la collateralisation axonale dans les ganglions de la ...

MARTIN PARENT

LA COLLATERALISATION AXONALE DANS LES GANGLIONS DE LA BASE CHEZ LE PRIMATE

Thèse présentée à la Faculté des études supérieures de l'Université Laval dans le cadre du programme de doctorat en neurobiologie pour l'obtention du grade de Philosophiae Doctor (Ph. D.)

FACULTE DE MEDECINE UNIVERSITÉ LAVAL QUÉBEC

2006

© Martin Parent, 2006

RESUME L'élucidation de la microcircuiterie liant les différentes composantes des ganglions de la base est d'une importance capitale afin d'améliorer notre compréhension de ce système neuronal hautement complexe impliqué notamment dans le contrôle de la motricité. C'est dans cette optique qu'ont été entrepris les travaux de recherche consolidés dans cet ouvrage qui rapporte des données neuroanatomiques nouvelles obtenues chez le singe cynomolgus {Macaca fascicularis) et le singe écureuil (Saimiri sciureus) à l'aide d'une technique de pointe permettant le marquage et la reconstruction tridimensionnelle complète de neurones individuels. L'injection par microiontophorèse d'un traceur antérograde, la biotine dextran aminé, dans le pallidum interne, le complexe centre médian/parafasciculaire du thalamus ainsi que le cortex moteur primaire, a permis de tracer en détail l'arborisation axonale des neurones composant ces structures. L'étude approfondie des neurones de projection du pallidum interne révèle que la majorité des axones pallidofuges sont fortement collatéralisés, un même neurone étant en mesure d'influencer à la fois le thalamus ventral, le

complexe

centre

médian/parafasciculaire

du

thalamus

ainsi

que

le

noyau

pédonculopontin du tegmentum mésencéphalique par le biais d'un jeu complexe de collatérales axonales. Il en est de même pour les neurones de projection du complexe thalamique centre médian/parafasciculaire dont le branchement axonal permet d'influencer individuellement et de façon variée, à la fois le cortex cérébral et le striatum. Nos travaux révèlent en plus que, contrairement à ce que l'on croyait, la projection corticostriée en provenance du cortex moteur primaire chez le primate n'est pas dédiée uniquement au striatum. En effet, la découverte de neurones corticaux projetant à la fois au striatum et vers le tronc cérébral via des collatérales axonales prouve que le cortex moteur primaire peut influencer le striatum de façon directe et indirecte. L'ensemble de ces résultats révèle que les ganglions de la base forment un réseau neuronal très vaste dont les éléments constitutifs possèdent un axone fortement collatéralisé. Ces données neuroanatomiques jettent un éclairage nouveau sur l'organisation anatomique et fonctionnelle des ganglions de la base chez le primate et doivent être prises en considération dans l'élaboration de nouvelles approches thérapeutiques visant à contrer les processus neurodégénératifs qui affectent les ganglions de la base, comme ceux associés aux maladies de Parkinson et de Huntington.

SUMMARY

A better knowledge of the neural wiring that links the major components of the basai ganglia is essential to understand the complex spatiotemporel séquence of neural events that ensures the correct flow of cortical information through this set of subcortical structures involved in the control of motor behavior. The présent thesis reports novel neuroanatomical findings gathered in both Old World (cynomolgus; Macaca fascicularis) and New Word (squirrel monkey, Saimiri sciureus) primates using a state-of-the-art method allowing a complète labelling and three-dimension reconstruction of single neurons. Microiontophoretic injections of biotin dextran aminé, an anterograde neuronal tracer, in the internai pallidum, the centre médian/parafascicular thalamic complex and the primary motor cortex allowed a detailed description of projection neurons within thèse forebrain structures. Our data show that the majority of pallidal neurons are endowed with a highly collateralized axon that projects to the ventral tiers thalamic nuclei, the centre médian/parafascicular thalamic complex and the brainstem pedunculopontin tegmental nucleus. Our results also reveal that single centre médian or parafascicular neurons are able to influence in a multifarious fashion both the cérébral cortex and the striatum. Furthermore, we hâve shown that the corticostriatal projection arising from primary motor cortex is not dedicated solely to the striatum, in contrast to current belief. We found neurons that project to the striatum indirectly through a thin collatéral emitted by a thick long-range axon heading towards the brainstem, a finding that indicates that the primary motor cortex also has an indirect access to the primate striatum providing this structure with a copy of the neuronal information that is being sent to the brainstem and/or spinal cord. Altogether, thèse data indicate that the main components of basai ganglia in primates harbour différent types of projection neurons, each endowed with a highly collateralized axons. In the light of thèse findings, the basai ganglia can now be viewed as a widely distributed neuronal network, whose éléments are endowed with a highly patterned set of axon collaterals. The understanding of this finely tuned network is a prerequisite for the development of new therapeutic avenues for the treatment of basai ganglia disorders, such as Parkinson's disease and Huntington's chorea.

