on glutamate terminals compared to those located on GABA terminals (Smith and
Villalba, 2008). In another population of GP tested cells, dopamine like quinpirole did not
affect their firing activity. This can be due to the absence of dopamine receptors on afferents
of these neurons. This result is consistent with data of a previous anatomical study showing
that D2Rs were not found in all GP neurons but only in a population of approximately 4050% (Floran et al., 1997).
We therefore postulate that dopamine acting at presynaptic D2Rs predominantly
reduces GABA release at GABAergic terminals in GP. Presynaptic rather than postsynaptic
85
dopaminergic modulation of GABAergic transmission in the GP is supported by the action of
dopamine on miniature GABAergic transmission, which can be mimicked by the use of
selective D2R agonists (Cooper and Stanford, 2001a). The firing rate increase of pallidal
neurons caused by dopamine and its D2R agonist, quinpirole, would lead to decreased firing
rate of their major basal ganglia efferent structures such as the STN and SNr.
In conclusion, our results support the hypothesis that dopamine released at the GP
modulates the activity of GP neurons via its action on D2 receptors and by increasing the
firing rate of GP neurons without changing its pattern. These results suggest that this
modulation may have a direct or indirect modulatory effect on its efferents structures such as
the STN and the SNr.
.
86
PART 2: The effect of local injection of dopamine and Quinpirole on the GP
on the activity of the STN and SNr neurons
In order to understand the importance of the role of dopamine in the modulation of the
pallido-thalamic activity, we studied the effects of intrapallidale injection of dopamine on
neuronal activity of the globus pallidus, then the effect of this modulation on the structure of
STN and SNr using an extracellular unit recording.
In this part of study, the recording was made in the STN and SNr then cured using an
electrode to sixteen channels (ext. Alpha Omega). For that we injected dopamine or
quinpirole into the GP (volume 200 nl), in order that all the neurons that project from the GP
to NST and SNr can receive the drugs. This manipulation profit for first validate the results
obtained during the injections and recordings made in the structure of the GP (part 1), then
study the response of the activity of neurons in the NST and SNr after local injection of
dopamine and quinpirole in the GP.
87
2.1 Effects of local injection of dopamine in the globus pallidus on the firing rate
of STN neurons
In basal conditions, the mean firing rate of STN neurons was 10.38±1.27 spikes/sec
(n=95 neurons in 27 rats) and most of the cells exhibited a tonic discharge pattern as shown
by the interspike intervals and density histograms (Figure 25A). Control microinjection of
saline (0.9% NaCl) into the GP revealed no significant effects on the firing rate and pattern of
neuronal activity in the STN (data not shown). However, dopamine injection into GP
predominantly induced an inhibitory effect on STN neurons. It significantly decreased the
firing rate of 9 out of 20 STN neurons (45%) with a percentage decrease of 33% (p<0.05,
paired t-test, Figure 25.A-D, Table 6). This effect occurred within 2-3 minutes after the
injection and lasted 30-40 minutes. In 5 out of 20 STN tested cells (25%), dopamine
significantly increased the firing rate (p<0.05, Figure 25.E-G, Table 6-8) and in 6 out of 20
STN tested neurons (30%), dopamine did not significantly change the firing rate (p>0.05,
Table 6). In all STN tested neurons, dopamine did not change the firing pattern as the
coefficient of variation of the interspike intervals did not significantly change after dopamine
injection into GP compared to control conditions (p>0.05, Figure 25, Table 7). Analysis of the
density histograms also showed the absence of changes of the firing pattern (p>0.05, Chi2
test).
88
Table 8: General assessment of the effect of dopamine and quinpirole on the activity of
neurons in the STN.
Structures/ Effects
Increase
No effect
Decrease
Totals
Dopamine GP-STN
5
6
9
20 neurons
Quinpirole GP-STN
11
23
41
75 neurons
Table 8: Overall assessment of the responses of GP neurons to intrapallidal injection of
dopamine and its D2R agonist (quinpirole). Note that majority of STN neurons
decreased their firing rate in response to dopamine or its D2R agonist
89
Figure 25: Intrapallidal microinjection of dopamine predominantly decreased the firing
rate without changing the tonic firing pattern of STN neurons.
(A-C) A representative example of STN neuron before (A) and after (C) microinjection of
dopamine into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B) raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same STN neuron.
(D) Circular plot representing the percentage of STN neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
E-G) A representative example of STN neuron before (EF) and after (FG) microinjection of
dopamine into the GP with spike train (E1G1), firing rate histogram (F), raster display of
random segments of recording (E2G2), insterspike interval histogram (E3G3) and density
histogram (E4G4) of the same STN neuron.
90
2.2 Effects of local injection of quinpirole in the globus pallidus on the
firing rate of STN neurons
Intrapallidal injection of quinpirole into the GP significantly affected the firing rate of
STN neurons. It decreased the firing rate of 41 out of 75 STN neurons (55%) with a
percentage decrease of 36% (p<0.05, Figure 26.A-D, Table 6). In only 11 out of 75 STN
tested cells (15%), quinpirole injection significantly increased the firing with a percentage
increase of 58% (p<0.05, Figure 26.E-G, Table 6). In 23 out of 75 STN tested neurons (31%),
quinpirole injection did not significantly change the firing rate (p>0.05, Figure 26, Table 6).
In all STN tested cells, quinpirole injection did not change the firing pattern as the coefficient
of variation of the interspike intervals did not significantly change after the injection of
dopamine compared to control conditions (p>0.05, Figure 26, Table 7). Analysis of the
density histograms also showed an absence of changes of the firing pattern (p>0.05, Chi2
test).
91
Figure 26: Intrapallidal microinjection of quinpirole predominantly decreased the firing
rate without changing the tonic firing pattern of STN neurons.
(A-C) A representative example of STN neuron before (A) and after (C) microinjection of
quinpirole into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B), raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same STN neuron.
(D) Circular plot representing the percentage of STN neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
E-G) A representative example of STN neuron before (E) and after (G) microinjection of
quinpirole into the GP showing an increase of its firing rate with spike train (E1G1), firing
rate histogram (F), raster display of random segments of recording (E2G2), insterspike
interval histogram (E3G3) and density histogram (E4G4) of the same STN neuron.
92
2.3 Effects of local injection of dopamine in the globus pallidus on the firing
rate of SNr neurons
In basal conditions, the mean firing rate of SNr neurons was 15.52±1.37 spikes/sec
(n=107 neurons in 15 rats) and most of the cells exhibited a regular discharge pattern as
shown by the coefficient of variation of the interspike intervals (Figure 27A). Control
microinjection of saline (0.9% NaCl) into the GP revealed no significant effects on the firing
rate and pattern of neuronal activity in the SNr (data not shown). However, dopamine
injection into GP predominantly induced an inhibitory effect on SNr neurons. It decreased the
firing rate of 13 out of 22 SNr neurons (59%) with a percentage decrease of 39% (p<0.05,
Figure 27A-D, Table 6). This effect occurred within 2-3 minutes after the injection and lasted
30-40 minutes. In 4 out of 22 SNr tested cells (18%), dopamine significantly increased the
firing rate with a percentage increase of 45% (p<0.05, Figure 27 E-G, Table 6 and in 5 out of
22 SNr tested neurons (23%) dopamine did not significantly change the firing rate (p>0.05,
Table 6-9). In all SNr tested neurons, dopamine did not change the firing pattern as the
coefficient of variation of the interspike intervals did not significantly change after dopamine
injection into GP compared to control conditions (p>0.05, Figure 27, Table 7). Analysis of the
density histograms also showed the absence of changes of the firing pattern (p>0.05, Chi2
test).
Table 9: General assessment of the effect of dopamine and quinpirole on the activity of
neurons in the SNr.
Structures/ Effects
Increase
No effect
Decrease
Totals
Dopamine GP-SNr
4
5
13
22 neurons
Quinpirole GP-SNr
15
24
46
85 neurons
Table 9: Overall assessment of the responses of GP neurons to intrapallidal injection of
dopamine and its D2R agonist (quinpirole). Note that majority of SNr neurons
decreased their firing rate in response to dopamine or its D2R agonist
93
Figure 27: Intrapallidal microinjection of dopamine predominantly decreased the firing
rate without changing the tonic firing pattern of SNr neurons.
(A-C) A representative example of SNr neuron before (A) and after (C) microinjection of
dopamine into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B), raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same SNr neuron.
(D) Circular plot representing the percentage of SNr neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
(E-G) A representative example of SNr neuron before (E) and after (G) microinjection of
dopamine into the GP showing an increase of its firing rate with spike train (D1F1), firing rate
histogram (F), raster display (E2G2), insterspike interval histogram (E3G3) and density
histogram (E4G4) of the same SNr neuron.
