UNIVERSITÉ MOHAMMED V
FACULTÉ DES SCIENCES
Rabat
N° d’ordre 2759THÈSE DE DOCTORAT
Présentée parMAMAD Omar
Discipline : Biologie Spécialité : NEUROSCIENCESDopaminergic 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 RATSoutenue 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
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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.
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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.
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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…
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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 pallido-subthalamic 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 pallido-subthalamic 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 pallido-subthalamic and pallido-nigral pathways by dopamine D2 receptors in the rat. 4th Conference of Mediterranean Neuroscience Society (MNS), Septembre 30- October 03,
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2012, Istanbul, Turquie.
Mamad O. Abedi.P, Delaville C., Benjelloun W. and Benazzouz A. Control of the pallido-subthalamic 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 pallido-subthalamic and pallido-nigral pathways by dopamine D2 receptors in the rat. Final meeting Neuromed Neuroscience, May 2-3,2013, Marseille France.
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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 ses agoniste/antagoniste), nous avons étudié chez le rat anesthésie à l‟uréthane, 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
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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
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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 «
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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.
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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.
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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
13 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
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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
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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.
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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.
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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).
18 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
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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).
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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 seven-transmembrane 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
21
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.
22 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
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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).
24 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
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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.
26 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 non-human 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).
27 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).
28 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
vivo and in vitro recording
Neuro-biochemical properties Electrophysiological and Neuro-biochemical properties
*Waveform of extracellular recording (Bergstrom et al., 1982,
1984).
* The sensitive to the amplitude of
injected current (Nambu and Llinas,
1997).
* Increases or decrease in activity (DeLong, 1971, Gardiner and Kitai, 1992, Goldberg and Bergman, 2011, Qi and Chen, 2011).
Two groups of neuronal population (around PV+:60%) & (PPE: 40%)(Kita, 1994, Hoover and Marshall, 1999, 2002).
3 calcium binding proteins:
PV,CB & CR) (Cooper and Stanford, 2002)
The visual inspection of current clamp and morphology (Cooper and
Stanford, 2001b), (Kita & Kitai, 1991).
Firing pattern (Mallet et al.,
2008)
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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,
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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
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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).
32 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
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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 GP-TI 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).
34 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.
35 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 goal-directed 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
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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, Fink-Jensen 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.
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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
38
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
39
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 membrane-bound 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: phosphate-activated glutaminase, SV: synaptic vesicle, and VATPase: vacuolar-type H+-ATPase. For other abbreviations, see the main text. From (Roth and Draguhn, 2012).
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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
41
of inhibitory receptors; GABABRs mediate a “slow” and “late” inhibition, or function as
auto-receptors 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
42
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).
43 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).