AVANT-PROPOS Je tiens d'abord à remercier mon directeur de recherche, mon père André Parent, pour m'avoir inspiré ainsi que pour m'avoir laissé suivre ses traces. Son appui et son encouragement ont été indispensables à l'aboutissement de ce projet. Qu'il trouve ici l'expression de ma profonde admiration.

Les travaux présentés dans cette thèse s'insèrent dans un vaste ensemble de données neuroanatomiques amassées depuis plusieurs années dans notre laboratoire. En ce sens, ils ne constituent qu'une pièce d'un casse-tête entamé il y a bien longtemps. J'aimerais profiter de l'occasion pour remercier mes prédécesseurs, plus particulièrement ceux qui, il y a déjà quelques années, se sont intéressés aux même problématiques que moi. Merci au Dr Luc De Bellefeuilles pour le travail remarquable qu'il a réalisé concernant les efférences du pallidum interne. Ses travaux sont d'ailleurs résumés dans le deuxième chapitre de cette thèse. Merci aussi au Dr Abbas Sadikot pour le travail colossal qu'il a effectué sur les efférences du complexe thalamique centre médian/parafasciculaire.

La plupart des résultats expérimentaux présentés dans cette thèse ont fait l'objet d'articles originaux parus dans des revues de calibre international, avec comité de lecture. Étant premier auteur des cinq articles incorporés, je peux affirmer avoir accompli la majeure partie des travaux : rédaction des projets de recherche (objectifs, hypothèses), manipulations expérimentales, collectes et analyses de données, revues de littérature, préparation des illustrations et écriture des textes. Mon directeur de recherche a évidemment joué un rôle substantiel dans l'élaboration de ces travaux, particulièrement en ce qui a trait à la correction ainsi qu'à la mise en forme des articles scientifiques.

Le premier chapitre de la thèse contient une introduction générale qui permet de situer les différentes composantes associées aux ganglions de la base du primate. Les principales relations hodologiques entre ces structures sous corticales y sont présentées de même que les différentes techniques utilisées pour identifier ces connexions. De plus, ce chapitre

IV

contient une description détaillée de la problématique de recherche, des objectifs de recherches ainsi que de l'approche méthodologique utilisée.

Le deuxième chapitre de la thèse est constitué d'un article de revue publié en 1999 dans The Journal of Chemical Neuroanatomy. J'ai effectué une revue de littérature complète des données accumulées dans notre laboratoire concernant les efférences du pallidum interne. M. Martin Lévesque, deuxième auteur, m'a grandement aidé afin de produire les illustrations.

Le troisième chapitre de la thèse présente une version allongée d'un article publié dans la revue intitulée The Journal of Comparative Neurology en 2001. J'ai effectué la majeure partie des travaux ayant mené à cette publication. J'aimerais remercier M. Martin Lévesque et M. René Boucher pour m'avoir enseigné l'ensemble des techniques stéréotaxiques adaptées aux primates. J'aimerais aussi remercier Mme Carole Émond pour m'avoir assisté dans les procédures immunohistologiques.

Dans le quatrième chapitre, on retrouve un article publié en 2004 dans Parkinsonism and Related Disorders. Mon directeur de recherche et moi-même étant les seuls auteurs de cet article, je peux affirmer avoir réalisé, avec sa collaboration, l'essentiel du projet de recherche. Le cinquième chapitre de cette thèse présente un article publié dans la revue The Journal of Comparative Neurology en 2006. J'ai réalisé, sous la direction du Dr André Parent, l'essentiel du projet de recherche. J'aimerais remercier Dons Côté, Cyntia Tremblay et Lucie Pelchat pour l'aide technique apportée au cours des nombreuses procédures immunohistochimiques. J'aimerais aussi remercier M. Martin Lévesque, Mme Catherine Couture et Mme Cynthia Moore pour m'avoir assité lors des chirurgies stéréotaxiques.