94
2.4 Effects of local injection of dopamine in the globus pallidus on the firing
rate of SNr neurons
Correspondingly, local microinjection of quinpirole into the GP significantly affected
the firing rate of SNr neurons. Quinpirole significantly decreased the firing rate of 46 out of
85 SNr neurons (55%) with a percentage decrease of 44% (p<0.05, Figure 28.A-D, Table 6).
In only 15 out of 85 SNr tested cells (18%), quinpirole injection significantly increased the
firing rate with a percentage increase of 47% (p<0.05, Figure 28. E-G, Table 6) and in 24 out
of 85 SNr tested neurons (28%), quinpirole injection did not significantly change the firing
rate (p>0.05, Table 6). In all SNr tested cells, quinpirole injection did not change the firing
pattern as the coefficient of variation of the interspike intervals did not significantly change
after the injection of quinpirole compared to control conditions (p>0.05, Figure 28, Table 7).
Analysis of the density histograms also showed an absence of changes of the firing pattern
(p>0.05, Chi2 test).
95
Figure 28: Intrapallidal microinjection of quinpirole predominantly decreased the firing
rate without changing the tonic firing pattern of SNr neurons.
(A-C) A representative example of SNr neuron before (A) and after (C) microinjection of
quinpirole into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B), raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same SNr neuron.
(D) Circular plot representing the percentage of SNr neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
(E-G) A representative example of SNr neuron before (E) and after (G) microinjection of
quinpirole into the GP showing an increase of its firing rate with spike train (E1G1), firing
rate histogram (F), raster display (E2G2), insterspike interval histogram (E3G3) and density
histogram (E4G4) of the same SNr neuron.
96
Discussion part 2: The effect of local injection of dopamine and Quinpirole
on the GP on the activity of NST and SNr neurons
The principal GABAergic input to the STN arises from the GP, which plays a key role
in the control of firing activity of STN neurons. In vitro electrophysiology studies reported
that spontaneous pallido-subthalamic activity influenced STN neuronal firing (Baufreton et
al., 2005) and that electrical stimulation of GP afferents evoked IPSP or IPSC through
activation of postsynaptic GABAA receptors (Bevan et al., 2002, Hallworth and Bevan, 2005,
Loucif et al., 2005). Here, we focused our study on the impact of dopaminergic modulation of
GP-STN neurotransmission and we showed that dopamine, like quinpirole, when injected into
the GP decreased the firing rate of most STN neurons. These results can be explained by the
fact that dopamine, through activation of D2Rs, predominantly increased the firing rate of GP
cells (present study), at the origin of GABA release in the STN, resulting in a reduction of the
firing activity of majority of STN recorded neurons. This is the first study showing that DA in
GP participates in the modulation of GP-STN neurotransmission and consequently controls
STN neuronal firing. The inhibitory effect is mediated by GABAARs, as they are concentrated
at GP–STN synapses and that GABAAR antagonists block spontaneous IPSCs (Bevan et al.,
2006). Furthermore, we showed that DA, via D2Rs, increased the firing rate of a minority of
STN neurons (25% for DA and 15% for quinpirole). This excitatory effect can be explained
by the fact that dopamine, via D2Rs, reduces the firing rate of a small sub-population of GP
cells inducing a decrease of GABA release in the STN, which in turn results in a disinhibition
of STN neurons.
In the two populations of STN responsive neurons, the firing rate changes were not
accompanied by a change in firing pattern. Together, our data show that DA participates in
the modulation of the GP-STN pathway, contributing to the control of firing rate but not
pattern of STN neurons. This is consistent with previous studies showing that the pattern of
97
GP inhibitory input to the STN is crucial in determining whether STN neurons fire in a tonic
or burst pattern (Bevan et al., 2002), and that burst activity in GP neurons is necessary to
generate sufficient hyperpolarization in STN neurons for rebound burst activity (Plenz and
Kital, 1999).
In addition to the control of GP-STN pathway, we show that dopamine in GP
modulates the neuronal activity of the principal output structure of basal ganglia network in
the rat, the SNr. We show that the responses of SNr neurons to pallidal microinjection of
dopamine, or its D2R agonist, are similar to those of STN neurons (including decreases,
increases and some neurons showing no change) with the same proportions. The changes
observed in SNr neurons can be due to i) the activation of GABAergic neurons of GP
projecting directly to the SNr or ii) to the deactivation of STN neurons projecting to the SNr
as majority of STN neurons are inhibited by dopamine when injected in the GP or iii) to a
combination of the two phenomena.
According to previous studies, it is likely that SNr cell responses to dopamine in GP
are a consequence of the two phenomena. The first hypothesis is supported by anatomical
tracing findings showing that individual GP neurons that project to the STN possess axon
collaterals innervating the SNr (for review, Smith et al. (1998), Deniau et al. (2007)).
Furthermore, in rat brain slices preparation, it has been shown that GP neurons have a
significant impact on the discharge of SNr cells (Deniau et al., 2007). Indeed, GP stimulation
evoked IPSPs of SNr neurons, which is strong enough to reset the firing of the neurons
(Deniau et al., 2007). The second hypothesis is supported by a previous electrophysiological
study showing that the STN lesions induced an attenuation of changes in mean firing rate of
SNr neurons in response to intrastriatal microinjection of apomorphine (Murer et al., 1997).
Based on these evidences, it is likely that SNr neuronal responses are due to changes in the
level of activity of inhibitory (GP) and excitatory (STN) afferents and that both GP-SNr and
98
GP-STN-SNr are important in the inhibitory response of SNr neurons to dopamine and
quinpirole injection into GP.
In conclusion, our data are the first to show that dopamine, through activation of D2Rs
located in the GP, plays a key role in modulating GP neuronal activity, which participates to
the control of its two principal efferent projections, the STN and SNr. The predominant effect
was an increase in the firing rate of GP neurons, resulting in the inhibition of GABA release
from presynaptic terminals in the STN and SNr, leading to decreased activity of its neurons.
In addition, the changes in STN neuronal activity may participate to the modulation of SNr
neurons. Our data provide evidence that dopamine in GP controls the firing rate but not the
pattern of GP neurons, which in turn control the firing rate, but not the pattern of STN and
SNr neurons.
99
IV. Conclusion and Perspectives
The first part of our work focused on the characterization of the electrical activity of
GP neurons through activation of D2Rs located in the GP, as demonstrated by the responses
of GP nurons to the local injections of dopamine, its D2R agonist and antagonist. The results
obtained showed that intrapallidal microinjection of dopamine resulted in a significant effect
at the level of GP neurons. This effect of dopamine on the D2Rs caused an increase in the
firing rate of GP neurons. We also showed that local injection of quinpirole (D2R agonist)
into the GP induced changes in the same way of the electrical activity of GP neurons. The
quinpirole significantly increased the firing rate of the majority of GP neurons. However, our
results demonstrated that neither dopamine nor quinpirole does significantly alter the firing
pattern of GP neurons.
In a second part of the study, we focused on the understanding of how dopamine and
quinpirole in the GP can modulate the electrical activity of STN and SNr neurons, two
structures that receive major and consistent inhibitory GABAergic projections from the GP.
Our electrophysiological results showed that intrapallidal microinjection of dopamine and
quinpirole in the GP induced significant changes in the firing rate of STN and SNr neurons.
Thus, in both the NST and SNr, dopamine and it‟s agonist significantly decreased the firing
rate of the majority of the recorded neurons with an absence of significant changes in the
firing patterm.
Our data show that dopamine, through activation of D2Rs plays a key role in
modulating GP neuronal activity, which participates to the control of its principal efferent
projection to the STN. The predominant effect was an increase in the firing rate of GP
neurons, resulting from the inhibition of local GABA release from presynaptic striato-pallidal
terminals in the GP. The firing rate increase of GP neurons leads to the release of GABA in
100
the STN and SNr resulting in a decreased activity of their neurons. Together, our data suggest
that, in physiological conditions, dopamine, which modulates only the firing rate, plays a key
role in maintaining the tonic discharge pattern characteristic of normal electrical activity of
GP neurons. This may explain at least in part why in physiological conditions in the presence
of dopamine, GP and STN neurons discharge in a tonic regular pattern and that when
dopamine is depleted they discharge in bursts in animals models (Bergman et al., 1994, Ni et
al., 2000, Vila et al., 2000, Meissner et al., 2005, Belujon et al., 2007) and in patients with
Parkinson‟s disease (Hutchison et al., 1998, Magnin et al., 2000, Benazzouz et al., 2002). The
oscillatory burst activity is considered as a pathological signature of GP and STN neurons
associated to Parkinson‟s disease.
Results of this work highlight new evidence in understanding the role of dopamine
in GP in the anatomo-functional organization of the basal ganglia network. This allows to
understand the involvement of extrastriatal dopamine in physiological conditions suggesting
that this extrastriatal dopamine may have an important implication in the emergence of motor
disorders associated to Parkinson‟s disease. It also makes light on the important role of
dopamine in regulating the function of the basal ganglia from structures other than the
striatum.