Le sixième chapitre renferme un article publié en 2005 dans The Journal of Comparative Neurology. Avec l'aide technique prodiguée par les personnes mentionnées au paragraphe

précédent, je peux affirmer

avoir réalisé la majorité

du travail nécessaire à

l'accomplissement de ce projet de recherche. J'aimerais profiter de l'occasion afin de remercier le Dr Cameron Mclntyre du « Cleveland Clinic Foundation » en Ohio pour avoir accepté de collaborer avec moi dans un projet excitant faisant le pont entre les données neuroanatomiques fondamentales recueillies lors de ma formation doctorale et le traitement clinique de la maladie de Parkinson.

Dr Pierre Blanchet, Dr Frédéric Calon, Dr Zhong-Wei Zhang et Dr André Parent ont accepté d'évaluer la présente thèse en tant que membres du jury. Leur présence m'honore et je les remercie. En terminant, j'aimerais remercier mes compagnons de route : Philippe Lavallée, Martin Lévesque, Martine Cossette, Andréanne Bédard, Julie-Christine Lévesque et tous les autres. Merci pour tous les bons moments passés ensemble.

Les travaux présentés dans le cadre de cette thèse se sont déroulés au Centre de recherche Université Laval Robert-Giffard (CRULRG) situé à Beauport. Ils ont été réalisés grâce à l'obtention de deux bourses de recherche, l'une provenant du Conseil en recherches en sciences naturelles et en génie du Canada (CRSNG) et l'autre provenant du Fonds pour la formation de chercheurs et l'aide à la recherche (FCAR) conjointement avec le Fonds de recherche en santé du Québec (FRSQ).

À Marie-Ève, Gabriel, Emile et Juliette...

"La complexité ne donne pas de la valeur aux choses, elle les rend seulement moins accessibles" Faya Dequoy

TABLE DES MATIERES

RESUME i SUMMARY ii AVANT-PROPOS iii TABLE DES MATIÈRES viii LISTE DES FIGURES xii LISTE DES ABRÉVIATIONS xvi CHAPITRE 1 2 Introduction générale 1.1 Préambule 2 1.1.1 Présentation des chapitres 4 1.2 Les ganglions de la base 6 1.2.1 Le modèle anatomo-fonctionnel des ganglions de la base 12 1.3 Pathologies associées aux ganglions de la base 16 1.3.1 La maladie de Parkinson 16 1.3.1.1 Traitement de la maladie de Parkinson 20 1.3.2 La chorée de Huntington 25 1.3.2.1 Traitement de la chorée de Huntington 29 1.4 L'importance de la collatéralisation axonale au sein des ganglions de la base du primate 30 1.5 Le pallidum interne 32 1.5.1 Anatomie 32 1.5.2 Cytologie 33 1.5.3 Hodologie 38 1.5.3.1 Afférences pallidales 38 1.5.3.2 Efférences pallidales 39 1.6 Le cortex moteur primaire 41 1.6.1 Anatomie et organisation fonctionnelle 41 1.6.2 Cytologie 43 1.6.3 Hodologie 44 1.6.3.1 Afférences du cortex moteur primaire 44 1.6.3.2 Efférences du cortex moteur primaire 46 1.7 Le complexe thalamique centre médian/parafasciculaire 49 1.7.1 Anatomie topographique 49 1.7.2 Cytologie 51 1.7.3 Hodologie 56 1.7.3.1 Afférences du complexe centre médian/parafasciculaire 56 1.7.3.2 Efférences du complexe centre médian/parafasciculaire 58 1.8 Problématique de recherche 62 1.9 Objectifs de recherche 63 1.9.1 Objectifs généraux 63 1.9.2 Objectifs spécifiques 63 1.10 Approche méthodologique 64