The present work focused only on the role of D2Rs in the dopaminergic modulation of
GP neuronal activity. In order to complete our knowledge, it is mandatory to investigate the
involvement of D1Rs. An interesting issue will be to investigate the responses of GP neurons
and also those of their efferent structures, the STN and SNr, to the intrapallidal injection of
D1R agonist and antagonist.
101
As the present study was carried out only in normal animals, we propose to investigate
the modulatory effect of dopamine in the rat model of Parkinson‟s disease obtained by the
stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle to mimic what
happened in the pathology. 6-hydroxydopamine is a neurotoxin selective of the degeneration
of dopamine neurons. Results of this project will allow to understand if the responses of GP
neurons to dopaminergic agents are similar or different compared to those obtained in normal
rats. Our hypothesis is that in the absence of dopamine, dopamine receptors are hypersensitive
and that the amplitude of responses should be higher that that obtained in normal conditions.
In order to complete the electrophysiological study, it should be interesting to
investigate the motor behavioral responses of dopaminergic agents injected locally into the
GP. To achieve this part, the “Open field actimeter” as well as the “Steping test” and the
“Rotarod” will be used to characterize the motor phenotype of dopaminergic modulation into
the GP. This part of the project will be done in normal rats as well as in 6-hydroxydopamine
rats.
102
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Annexe 1 Mamad O., Delaville C., Benjelloun W. and Benazzouz A. Dopaminergic control of the
globus pallidus through activation of D2 receptors and its impact on the electrical activity of
subthalamic nucleus and substantia nigra reticulata neurons. PlosOne, 2015, In Press.
1 PloSOne
Dopaminergic control of the globus pallidus through activation of D2
receptors and its impact on the electrical activity of subthalamic nucleus
and substantia nigra reticulata neurons
Omar Mamad1,2,3, Claire Delaville1,2, Wail Benjelloun3 and Abdelhamid Benazzouz1,2 *
1. Univ. de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000
Bordeaux, France.
2. CNRS, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France.
3. Université Mohamed V-Agdal, Faculté des Sciences, Equipe Rythmes Biologiques,
Neurosciences et Environnement, 10000 Rabat, Morocco.
* Corresponding author: Dr. Abdelhamid Benazzouz, Institut des Maladies
Neurodégénératives (IMN), CNRS UMR 5293, Université de Bordeaux, 146, Rue LéoSaignat, 33076 Bordeaux Cedex, France.
E-mail: [email protected]
Tel: +33 557 57 46 25
Fax: +33 556 90 14 21
Abstract
The globus pallidus (GP) receives dopaminergic afferents from the pars compacta of
substantia nigra and several studies suggested that dopamine exerts its action in the GP
through presynaptic D2 receptors (D2Rs). However, the impact of dopamine in GP on the
pallido-subthalamic and pallido-nigral neurotransmission is not known. Here, we investigated
the role of dopamine, through activation of D2Rs, in the modulation of GP neuronal activity
and its impact on the electrical activity of subthalamic nucleus (STN) and substantia nigra
reticulata (SNr) neurons. Extracellular recordings combined with local intracerebral
microinjection of drugs were done in male Sprague-Dawley rats under urethane anesthesia.
We showed that dopamine, when injected locally, increased the firing rate of the majority of
neurons in the GP. This increase of the firing rate was mimicked by quinpirole, a D2R
agonist, and prevented by sulpiride, a D2R antagonist. In parallel, the injection of dopamine,
as well as quinpirole, in the GP reduced the firing rate of majority of STN and SNr neurons.
However, neither dopamine nor quinpirole changed the tonic discharge pattern of GP, STN
and SNr neurons. Our results are the first to demonstrate that dopamine through activation of
D2Rs located in the GP plays an important role in the modulation of GP-STN and GP-SNr
neurotransmission and consequently controls STN and SNr neuronal firing. Moreover, we
provide evidence that dopamine modulate the firing rate but not the pattern of GP neurons,
which in turn control the firing rate, but not the pattern of STN and SNr neurons.
2 Introduction
The globus pallidus (GP, the rodent homologue of the primate globus pallidus externus,
GPe) is a basal ganglia structure playing a key role in the control of movement. It is
considered as an inhibitory GABAergic relay in the indirect pathway, linking the striatum to
the pars reticulata of substantia nigra (SNr), directly or indirectly via the subthalamic nucleus
(STN) [1]. Major GP afferents originate from the striatum and use GABA as a
neurotransmitter, while its glutamatergic afferents arise from the STN [2] and the
parafascicular nucleus of the thalamus [3]. In addition, GP receives dopaminergic projections
from the pars compacta of substantia nigra (SNc) [4]. While the striatum is by far the main
target of SNc dopamine neurons, dopamine also mediates its regulatory function at the level
of GP [5,6]. Both dopamine D1 (D1R) and D2 (D2R) receptor families are expressed in the
GP with a predominance of D2Rs [7]. Most of the presynaptic dopamine receptors are
thought to be D2Rs, and are located on terminals of the GABAergic and glutamatergic
afferents with lower levels of D1Rs detected in axons and terminal buttons in GP [6-8]. From
a functional point of view, a major role of dopamine in the modulation of GP neuronal
activity has been suggested by studies demonstrating that intrapallidal dopamine receptor
blockade [9] or dopamine depletion [10] produced motor deficits in rodents that can be
associated with a reduction of the firing rate of GP neurons [11,12].
Given the predominance of D2Rs in GP and that most actions of dopamine in the GP
are mediated by D2Rs [5], we investigated the role of dopamine, through activation of D2Rs,
in the control of GP neuronal activity using in vivo extracellular recordings in the rat. Then, as
the GP is tightly interconnected with the STN and SNr, we studied the impact of dopamine in
the GP on the control of pallido-subthalamic and pallido-nigral neurotransmission.
Materials and Methods
Ethics Statement
All animal experiments were performed in accordance with European Communities Council
Directive 2010/63/UE. The study received approval from the local Ethics Committee under
the number 50120136-A (Comité d’éthique pour l’expérimentation animale Bordeaux,
France). All efforts were made to minimize the number of animals used and their suffering.
Animals
Adult male Sprague Dawley rats, weighing 280–380 g, were used for in vivo
electrophysiological experiments under anesthesia. Animals were provided by the “Centre
d’Elevage Depré” (Saint Doulchard, France) and arrived at least 1 week before use. They
were housed four per cage under artificial conditions of light (12/12 light/dark cycle; lights on
at 7:00 A.M.), temperature (24°C), and humidity (45%) with food and water available ad
libitum.
Drugs
Drugs were chosen on the basis of affinity for their preferential receptors. Dopamine,
purchased from Sigma (Saint-Quentin Fallavier, France), was used at the dose of 2 µg
3 dissolved in 200 nl of 0.9% NaCl. Quinpirole (Sigma) was chosen as a D2R agonist and three
doses were tested (0.2, 0.4 and 0.8 µg) to investigate the dose-response on GP neuronal
activity. These doses were also dissolved in 200 nl of 0.9% NaCl. Concerning the
experiments with the injection and recording in the GP, a small volume of 20 nl of each
solution was used to avoid the risk of losing the recorded cell due to the local pressure
injection. When the injection was done in GP and recordings in the STN or SNr, a volume of
200 nl was used to have a larger diffusion into the nucleus. 200 nl was selected after a series
of control tests, using the pontamine sky blue, in which this volume showed a diffusion of the
solution into the GP without a spread outside the nucleus. By using this volume, the injected
drug exerts its effect everywhere in the nucleus to affect GP cells projecting to the recorded
neurons in the STN and SNr. It is unlikely that this volume may exert different effects
compared to 20 nl as the concentration is the same and the proportions of the responsive
excited and inhibited STN and SNr neurons were concordant with those of GP neurons (see
results). Dopamine D2Rs were blocked by intra-peritoneal injection of sulpiride, a selective
D2R antagonist (40 mg/kg), which was dissolved in 2 ml sterile injectable water to which was
added HCl and the final pH of 6.5–7.2 was titrated with NaOH. The final pH of dopamine and
quinpirole solutions was between 7.0 and 7.2.