IX

CHAPITRE 2

71

The pallidofugal projection System in primates: évidence for neurons branching ipsilaterally and contralaterally to the thalamus and brainstem 2.1 RÉSUMÉ 72 2.2 ABSTRACT 73 2.3 INTRODUCTION 74 2.4 MATERIALS AND METHODS 75 2.4.1 Fluorescence double rétrograde labeling experiments 75 2.4.2 Anterograde labeling experiments 76 2.4.3 Single-axon tracing experiments 77 2.5 RESULTS 78 2.5.1 Rétrograde tracer experiments: ipsilateral cell labeling 78 2.5.1.1 Thalamus (VA/VL) and habenula (HL) injections 78 2.5.1.2 Thalamus (VA/VL) and intralaminar nuclei (CM/Pf) injections 79 2.5.1.3 Thalamus (VA/VL) and brainstem (PPN) injections 79 2.5.1.4 Intralaminar nuclei (CM/Pf) and brainstem (PPN) injections 79 2.5.1.5 Substantia nigra (SN) injections 80 2.5.2 Rétrograde tracer experiments: bilatéral cell labeling 80 2.5.3 Anterograde tracer experiments: ipsilateral axonal labeling 81 2.5.4 Anterograde tracer experiments: axonal arborization 82 2.5.5 Anterograde tracer experiments: contralateral axonal labeling 83 2.5.6 Single-axon tracing experiments 84 2.6 DISCUSSION 84 2.6.1 Origin and collateralization of the pallidofugal projections 84 2.6.2 Contralateral pallidofugal projections 86 2.6.3 The reticular thalamic nucleus 88 2.6.4. Concluding remarks 89 CHAPITRE 3 104 Motor and limbic neurons in the internai pallidum of primates: single-axon tracing and three-dimensional reconstruction 3.1 RÉSUMÉ 105 3.2 ABSTRACT 106 3.3 INTRODUCTION 107 3.4 MATERIALS AND METHODS 108 3.4.1 Préparation of the animais 108 3.4.2 Anterograde labeling 109 3.4.2.1 Injection procédures 109 3.4.2.2 Tracer visualization and cytochrome oxidase staining 110 3.4.2.3 Calbindin immunohistochemistry 111 3.4.2.4 Material analysis 111 3.4.3 Rétrograde labeling 112 3.5 RESULTS 112 3.5.1 General labeling features 112 3.5.2 Axonal trajectory 114 3.5.2.1 General features 114 3.5.2.2 Type la neurons 115 3.5.2.3 Type Ib neurons 117

X

3.5.2.4 Type Ha neurons 3.5.2.5 Type Ilb neurons 3.5.3 Topographical distribution 3.6 DISCUSSION 3.6.1 The ansa lenticularis or the lenticular fasciculus 3.6.2 Pallidothalamic projection 3.6.3 Pallidointralaminar projection 3.6.4 Pallidotegmental projection 3.6.5 Pallidohabenular projection 3.6.6 Contralateral pallidofugal projections 3.6.7 Pallidal inputs to Forel's H field and zona incerta 3.6.8 Comparison with other basai ganglia components CHAPITRE 4 The pallidofugal motor fiber System in primates 4.1 RÉSUMÉ 4.2 ABSTRACT 4.3 INTRODUCTION 4.4 MATERIALS AND METHODS 4.4.1 Préparation of the animais 4.4.2 Injection procédures 4.4.3 Tracer révélation and cytochrome oxidase staining 4.4.4 Material analysis 4.5 RESULTS 4.6 DISCUSSION 4.6.1 Définition of motor and limbic pallidal neurons 4.6.2 Organization of the pallidofugal fiber system 4.6.3 Functional considérations CHAPITRE 5 Single-axon tracing study of corticostriatal projection arising from the primary motor cortex in primates 5.1 RÉSUMÉ 5.2 ABSTRACT 5.3 INTRODUCTION 5.4 MATERIALS AND METHODS 5.4.1 Injection procédures 5.4.2 Tracer visualization and cytochrome oxidase staining 5.4.3 Material analysis 5.5 RESULTS 5.5.1 General labeling features 5.5.2 Axonal trajectory 5.5.3 Contralateral corticostriatal projections 5.5.4 Corticosubthalamic and corticorubral projections 5.6 DISCUSSION 5.6.1 The corticostriatal projection: direct, indirect, orboth? 5.6.2 The corticostriatal projection: cellular origin and termination pattern 5.6.3 A comparison between corticostriatal and thalamostriatal projections 5.6.4 Corticosubthalamic and corticoclaustral projections

120 122 122 123 124 125 126 128 129 131 132 133 154 155 156 157 159 159 159 160 161 161 164 164 165 166 178 179 180 181 182 182 183 184 186 186 187 189 190 191 191 193 195 196

XI

5.6.5 Concluding remarks 198 CHAPITRE 6 208 Single-axon tracing and three-dimensional reconstruction of centre médianparafascicular thalamic neurons in primate 6.1 RÉSUMÉ 209 6.2 ABSTRACT 210 6.3 INTRODUCTION 211 6.4 MATERIALS AND METHODS 213 6.4.1 Préparation of the animais 213 6.4.2 Injection procédures 213 6.4.3 Tracer visualization and cytochrome oxidase staining 214 6.4.4 Material analysis 215 6.5 RESULTS 216 6.5.1 Nuclei délimitation and gênerai labeling features 216 6.5.2 Axonal trajectory 218 6.5.2.1 Centre médian thalamic nucleus 218 6.5.2.2 Parafascicular thalamic nucleus 221 6.5.3 Topographical distribution 222 6.6 DISCUSSION 222 6.6.1 Somatodendritic morphology 223 6.6.2 Thalamostriatal projections 223 6.6.3 Thalamocortical projections 226 6.6.4 Other CM/Pf projections 227 6.6.5 Functional considérations 228 CHAPITRE 7 250 Conclusions générales 7.1 Les axones pallidofuges et la microstimulation intracérébrale à haute fréquence 251 7.2 L'innervation glutamatergique striatale 256 7.3 Orientation spatiale des arborisations axonales et dendritiques 257 7.4 Différences interspécifiques 258 7.5 La ségrégation de l'information neuronale 259 7.6 Rôles fonctionnels de la collatéralisation axonale 261 7.6.1 Le concept de divergence 261 7.6.2 La plasticité 262 7.7 La collatéralisation axonale et la vulnérabilité face aux processus neurotoxiques et neurodégénératifs 262 7.8 Hypothèse d'un nombre maximal de varicosités axonales 263 7.9 Conclusions 265 LISTE DES OUVRAGES CITÉS 266 ANNEXE 1 291 Liste des contributions

LISTE DES FIGURES CHAPITRE 1 Figure 1.1

Les ganglions de la base

9

Figure 1.2

Territoires fonctionnels du striatum

11

Figure 1.3

Modèle des ganglions de la base en condition normale

15

Figure 1.4

Modèle des ganglions de la base en conditions pathologiques

19

Figure 1.5

Interventions neurochirurgicales afin d'atténuer les symptômes de la maladie de Parkinson

24

Figure 1.6

Caractéristique pathologique de la chorée de Huntington

28

Figure 1.7

Organisation somatodenritique des neurones du pallidum interne

35

Figure 1.8

Organisation en disque de l'arborisation dendritique des neurones du pallidum

37

Figure 1.9

Le thalamus selon Jules Bernard Luys

53

Figure 1.10

Organisation somatodenritique des neurones du complexe thalamique centre médian/parafasciculaire Organisation ultrastructurale des principales afférences des neurones épineux de taille moyenne du striatum La ventriculographie

Figure 1.11 Figure 1.12

55 61 67

CHAPITRE 2 Figure 2.1 Figure 2.2

Distribution and relative proportion of retrogradely labeled neurons in each of the five rétrograde double-labeling experiments

92

Distribution and relative proportion of retrogradely labeled neurons following double rétrograde tracer injections in the thalamus and brainstem

94

Xlll

Figure 2.3

Figure 2.4 Figure 2.5

Figure 2.6

Photomicrographs showing examples of singly and doubly labeled neurons disclosed after double rétrograde tracer injections in the thalamus and brainstem

96

Anterogradely labeled axons observed after PHA-L injections into theGPi

98

Photomicrographs of anterogradely labeled axons and terminal arborization observed ipsilaterally after PHA-L inj ection into the GPi

100

Reconstruction of a single axon of a GPi neuron that was microiontophoretically injected with BDA