Extracellular recordings and drugs microinjections
Extracellular single-unit recordings were made in rats anesthetized with urethane (1.2 g/kg,
i.p.). For recording and simultaneous microinjection of drugs in the GP, a double-barreled
pipette assembly, similar to that described previously [13,14], was used. The tips of recording
and injection micropipettes were separated by 150 to 200 µm. For recording in the STN or
SNr and simultaneous microinjection of drugs in the GP, the injection micropipette was
placed in the GP and the recording electrode was placed in the STN or SNr. The injection
micropipette was filled either with dopamine or with quinpirole drugs and the recording
electrode, with an impedance of 8 to 12 MΩ, was filled with 4% pontamine sky blue in 0.9%
NaCl. The micropipettes were placed into the targeted nuclei according to the stereotactic
coordinates given in the brain atlas of Paxinos and Watson [15] for the GP (AP: 0.9 mm
posterior to bregma, L: 3 mm from the midline, DV: 5.5 - 7.5 mm from the dura), STN (AP: 3.8 mm posterior to bregma, L: 2.5 mm from the midline, DV: 7.5-8.5 mm from the dura) and
SNr (AP: -5.3 mm posterior to bregma; L: 2.5 mm from the midline; DV: 7.5-8.5 mm from
the dura). Extracellular neuronal activity was amplified, bandpass-filtered (300–3000 Hz)
using the Neurolog system (Digitimer, Hertfordshire, UK), displayed on an oscilloscope and
transferred via a Powerlab interface to a computer equipped with Chart software (AD
Instruments, Charlotte, USA). Only neuronal activity with a signal-to-noise ratio of 3:1 was
recorded and used for additional investigation. Basal firing of GP, STN and SNr neurons was
recorded for 20 min. before drug injection to ascertain the stability of the discharge activity. A
dopaminergic drug or the saline vehicle was then injected into the GP at a volume of 20 nl,
using brief pulses (200 ms) of pneumatic pressure (Picospritzer III, Royston Herts, UK). In all
rats, the central part of the nucleus was targeted. At the end of each session, the recording site
was marked by electrophoretic injection (Iso DAM 80, WPI, Hertfordshire, UK) of
Pontamine sky blue through the micropipette at a negative current of 20 µA for 8 min.
Recording sequences of 10 minutes each were used for off-line data analysis of GP, STN and
4 SNr neuronal activity recorded before and after drug injection. We used a spike discriminator
program (spike histogram program, AD Instruments, Charlotte, USA), and firing parameters
were determined using Neuroexplorer (Alpha Omega, Nazareth, Israel). After the drug
injection, the start of an excitatory effect was considered when the firing rate was higher than
the mean+(2xSD) of the baseline and the start of an inhibitory effect was considered when the
firing rate was lower than the mean-(2xSD) (SD= standard deviation). The minimum period
of time accepted as a significant effect was 10 seconds and the end of an effect was defined
when the firing rate returned to the same value relative to baseline. The firing patterns of GP,
STN and SNr neurons were analyzed using the coefficient of variation of the interspike
intervals as well as the density histograms according to the method developed by Kaneoke
and Vitek [16], as previously described [13,17]. An algorithm using Matlab computer
software was used allowing the discrimination of tonic regular, irregular and burst firing.
Validation of the recording sites
After completion of the experiments, animals were sacrificed by an overdose of urethane, the
brains were removed, frozen in isopentane at –45 °C and stored at –80 °C. Fresh-frozen brains
were cryostat-cut into 20 µm coronal sections and acetylcholine esterase staining was used as
previously described [18] to determine the location of the Pontamine sky blue dots marking
the recording site in the recorded structure. Only brains in which both the recording and drug
injection were shown to be in the targeted structure were used for data analysis.
Statistical analysis
Data are presented as mean ± S.E.M. Statistical analyses were performed using Sigmaplot
(Systat Software, San Jose, USA). Firing rates and coefficients of variation of interspike
intervals during the 20 minutes before and the 20 minutes after drug injection were compared
using paired student t-test. The effects of DA alone or combined with the injection of
sulpiride were compared using One Way ANOVA followed, when significant, by a multiple
comparison procedures using student Newman-Keuls test. The distribution of the firing
patterns was assessed using a Chi2 test.
Results
Effects of intrapallidal injection of dopamine and quinpirole on the spontaneous firing
of GP neurons
The effects of local injection of dopamine and the D2R agonist, quinpirole, on the
spontaneous discharge of GP neurons were investigated in 40 animals under urethane
anesthesia. In control conditions, the mean firing rate of GP neurons was 19.32±1.05
spikes/sec (n=84) and all GP cells exhibited a tonic discharge pattern as shown by the
interspike intervals and density histograms (Fig. 1A). Control microinjection of saline (0.9%
NaCl) into the GP revealed no significant effects on the firing rate and pattern of neuronal
activity in the GP (data not shown). However, intrapallidal injection of dopamine
predominantly induced an excitatory effect on GP neurons (Fig. 1BC). It significantly
increased the firing rate of 34 out of 50 GP neurons (68%, Fig. 1D) with a percentage increase
5 of 45% (p<0.001, paired t-test, Fig. 1B-D, Table 1). This effect occurred within 2-3 minutes
after the injection and lasted 30-40 minutes. In only 2 GP tested cells (4%), dopamine
decreased the firing rate with a percentage decrease of 28% and in 14 GP neurons (28%)
dopamine did not alter the firing rate (p>0.05, Fig. 1D, Table 1). In all GP tested neurons,
local dopamine injection did not change the firing pattern as the coefficient of variation of the
interspike intervals did not significantly change compared to before injection (p>0.05, Table
2). Analysis of the density histograms (Fig. 1AC) (16) showed similar results, e.g. the absence
of significant changes of the firing pattern (p>0.05, Chi2 test).
Table 1. Firing rates of GP, STN and SNr neurons before and after dopamine or quinpirole injection
into the GP.
FR increase cells
FR decrease cells
Non responsive cells
Before
After
Before
After
Before
After
Dopamine
18.64±1.62
27.03±2.44***
21.15±8.95
15.20±9.43*
22.63±2.07
22.63±2.12
ns
Quinpirole
14.31±2.14
19.44±2.72**
20.22±3.31
10.87±1.56**
24.68±3.96
25.61±3.99
ns
Dopamine
4.72±0.60
6.66±0.96*
5.12±0.79
3.43±0.56*
6.39±0.72
6.22±0.60
Quinpirole
11.92±6.69
18.86±4.10*
11.36±2.51
7.23±1.43*
12.91±2.48
13.33±2.67
ns
Dopamine
9.03±2.90
13.10±3.78*
9.41±1.24
5.73±1.03*
12.30±4.16
12.54±4.64
ns
Quinpirole
21.82±6.00
32.12±7.40*
16.10±2.11
8.99±1.60*
12.91±2.48
13.33±2.67
ns
GP neurons
STN neurons
ns
SNr neurons
FR: firing rate in spikes/sec; values are presented as the mean ± SEM. Statistical analysis using
paired t-test was performed; *: p<0.05, **: p<0.01, ***: p<0.001 in comparison with before drug
injection.
Table 2. Coefficient of variations of GP, STN and SNr neurons before and after dopamine or quinpirole
injection into the GP.
GP neurons
Before
STN neurons
After
Before
SNr neurons
After
Before
After
Dopamine
0.32±0.06
0.27±0.04
ns
1.12±0.02
1.10±0.02
ns
1.16±0.08
1.15±0.07
ns
Quinpirole
0.34±0.06
0.36±0.06
ns
1.12±0.03
1.12±0.03
ns
1.18±0.06
1.16±0.05
ns
Values are presented as the mean ± SEM. Statistical analysis using paired t-test was performed; ns: non
significant difference in comparison with before drug injection.
6 To test the hypothesis that the increased firing rate observed in GP cells after local
microinjection of dopamine results from the activation of dopamine D2Rs, these receptors
were blocked by the injection of sulpiride, a selective D2R antagonist. In all GP tested cells,
sulpiride blocked the excitatory effect induced by dopamine (Fig. 1E). To confirm the
importance of the D2Rs in the effects induced by dopamine, we tested the effect of
intrapallidal injection of quinpirole, a selective D2R agonist. First, in a set of experiments, we
investigated the dose response of quinpirole at the doses of 0.2, 0.4, and 0.8 µg. Local
microinjection of quinpirole significantly affected the firing rate of GP neurons in a dosedependent manner (F=26.42, p<0.001, Fig. 2A). In contrast to the doses of 0.2 and 0.4 µg,
which did not affect the firing rate (p>0.05 for the two doses), 0.8 µg significantly increased
the firing rate of the majority of GP neurons (19 out of 29, 66%) with a percentage increase of
36% (p<0.01, paired t-test, Fig. 2A-E, Table 1). In 5 out of 29 GP tested cells (17%),
quinpirole significantly decreased the firing rate (-46.24%, p<0.01, Fig. 2F-H, Table 1) and in
5 out of 29 GP tested neurons (17%) dopamine did not significantly alter the firing rate
(p>0.05, Table 1). In all GP tested neurons, quinpirole did not change the firing pattern as the
coefficient of variation of the interspike intervals did not significantly change after the
injection of quinpirole compared to control conditions (p>0.05, Fig. 2, Table 2). Analysis of
the density histograms showed similar results, e.g. the absence of significant changes in the
firing pattern (p>0.05, Chi2 test).