102

CHAPITRE 3 Figure 3.1

Patterns of neural activity that characterizes the external and internai segment of the pallidum. Photomicrograph of an injection site in the GPi with two distinctly labeled neurons

136

Figure 3.2

Axon trajectories of type I neurons within the GPi

138

Figure 3.3

Axonal arborization of atype la GPi neuron

140

Figure 3.4

Axonal arborization of a type Ib GPi neuron

142

Figure 3.5

Photomicrographs of terminal arborization of GPi axons

144

Figure 3.6

Axonal arborization of a type Ha GPi neuron

146

Figure 3.7

Localization of GPi neurons whose axons were connected to their parent cell bodies

148

Distribution of retrogradely labeled neurons in GPi after injection into the HL

150

Typical columnar arrangement of the axonal arborizations of GPi and GPe neurons

152

Figure 3.8 Figure 3.9

XIV

CHAPITRE 4 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4

Axonal arborization of a typical GPi motor neuron. Photomicrographs of Golgi-like labeled neurons in the GPi

170

Terminal axonal arborization of an individually labeled GPi motor neuron

172

Three-dimensional reconstruction of the initial axonal trajectory of four GPi motor neurons

174

Current model of the organization of the pallidofugal fïber compared with a revised view based on the data obtained in the présent study

176

CHAPITRE 5 Figure 5.1 Figure 5.2

Figure 5.3

Figure 5.4

Injection sites and Golgi-like labeled neurons in the primary motor cortex

200

Reconstructions of single BDA-labeled Ml neurons projecting directly to the putamen

202

Reconstruction of a Ml neuron that projects to the striatum via a thin collatéral emitted in the corona radiata by a thick long-range axon that courses toward the brainstem

204

Reconstructions of axonal branches that cross the midline by passing through the corpus callosum and innervate the contralateral putamen. Photomicrograph of terminal arborization in the dorsolateral part of the putamen 206

CHAPITRE 6 Figure 6.1 Figure 6.2

Patterns of neural activity that characterizes the central latéral nucleus and the centre médian nucleus

231

BDA injection site and Golgi-like BDA-labeled neurons in the primate CM nucleus

233

Figure 6.3

Reconstruction of a single BDA-labeled CM neuron that projects densely to the latéral sector of the putamen, but not to the cortex. Photomicrograph of dense terminal fields in the putamen 235

Figure 6.4

Three-dimensional reconstruction of a CM neuron that projects only to the striatum

237

XV

Figure 6.5

Three-dimensional reconstruction of a CM neuron that projects to motor cortex, but not to the striatum

239

Figure 6.6

Caméra lucida drawing of a neuron whose axon projects to motor cortex and to the reticular thalamic nucleus, but not to the striatum 241

Figure 6.7

Caméra lucida reconstructions of two labeled CM neurons projecting to striatum and cortex

243

Figure 6.8

Three-dimensional reconstruction of a CM neuron that projects to both the dorsolateral sector of the putamen and the cérébral cortex 245

Figure 6.9

Caméra lucida reconstructions on sagittal plane of two BDA-labeled Pf neurons, one projecting to the striatum only and the other to both the striatum and the cérébral cortex

Figure 6.10

247

Localization of neurons within the CM/Pf complex, the axon of which was connected to parent cell body 249

CHAPITRE 7 Figure 7.1

Modèle informatisé de l'effet d'une stimulation intracérébrale à haute fréquence du noyau subthalamique sur les axones pallidofuges et les neurones du noyau subthalamique

255

LISTE DES ABREVIATIONS

ABC: AC: ACh: ad: AL: am: ami: as: AS: av: BDA: C: CA: CB: ce: CD: CI: CL: CM: CP: CX: DA: DAB: DBS: DP: DSP: DYN: EB: eml: EN: ENK: FB: FH: FR: FX: GABA: Glu: GP: GPe: GPi: GPi-1: GPi-m: HL: HM: HRP: HY: Hl : H2: IC: iml:

complexe avidine-biotine-peroxidase commissure antérieure acétylcholine noyau atérodorsal du thalamus anse lenticulaire noyau antéromédian du thalamus lame médullaire accessoire territoire associatif aqueduc cérébral (de Sylvius) noyau antéroventral du thalamus biotine dextran aminé cervelet noyau central de l'amygdale calbindine D-28K corps calleux noyau caudé claustrum noyau central latéral du thalamus noyau centre médian du thalamus pédoncule cérébral cortex cérébral dopamine 3,3'-diaminobenzidine tetrahydrochloride deep brain stimulation dapi-primuline décussation des pédoncules cérébelleux supérieurs dynorphine evans blue lame médullaire externe noyau entopédonculaire enképhaline fastblue champ H de Forel fasciculus retroflexus fornix acide y-aminobutyrique glutamate globus pallidus globus pallidus, segment externe globus pallidus, segment interne portion latérale du GPi portion médiane du GPi habenula, noyau latéral habenula, noyau médian horseradish peroxidase hypothalamus faisceau lenticulaire, champ H2 de Forel faisceau lenticulaire capsule interne lame médullaire interne

XV11

LD: LF: LG: LH: li: LO: LP: MD: MLF: MPTP: Ml : NA: NRS: NY: OT: OTU: PB: PBS: Pf: PH: PHA-L: pm: PO: PPN: Pul: PUT: R: RRA: se: SCP: SI: sm: smt: SN: SNc: SNr: SP: STN: STR: TB: TBS: th: VA: VAdc : VApc: VL: VLo: VM: vp: VP: VPi: VPm: wm: Zi: III:

noyau latérodorsal du thalamus faisceau lenticulaire, champ H2 de Forel corps genouillé latéral aire hypothalamique latérale territoire limbique du striatum noyau lateralis oralis du thalamus noyau latéropostérieur du thalamus noyau médiodorsal du thalamus faisceau longitudinal médian l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine cortex moteur primaire noyau accumbens normal rabbit sérum nuclear yellow tractus optique tubercule olfactif tampon phosphate tampon phosphate salin noyau parafascicualaire du thalamus noyau paraventriculaire de l'hypothalamus Phaseolus vu/gflrà-leucoagglutinin cortex prémoteur noyaux pontins noyau pédonculopontin du tegmentum mésencéphalique pulvinar putamen noyau réticulaire du thalamus aire rétrorubrale collicule supérieur pédoncule cérébelleux supérieur substantia innominata territoire sensorimoteur strie médullaire thalamique substance noire pars compacta de la SN pars reticulata de la SN substance P noyau subthalamique striatum true blue tampon tris salin thalamus noyau ventral antérieur du thalamus noyau ventral antérieur du thalamus, partie densicellulaire noyau ventral antérieur du thalamus, partie parvicellulaire noyau ventral latéral du thalamus noyau ventral latéral du thalamus, pars oralis noyau ventromédian du thalamus pallidum ventral noyau ventral postérieur du thalamus noyau ventral postéroinférieur du thalamus noyau ventral postéromédian du thalamus substance blanche sous-corticale zona incerta troisième ventricule

CHAPITRE 1 INTRODUCTION GENERALE

CHAPITRE 1

1.1

Préambule

Les années que j'ai passé au Centre de recherche Université Laval Robert-Giffard à étudier l'organisation anatomique des ganglions de la base des primates m'ont permis de me rendre à l'évidence : la complexité des connexions anatomiques sur lesquelles reposent les interactions entre les différentes composantes de ces structures occupant la base des hémisphères cérébraux dépasse presque l'entendement humain. Il existe quand même un espoir d'en arriver à une meilleure compréhension de l'organisation anatomique et fonctionnelle de cet ensemble de structures qui jouent un rôle important dans le contrôle du comportement moteur et cet espoir est généré, entre autres, par les études hodologiques, comme celles présentées dans cette thèse. Ce type d'analyse utilise des approches de plus en plus puissantes afin de décoder le substratum anatomique sur lequel reposent l'intégration et l'acheminement de l'information neuronale au sein des ganglions de la base. La connaissance que génèrent de telles études morphologiques est essentielle à une meilleure compréhension du fonctionnement des ganglions de la base en condition normale ou pathologique.