Effects of intrapallidal injection of dopamine and quinpirole on the spontaneous firing
of STN neurons
In basal conditions, the mean firing rate of STN neurons was 10.38±1.27 spikes/sec (n=95
neurons in 27 rats) and most of the cells exhibited a tonic discharge pattern as shown by the
interspike intervals and density histograms (Fig. 3A). Control microinjection of saline (0.9%
NaCl) into the GP revealed no significant effects on the firing rate and pattern of neuronal
activity in the STN (data not shown). However, dopamine injection into GP predominantly
induced an inhibitory effect on STN neurons. It significantly decreased the firing rate of 9 out
of 20 STN neurons (45%) with a percentage decrease of 33% (p<0.05, paired t-test, Fig. 3AD, Table 1). This effect occurred within 2-3 minutes after the injection and lasted 30-40
minutes. In 5 out of 20 STN tested cells (25%), dopamine significantly increased the firing
rate (p<0.05, Fig. 3E-G, Table 1) and in 6 out of 20 STN tested neurons (30%), dopamine did
not significantly change the firing rate (p>0.05, Table 1). In all STN tested neurons,
dopamine did not change the firing pattern as the coefficient of variation of the interspike
intervals did not significantly change after dopamine injection into GP compared to control
conditions (p>0.05, Fig. 3, Table 2). Analysis of the density histograms also showed the
absence of changes of the firing pattern (p>0.05, Chi2 test).
Intrapallidal injection of quinpirole into the GP significantly affected the firing rate of STN
neurons. It decreased the firing rate of 41 out of 75 STN neurons (55%) with a percentage
decrease of 36% (p<0.05, Fig. 4A-D, Table 1). In only 11 out of 75 STN tested cells (15%),
quinpirole injection significantly increased the firing with a percentage increase of 58%
(p<0.05, Fig.4E-G, Table 1). In 23 out of 75 STN tested neurons (31%), quinpirole injection
did not significantly change the firing rate (p>0.05, Fig. 4, Table 1). In all STN tested cells,
7 quinpirole injection did not change the firing pattern as the coefficient of variation of the
interspike intervals did not significantly change after the injection of dopamine compared to
control conditions (p>0.05, Fig. 4, Table 2). Analysis of the density histograms also showed
an absence of changes of the firing pattern (p>0.05, Chi2 test).
Effects of intrapallidal injection of dopamine and quinpirole on the spontaneous firing
of SNr neurons
In basal conditions, the mean firing rate of SNr neurons was 15.52±1.37 spikes/sec (n=107
neurons in 15 rats) and most of the cells exhibited a regular discharge pattern as shown by the
coefficient of variation of the interspike intervals (Fig. 5A). Control microinjection of saline
(0.9% NaCl) into the GP revealed no significant effects on the firing rate and pattern of
neuronal activity in the SNr (data not shown). However, dopamine injection into GP
predominantly induced an inhibitory effect on SNr neurons. It decreased the firing rate of 13
out of 22 SNr neurons (59%) with a percentage decrease of 39% (p<0.05, Fig. 5A-D, Table
1). This effect occurred within 2-3 minutes after the injection and lasted 30-40 minutes. In 5
out of 22 SNr tested cells (18%), dopamine significantly increased the firing rate with a
percentage increase of 45% (p<0.05, Fig. 5E-G, Table 1) and in 5 out of 22 SNr tested
neurons (23%) dopamine did not significantly change the firing rate (p>0.05, Table 1). In all
SNr tested neurons, dopamine did not change the firing pattern as the coefficient of variation
of the interspike intervals did not significantly change after dopamine injection into GP
compared to control conditions (p>0.05, Fig. 5, Table 2). Analysis of the density histograms
also showed the absence of changes of the firing pattern (p>0.05, Chi2 test).
Correspondingly, local microinjection of quinpirole into the GP significantly affected the
firing rate of SNr neurons. Quinpirole significantly decreased the firing rate of 46 out of 85
SNr neurons (55%) with a percentage decrease of 44% (p<0.05, Fig. 6A-D, Table 1). In only
15 out of 85 SNr tested cells (18%), quinpirole injection significantly increased the firing rate
with a percentage increase of 47% (p<0.05, Fig. 6E-G, Table 1) and in 24 out of 85 SNr tested
neurons (28%), quinpirole injection did not significantly change the firing rate (p>0.05, Table
1). In all SNr tested cells, quinpirole injection did not change the firing pattern as the
coefficient of variation of the interspike intervals did not significantly change after the
injection of quinpirole compared to control conditions (p>0.05, Fig. 6, Table 2). Analysis of
the density histograms also showed an absence of changes of the firing pattern (p>0.05, Chi2
test).
Discussion
Dopamine, through activation of D2Rs, modulates the firing rate but not the pattern of
GP neurons
In the present study, we provide evidence that dopamine, through activation of D2Rs, exerts
an excitatory effect on the majority of GP neurons in vivo. Thus, dopamine-induced firing rate
increase was mimicked by the selective D2R agonist, quinpirole, and prevented by the
8 selective D2R antagonist, sulpiride. However, neither dopamine nor quinpirole changed the
discharge pattern, demonstrating that dopamine, through activation of D2Rs, modulates the
firing rate but not the pattern of GP neurons under physiological conditions. The possibility
that the absence of change of the firing pattern may be influenced by the use of urethane
anesthesia cannot be completely rule out. However, this possibility may be minimized, as
dopamine depletion in the rat, under the same conditions of anesthesia, has been shown to
induce burst activity in GP neurons (19) and also in STN neurons (13, 17, 20, 21).
Furthermore, similar burst activity has been reported in non-anesthetized MPTP-treated
monkeys (22) and in patients with Parkinson’s disease (23, 24). Our results are consistent
with previous studies showing that quinpirole increased the firing rate of GP neurons in the
rat (11) and of GPe neurons in non-human primate (25). Quinpirole also increased the
expression of the immediate early gene c-fos, which is a marker of neuronal activity (26). The
firing rate increase, which represents the major effect of dopamine on GP neurons, may be
explained by the action of this neurotransmitter on D2Rs located pre-synaptically on GABA
striato-pallidal fibers (6). Thus, dopamine, like quinpirole, reduces GABA release by
activating D2Rs, resulting in a disinhibition of GP neurons. This is consistent with an early
study, which showed that iontophoretic injection of dopamine or amphetamine reduced
GABA transmission in the GP (27). Accordingly, in vitro data demonstrated that dopamine,
through activation of D2Rs on striato-pallidal terminals, exerts an inhibitory effect on GABA
release in the rat GP (28, 29). These studies, together with ours, suggest that the striatopallidal GABAergic inhibition is under the control of presynaptic D2Rs and that local
depletion of dopamine may contribute to the changes in GP neuronal activity observed in
animal models of Parkinson's disease. Thus, intrapallidal injection of 6-hydroxydopamineinduced dopamine depletion in GP resulted in a decrease of the firing rate of GP neurons (12).
This supports the key role played by dopamine at extrastriatal sites, suggesting that
dopaminergic drugs may play their anti-parkinsonian effects through activation of D2Rs
located in GP in addition to their action in the striatum.
In addition to their localization on GABA fibers, D2Rs are also located presynaptically on
glutamatergic afferents originating from the STN and the parafascicular nucleus of the
thalamus (6). Their activation has been suggested to reduce glutamatergic release in GP of in
vitro slices (30). This may explain why in some of our GP tested cells dopamine, like
quinpirole, reduced their firing rate. The fact that this effect was observed in only a minority
of GP neurons compared to those showing an increase in their firing rate, is consistent with
the reduced number of D2Rs located on glutamate terminals compared to those located on
GABA terminals (6). In another population of GP tested cells, dopamine like quinpirole did
not affect their firing activity. This can be due to the absence of dopamine receptors on
afferents of these neurons. This result is consistent with data of a previous anatomical study
showing that D2Rs were not found in all GP neurons but only in a population of
approximately 40-50% (29).
We therefore postulate that dopamine acting at presynaptic D2Rs predominantly reduces
GABA release at GABAergic terminals in GP. Presynaptic rather than postsynaptic
dopaminergic modulation of GABAergic transmission in the GP is supported by the action of
9 dopamine on miniature GABAergic transmission, which can be mimicked by the use of
selective D2R agonists (28). The firing rate increase of pallidal neurons caused by dopamine
and its D2R agonist, quinpirole, would lead to decreased firing rate of their major basal
ganglia efferent structures such as the STN and SNr.
Dopamine, through activation of D2Rs in GP, modulates the GP-STN and GP-SNr
neurotransmission by controlling the firing rate but not the pattern of STN and SNr
neurons
The principal GABAergic input to the STN arises from the GP, which plays a key role in the
control of firing activity of STN neurons. In vitro electrophysiology studies reported that
spontaneous pallido-subthalamic activity influenced STN neuronal firing (31) and that
electrical stimulation of GP afferents evoked IPSP or IPSC through activation of postsynaptic
GABAA receptors (32-34). Here, we focused our study on the impact of dopaminergic
modulation of GP-STN neurotransmission and we showed that dopamine, like quinpirole,
when injected into the GP decreased the firing rate of most STN neurons. These results can be
explained by the fact that dopamine, through activation of D2Rs, predominantly increased the
firing rate of GP cells (present study), at the origin of GABA release in the STN, resulting in a
reduction of the firing activity of majority of STN recorded neurons. This is the first study
showing that DA in GP participates in the modulation of GP-STN neurotransmission and
consequently controls STN neuronal firing. The inhibitory effect is mediated by GABAARs,
as they are concentrated at GP–STN synapses and that GABAAR antagonists block
spontaneous IPSCs (35). Furthermore, we showed that DA, via D2Rs, increased the firing rate
of a minority of STN neurons (25% for DA and 15% for quinpirole). This excitatory effect
can be explained by the fact that dopamine, via D2Rs, reduces the firing rate of a small subpopulation of GP cells inducing a decrease of GABA release in the STN, which in turn results
in a disinhibition of STN neurons.
In the two populations of STN responsive neurons, the firing rate changes were not
accompanied by a change in firing pattern. Together, our data show that DA participates in
the modulation of the GP-STN pathway, contributing to the control of firing rate but not
pattern of STN neurons. This is consistent with previous studies showing that the pattern of
GP inhibitory input to the STN is crucial in determining whether STN neurons fire in a tonic
or burst pattern (32), and that burst activity in GP neurons is necessary to generate sufficient
hyperpolarization in STN neurons for rebound burst activity (36).
In addition to the control of GP-STN pathway, we show that dopamine in GP modulates the
neuronal activity of the principal output structure of basal ganglia network in the rat, the SNr.
We show that the responses of SNr neurons to pallidal microinjection of dopamine, or its D2R
agonist, are similar to those of STN neurons (including decreases, increases and some neurons
showing no change) with the same proportions. The changes observed in SNr neurons can be
due to i) the activation of GABAergic neurons of GP projecting directly to the SNr or ii) to
the deactivation of STN neurons projecting to the SNr as majority of STN neurons are
inhibited by dopamine when injected in the GP or iii) to a combination of the two phenomena.
10 According to previous studies, it is likely that SNr cell responses to dopamine in GP are a
consequence of the two phenomena. The first hypothesis is supported by anatomical tracing
findings showing that individual GP neurons that project to the STN possess axon collaterals
innervating the SNr (for review, Smith, Bevan (37), Deniau, Mailly (38)). Furthermore, in rat
brain slices preparation, it has been shown that GP neurons have a significant impact on the
discharge of SNr cells (38). Indeed, GP stimulation evoked IPSPs of SNr neurons, which is
strong enough to reset the firing of the neurons (38). The second hypothesis is supported by a
previous electrophysiological study showing that the STN lesions induced an attenuation of
changes in mean firing rate of SNr neurons in response to intrastriatal microinjection of
apomorphine (39). Based on these evidences, it is likely that SNr neuronal responses are due
to changes in the level of activity of inhibitory (GP) and excitatory (STN) afferents and that
both GP-SNr and GP-STN-SNr are important in the inhibitory response of SNr neurons to
dopamine and quinpirole injection into GP.
In conclusion, our data are the first to show that dopamine, through activation of D2Rs
located in the GP, plays a key role in modulating GP neuronal activity, which participates to
the control of its two principal efferent projections, the STN and SNr. The predominant effect
was an increase in the firing rate of GP neurons, resulting in the inhibition of GABA release
from presynaptic terminals in the STN and SNr, leading to decreased activity of its neurons.
In addition, the changes in STN neuronal activity may participate to the modulation of SNr
neurons. Our data provide evidence that dopamine in GP controls the firing rate but not the
pattern of GP neurons, which in turn control the firing rate, but not the pattern of STN and
SNr neurons.
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2396(199712)27:4<278::AID-­‐SYN2>3.0.CO;2-­‐9. PubMed PMID: 9372551. 14 Figure 1: Intrapallidal microinjection of dopamine predominantly increased the firing
rate without changing the tonic firing pattern of GP neurons.
(A-C) A representative example of GP neuron before (AB) and after (BC) microinjection of
dopamine (DA) into the GP showing an increase of its firing rate with spike train (A1C1),
firing rate histogram (B), raster display of random segments of recording (A2C2), insterspike
interval histogram (A3C3) and density histogram (A4C4) of the same GP neuron.
(D) Circular plot representing the percentage of GP neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
(E) Representative firing rate histogram with the response of a GP neuron showing an
increase of its firing rate after dopamine injection corresponding to the effect observed in the
majority of GP neurons. The excitatory effect of dopamine (DA) was prevented by the
selective D2R antagonist, sulpiride (Sulp). Note that DA increased the firing rate (E1E2)
without changing the coefficient of variation (E3) of GP neurons and that after the injection of
sulpiride (DA+Sulp), DA had no effect on the firing rate (E1E2). *p<0.05
15 Figure 2: Intrapallidal microinjection of quinpirole predominantly increased the firing
rate of GP neurons in a dose-dependent manner without changing the tonic firing
pattern.
(A) Histograms showing the dose response effects of quinpirole (Quin 0.2, 0.4 and 0.8 µg) on
the firing rate (A1) and the coefficient of variation of the interspike intervals (A2) of GP
neurons. ***p<0.001.
(B-D) A representative example of GP neuron before (BC) and after (CD) microinjection of
quinpirole (Quin) into the GP showing an increase of its firing rate with spike train (B1D1),
firing rate histogram (C), raster display of random segments of recording (B2D2), insterspike
interval histogram (B3D3) and density histogram (B4D4) of the same GP neuron.
(E) Circular plot representing the percentage of GP neurons showing an increase, a decrease
or no change of their firing rate after the local injection of quinpirole.
(F-H) A representative example of GP neuron before (FG) and after (GH) microinjection of
quinpirole (Quin) into the GP showing a decrease of its firing rate with spike train (F1H1),
firing rate histogram (G), raster display of random segments of recording (F2H2), insterspike
interval histogram (F3H3) and density histogram (F4H4) of the same GP neuron.
16 Figure 3: Intrapallidal microinjection of dopamine predominantly decreased the firing
rate without changing the tonic firing pattern of STN neurons.
(A-C) A representative example of STN neuron before (A) and after (C) microinjection of
dopamine into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B) raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same STN neuron.
(D) Circular plot representing the percentage of STN neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
(E-G) A representative example of STN neuron before (EF) and after (FG) microinjection of
dopamine into the GP with spike train (E1G1), firing rate histogram (F), raster display of
random segments of recording (E2G2), insterspike interval histogram (E3G3) and density
histogram (E4G4) of the same STN neuron.
17 Figure 4: Intrapallidal microinjection of quinpirole predominantly decreased the firing
rate without changing the tonic firing pattern of STN neurons.
(A-C) A representative example of STN neuron before (A) and after (C) microinjection of
quinpirole into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B), raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same STN neuron.
(D) Circular plot representing the percentage of STN neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
E-G) A representative example of STN neuron before (E) and after (G) microinjection of
quinpirole into the GP showing an increase of its firing rate with spike train (E1G1), firing
rate histogram (F), raster display of random segments of recording (E2G2), insterspike
interval histogram (E3G3) and density histogram (E4G4) of the same STN neuron.
18 Figure 5: Intrapallidal microinjection of dopamine predominantly decreased the firing
rate without changing the tonic firing pattern of SNr neurons.
(A-C) A representative example of SNr neuron before (A) and after (C) microinjection of
dopamine into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B), raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same SNr neuron.
(D) Circular plot representing the percentage of SNr neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
(E-G) A representative example of SNr neuron before (E) and after (G) microinjection of
dopamine into the GP showing an increase of its firing rate with spike train (D1F1), firing rate
histogram (F), raster display (E2G2), insterspike interval histogram (E3G3) and density
histogram (E4G4) of the same SNr neuron.
19 Figure 6: Intrapallidal microinjection of quinpirole predominantly decreased the firing
rate without changing the tonic firing pattern of SNr neurons.
(A-C) A representative example of SNr neuron before (A) and after (C) microinjection of
quinpirole into the GP showing a decrease of its firing rate with spike train (A1C1), firing rate
histogram (B), raster display of random segments of recording (A2C2), insterspike interval
histogram (A3C3) and density histogram (A4C4) of the same SNr neuron.
(D) Circular plot representing the percentage of SNr neurons showing an increase, a decrease
or no change of their firing rate after the local injection of dopamine.
(E-G) A representative example of SNr neuron before (E) and after (G) microinjection of
quinpirole into the GP showing an increase of its firing rate with spike train (E1G1), firing
rate histogram (F), raster display (E2G2), insterspike interval histogram (E3G3) and density
histogram (E4G4) of the same SNr neuron.
20 Annexe 2
Benazzouz A., Mamad O., Abedi P., Bouali-Benazzouz R. and Chetrit J. Involvement of dopamine
loss in extrastriatal basal ganglia nuclei in the pathophysiology of Parkinson’s disease. Frontiers in
Aging Neuroscience, 2014, 13, 6:87. doi: 10.3389/fnagi.2014.00087. eCollection 2014.
21 MINI REVIEW ARTICLE
AGING NEUROSCIENCE
published: 13 May 2014
doi: 10.3389/fnagi.2014.00087
Involvement of dopamine loss in extrastriatal basal ganglia
nuclei in the pathophysiology of Parkinson’s disease
Abdelhamid Benazzouz 1,2 *, Omar Mamad 1,2,3 , Pamphyle Abedi 1,2,3 , Rabia Bouali-Benazzouz 4 and
Jonathan Chetrit 1,2
1
Institut des Maladies Neurodégénératives, Université Bordeaux Segalen, UMR 5293, Bordeaux, France
CNRS, Institut des Maladies Neurodégénératives, Université Bordeaux Segalen, UMR 5293, Bordeaux, France
3
Faculté des Sciences, Equipe Rythmes Biologiques, Neurosciences et Environnement, Université Mohamed V-Agdal, Rabat, Morocco
4
Institut Interdisciplinaire des Neurosciences, Université Bordeaux Segalen, UMR 5297, Bordeaux, France
2
Edited by:
Isidro Ferrer, University of
Barcelona, Spain
Reviewed by:
Nicola Pavese, Imperial College
London, UK
Concepcio Marin, Institut
d’Investigacions Biomèdiques
August Pi i Sunyer, Spain
*Correspondence:
Abdelhamid Benazzouz, Institut des
Maladies Neurodégénératives and
CNRS, Université Bordeaux
Segalen, UMR 5293, 146 Rue
Léo-Saignat, 33076 Bordeaux
Cedex, France
e-mail: [email protected]
Parkinson’s disease (PD) is a neurological disorder characterized by the manifestation of
motor symptoms, such as akinesia, muscle rigidity and tremor at rest. These symptoms
are classically attributed to the degeneration of dopamine neurons in the pars compacta
of substantia nigra (SNc), which results in a marked dopamine depletion in the striatum.
It is well established that dopamine neurons in the SNc innervate not only the striatum,
which is the main target, but also other basal ganglia nuclei including the two segments of
globus pallidus and the subthalamic nucleus (STN). The role of dopamine and its depletion
in the striatum is well known, however, the role of dopamine depletion in the pallidal
complex and the STN in the genesis of their abnormal neuronal activity and in parkinsonian
motor deficits is still not clearly determined. Based on recent experimental data from
animal models of Parkinson’s disease in rodents and non-human primates and also from
parkinsonian patients, this review summarizes current knowledge on the role of dopamine
in the modulation of basal ganglia neuronal activity and also the role of dopamine depletion
in these nuclei in the pathophysiology of Parkinson’s disease.
Keywords: dopamine, extrastriatal dopamine, basal ganglia, globus pallidus, subthalamic nucleus, Parkinson’s
disease
INTRODUCTION
Parkinson’s disease (PD) is a neurological disorder characterized by the manifestation of motor symptoms such as akinesia,
muscle rigidity and tremor at rest. These motor deficits are
classically attributed to the degeneration of dopamine neurons
in the pars compacta of substantia nigra (SNc), which result
in a marked dopamine depletion in the striatum, the primary
projection region of the SNc. Furthermore, it is well established
now that dopamine neurons in the SNc innervate not only the
striatum but also other basal ganglia nuclei including the two
segments of globus pallidus, the external part (GPe in primate,
the equivalent of GP in rodents) and the internal part (GPi in
primate, the equivalent of entopeduncular nucleus in rodents),
as well as the subthalamic nucleus (STN; Smith and Villalba,
2008). Dopamine has been shown to modulate the neuronal
electrical activity of all these basal ganglia nuclei (Rommelfanger
and Wichmann, 2010).
Dopamine cell degeneration in the pathophysiology of PD
is considered as the main hallmark of the disease (Agid and
Blin, 1987; Hornykiewicz, 1998). Indeed, dopamine depletion
by stereotaxic injection of 6-hydroxydopamine (6-OHDA) in
the rat or by systemic injections of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) in the non-human primate resulted
in alterations of the firing rate and/or patterns of GPe, GPi and
STN neurons. The tonic regular pattern in the normal condition
Frontiers in Aging Neuroscience
changed toward a pathological exaggerated burst firing with oscillations after dopamine cell lesions in the substantia nigra (SNc;
Albin et al., 1989; DeLong, 1990; Bergman et al., 1994; Wichmann
et al., 1994; Boraud et al., 1998; Ni et al., 2000, 2001b; Magill et al.,
2001; Breit et al., 2007; Rivlin-Etzion et al., 2010). Similar bursty
pattern has been reported in PD patients when microrecordings
have been done during surgery for the implantation of deep
brain stimulation electrodes (Hutchison et al., 1998; Benazzouz
et al., 2002). According to the classical model of the anatomofunctional organization of the basal ganglia, the pathological
activity recorded in basal ganglia nuclei has been identified as a
consequence of dopamine depletion in the striatum (Albin et al.,
1989).
In normal physiological conditions, dopamine has long been
known to be a crucial neuromodulator of striatal processing
of cortical informations carried by glutamatergic synapses on
medium spiny neurons, which represents the principal projection neurons of the striatum. Dopamine excites medium spiny
neurons of the “direct” pathway through dopamine D1 receptors,
while it inhibits striatal neurons of the “indirect” pathway through
dopamine D2 receptors (Alexander and Crutcher, 1990; Surmeier
et al., 2007). In the context of PD, studies of the neuronal activity
in the basal ganglia of MPTP monkeys and 6-OHDA rat models
of the disease suggested that the direct and the indirect pathways
are differentially affected by the loss of dopamine in the striatum.
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Benazzouz et al.
Extrastriatal dopamine and Parkinson’s disease
The GABAergic inhibitory direct striato-GPi pathway becomes
underactive, whereas the GABAergic projection from the striatum
to the GPe of the indirect pathway becomes overactive, leading
to the reduced activity along the inhibitory GPe-GPi and GPeSTN pathways. Thus, it is suggested that exaggerated oscillatory
bursts in STN and in GPi may have been secondary to tonic
disinhibition of both structures from GPe after loss of dopamine
in the striatum. However, the role of dopamine depletion in these
extrastraiatal basal ganglia nuclei in the pathophysiology of PD is
still not clearly defined. Nevertheless, in view of the demonstrated
physiologic actions of dopamine on pallidal and STN neuronal
activity as well as the effects on motor behavior of local injection
of dopamine drugs, it is assumed that the loss of pallidal and
subthalamic dopaminergic control would contribute to the motor
symptoms in PD (Rommelfanger and Wichmann, 2010; Wilson
and Bevan, 2011).
The pallidal complex and the STN are innervated by nigral
dopamine fibers, by separate fiber system and also by collaterals
of nigrostriatal fibers. This has been shown in rodents (Lindvall
and Bjorklund, 1979; Debeir et al., 2005; Anaya-Martinez et al.,
2006), in non-human primate (Nobin and Bjorklund, 1973;
Parent and Smith, 1987; Lavoie et al., 1989; Parent et al., 1989;
François et al., 1999; Hedreen, 1999; Jan et al., 2000) and in
human brains (Nobin and Bjorklund, 1973; Cossette et al., 1999;
François et al., 1999; Jan et al., 2000). Dopamine acts via five
receptor subtypes subdivided into two receptor families: D1 (D1
and D5 subtypes) and D2 (D2, D3 and D4 subtypes). All are
prototypic of G-protein-coupled receptors with dopamine D1
receptors being positively linked to adenylate cyclase and D2
receptors had negative coupling to the enzyme (Kebabian and
Calne, 1979). A large number of experimental studies reported
that functional dopamine receptors are expressed in the striatum
and also in the GPe, GPi and STN and that dopamine modulates
their neuronal activity through a variety of mechanisms via preand post-synaptic sites (Smith and Villalba, 2008; Rommelfanger
and Wichmann, 2010).
Dopamine, through D1 and D2 family receptors, in the pallidal
complex and the STN may modulate the motor circuit and
consequently dopamine depletion in these structures may play a
role in the pathophysiology of PD. Lesions of dopamine neurons
in the SNc in rodents and monkeys have been shown to reduce
dopamine levels in GPe, GPi and STN in addition to the striatum
(Parent et al., 1990; François et al., 1999; Jan et al., 2000; Fuchs
and Hauber, 2004).
DOPAMINE DEPLETION IN THE GLOBUS PALLIDUS
Rajput et al. (2008) have recently reported a marked loss of
dopamine in the GPe (−82%) of PD patients with a severe loss
of dopamine in the caudate (−89%) and the putamen (−98.4%).
Based on the conclusions of a previous experimental study (Pifl
et al., 1991), the authors suggested that pallidal dopamine participates in the functional compensation against the severe loss
of dopamine in the striatum at the early stage of the disease.
It has been shown that in the MPTP-treated primate model
of parkinsonism in which the animals with stable parkinsonian symptoms showed a marked pallidal dopamine depletion,
asymptomatic animals showed normal pallidal dopamine levels
Frontiers in Aging Neuroscience
but had very marked striatal dopamine deficit (Pifl et al., 1991).
Similarly, imaging studies using positron emission tomography
in PD patients reported that while patients with severe advanced
stage of the disease had significantly reduced 18F-dopa uptake in
the striatum, GPe and GPi, patients at mild stage of the disease
demonstrated a severely reduced 18F-dopa uptake in the striatum
but normal uptake in GPe and GPi (Whone et al., 2003; Pavese
et al., 2011). Furthermore, an increase in 18F-dopa uptake in
GPi has been reported in early stage PD (Rakshi et al., 1999;
Whone et al., 2003; Moore et al., 2008; Pavese et al., 2011).
Together, these studies postulate that dopamine plays a key role in
the compensatory up-regulation of the nigro-pallidal dopamine
projection in the early stages of PD representing a compensatory adaptive mechanism to preserve functionality. In contrary,
dopamine depletion in the two pallidal segments (GPe and GPi)
may participate in the aggravation of motor symptoms in the late
stages of PD.
Studies on the expression of dopamine receptors in the
pallidal complex of parkinsonian brains reported conflicting
data. While some studies found no difference in dopamine
D1R expression in the GPe and GPi (Rinne et al., 1985;
Cortés et al., 1989), others found dopamine D1R expression
unchanged in the GPi and decreased in GPe (Hurley et al.,
2001). Dopamine D2 receptors, including D3 receptors, were
unchanged in both GPe and GPi (Bokobza et al., 1984; Cortés
et al., 1989; Ryoo et al., 1998). The absence of changes in
the expression of D1 and D2 receptors can be explained by
the fact that patients were under dopaminergic medication
before death and that the treatment is likely to normalize the
expression of these receptors. This may be true for the striatum but not for the pallidal complex as in MPTP monkeys,
the expression of dopamine D3 receptors was reduced in the
caudate nucleus but not in the GPe and GPi and that L-Dopa
treatment normalized the hypoexpression of dopamine D3 receptors in the caudate nucleus and increased to a level higher than
normal in GPi without any change in the GPe (Bézard et al.,
2003).
The contribution of pallidal dopamine in the pathophysiology
of PD has also been demonstrated in rodents. Activation of D1 or
D2 dopamine receptors in the GP induced movement facilitation
(Sañudo-Peña and Walker, 1998). In contrast, local blockade of
D1 and/or D2 receptors by intra-pallidal infusions of specific
antagonists induced akinesia in rats (Hauber et al., 1998; Hauber
and Lutz, 1999). Similarly, in rats bearing a unilateral 6-OHDA
lesion, it has been shown that blockade of either dopamine D1 or
D2 receptors reduced apormorphine-induced turnings and that
dopamine infusion into the GP improved motor deficits in the
same animal model (Galvan et al., 2001). Furthermore, we have
recently shown that intra-pallidal injection of 6-OHDA produced
deficits of dopaminergic transmission that caused asymmetrical
motor impairment and reduction of locomotor activity in the rat
(Bouali-Benazzouz et al., 2009; Abedi et al., 2013). Together, these
studies provide arguments that dopamine transmission within
the globus pallidus is necessary to achieve motor control and
that its lack plays a role in the pathophysiology of parkinsonian
motor symptoms, in addition to dopamine depletion in the
striatum.
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Extrastriatal dopamine and Parkinson’s disease
DOPAMINE DEPLETION IN THE SUBTHALAMIC NUCLEUS
STN holds a pivotal position in basal ganglia circuitry exerting an excitatory drive on the output structures of the system (Albin et al., 1989; Alexander and Crutcher, 1990). The
STN has been shown to play a key role in motor control,
as its hyperactivity with oscillatory bursts has been associated
to parkinsonian motor deficits. The motor symptoms can be
reversed by selective STN lesion or high frequency stimulation
(Bergman et al., 1990; Benazzouz et al., 1993; Limousin et al.,
1995; Krack et al., 2003). Several studies have suggested the
implication of SNc-STN dopaminergic projection in the pathophysiology of PD. Thus, bilateral infusions of D1 but not D2
receptor antagonists into the STN induced catalepsy in normal
rats (Hauber, 1998) and that activation of D1 receptors resulted
in orofacial dyskinesias in normal and dopamine-depleted rats
(Parry et al., 1994; Mehta et al., 2000). From these studies it
is suggested that dopaminergic agents acting at D1 receptors
have stronger functional and behavioral effects than agents acting
at D2 receptors. Several studies have shown that dopamine is
reduced in the STN in experimental Parkinsonism and also in
patients with PD (Pifl et al., 1990; Hornykiewicz, 1998; François
et al., 2000) and such dopamine loss in the STN may contribute
to increase abnormal neuronal activity. Accordingly, selective
lesions of the dopaminergic fibers located in the STN, by intrasubthalamic infusion of 6-OHDA, resulted in contralateral muscle
rigidity and ipsilateral turning in response to systemic administration of DL-methamphetamine (Flores et al., 1993). These
motor deficits could be explained by the fact that 6-OHDA
injection into the STN resulted in retrograde degeneration of
dopamine cell bodies in the lateral part of the SNc (Ni et al.,
2001a) and was at the origin of the significant increase in the
percentage of STN neurons exhibiting burst pattern (Ni et al.,
2001b). These results provide evidence that the degeneration
of SNc-STN dopaminergic projections plays, at least in part,
a role in the development of the pathological burst pattern
of STN neurons and therefore to the manifestation of PD-like
motor deficits. In the same 6-OHDA rat model, it has been
shown that lesions of the SNc dopaminergic cells increased the
level of dopamine D2 receptor mRNA, decreased D3 receptor mRNA levels, and did not induce significant changes in
Dl receptor mRNA in the STN (Flores et al., 1999). Furthermore, several studies have shown that dopamine D5 receptors,
which display a high agonist-independent constitutive activity
in vitro (Tiberi and Caron, 1994; Demchyshyn et al., 2000),
are located in the STN and are able to potentiate burst firing
in STN neurons in in vitro rat brain slices (Baufreton et al.,
2003, 2005). These authors suggested that in the parkinsonian
state, the reduction of dopaminergic transmission in the STN
results in a lack of activation of dopamine D2 receptors, and
as D5 receptors are constitutively active even in the absenc of
dopamine, they contribute to the development of burst discharges
of STN neurons (Baufreton et al., 2005). This assumption has
been demontsrated by our recent in vivo study, in which we
have shown that local microinjection of an inverse agonist of D5
receptors, flupenthixol, reduced burst activity of STN neurons
and therefore improved the motor deficits in the 6-OHDA rat
model of PD (Chetrit et al., 2013). Moreover, STN dopaminergic
Frontiers in Aging Neuroscience
afferents have also been suggested to play a relevant role in the
expression of dyskinesias. Indeed, dopamine depletion in the
STN attenuated levodopa-induced dyskinesia in rats bearing a
concomitant lesion of the nigrostriatal pathway (Marin et al.,
2013).
CONCLUSION
While the degeneration of the dopaminergic nigrostriatal pathway
is the hallmark of PD, there is strong evidence about the key role
played by dopamine loss at extrastriatal sites, especially in the pallidal complex and the STN, in the pathophysiology of the disease.
Activation of dopamine receptors in these important basal ganglia nuclei modulates their neuronal activity, and consequently
participates in the beneficial effects, and may be in the adverse
effects, of the classical dopaminomimetic antiparkinsonian drugs.
Together, we can assume that dopamine transmission within the
globus pallidus and the STN is necessary to achieve normal motor
control.
ACKNOWLEDGMENTS
This study was supported by grants from the “Centre national
de la Recherche Scientifique”, “Université Bordeaux Segalen” and
exchange grants of the “Groupement de Recherche International”
(GDRI N198, CNRS and INSERM France, and CNRST Morocco),
Egide-Volubilis N◦ 20565ZM, CNRS-CNRST Convention Adivmar 22614 and NEUROMED.
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 11 April 2014; accepted: 23 April 2014 ; published online: 13 May 2014.
Citation: Benazzouz A, Mamad O, Abedi P, Bouali-Benazzouz R and Chetrit J (2014)
Involvement of dopamine loss in extrastriatal basal ganglia nuclei in the pathophysiology of Parkinson’s disease. Front. Aging Neurosci. 6:87. doi: 10.3389/fnagi.2014.00087
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