Depuis toujours, les méthodes de fixation et de coloration ont été des facteurs limitants à l'étude morphologique du tissu cérébral. Après les balbutiements du XVIIIe siècle, les procédures ont commencé à être standardisées au début du XIXe siècle. À l'époque, l'une des méthodes de coloration les plus répandues faisait usage du carmin, un colorant d'un rouge éclatant extrait de certains insectes. L'anatomiste allemand Joseph von Gerlach (1820-1886) fut le premier à utiliser cette substance pour colorer le tissu nerveux. En 1873, l'Italien Camillo Golgi (1843-1926) mit au point une méthode de coloration à l'argent permettant d'imprégner complètement mais de façon non spécifique les cellules nerveuses. Sa découverte fut résumée dans un article intitulé Sulla struttura délia grigia del

cervello (De la structure de la matière grise du cerveau) qui parut dans la Gazetta Medica Italiana Lombardia (Golgi, 1873). La morphologie des neurones y est présentée avec une clarté remarquable; Golgi parle du corps cellulaire et de ses prolongements, en spécifiant bien qu'il n'existe qu'un seul axone (cylindraxe) mais plusieurs dendrites ramifiées. Il mentionne même, et ce pour la première fois, l'existence de collatérales axonales, mais il ne les illustre pas. Le célèbre Santiago Ramôn y Cajal (1852-1934) allait le faire dans son remarquable traité intitulé Histologie du système nerveux (1909-1911) qui contient un très grand nombre de données obtenues grâce à la méthode de Golgi. Inspiré par la méthode de Golgi, l'Italien Vittorio Marchi (1851-1908) développa, à la fin du XIXe siècle, une procédure faisant usage de l'imprégnation par l'argent pour marquer les fibres nerveuses myélinisées en dégénérescence suite à des lésions cérébrales; il s'agissait là de la toute première méthode pour tracer les faisceaux nerveux dans le système nerveux central (Marchi et Algeri, 1885-1886). L'idée d'utiliser l'imprégnation argentique pour marquer les fibres nerveuses en dégénérescence donna origine à de nombreuses variantes méthodologiques et cette approche demeura en usage jusqu'au milieu du XXe siècle (Nauta et Gygax, 1954; Albrecht et Fernstrom, 1959; Alksne et al, 1966; Fink et Heimer, 1967). Au cours de cette même période on découvrait l'existence du flot axonal ainsi que la possibilité d'utiliser des traceurs radioactifs pour marquer les fibres nerveuses (Droz et Leblond, 1963). La découverte du flot axonal permit la mise sur pied de différentes méthodes afin d'étudier le trajet des fibres nerveuses sans avoir recours à des lésions. On procéda d'abord à l'injection, dans des régions circonscrites du cerveau, d'acides aminés tritiés pouvant migrer antérogradement vers les terminaisons axonales et être visualisés par autoradiographie; cette approche permit de décrire des voies neuronales dont l'existence était, jusqu'alors, insoupçonnée. On poursuivit avec l'injection massive de divers traceurs antérogrades et rétrogrades, fluorescents ou non, qui se sont avérés des outils fort utiles pour étudier, entre autres, les connexions entre les différentes composantes des ganglions de la base. Cependant, la possibilité de marquer des fibres de passage ainsi que la trop grande quantité d'axones marqués rendant impossible l'étude de la microcircuiterie au niveau neuronal unitaire constituent les principales limites de ces techniques.

Les travaux présentés dans cette thèse avaient comme objectif initial l'approfondissement de notre connaissance de la microcircuiterie des ganglions de la base chez le primate en utilisant

une

méthode

originale

d'analyse

quantitative

et

de

reconstruction

tridimensionnelle des arborisations axonales et dendntiques de neurones individuels dont l'activité élelctrophysiologique a été enregistrée au préalable. À notre grand étonnement, la mise en application de cette technique de pointe à l'étude du cerveau des primates a considérablement changé notre façon de voir l'organisation anatomo-fonctionnelle des ganglions de la base. Des reconstructions neuronales individuelles, complètes et détaillées ont permis de caractériser de façon précise les types neuronaux présents dans certaines composantes des ganglions de la base. Ces reconstructions ont révélé la présence de neurones

de projection

dont

l'axone

est fortement

collatéralisé,

un

caractère

neuromorphologique qui permet à un même neurone d'acheminer une copie de l'information neuronale qu'il traite à plusieurs structures cibles.

1.1.1

Présentation des chapitres

Le premier volet de mon doctorat a été dédié à l'étude des projections pallidofuges chez le primate et les résultats obtenus sont consignés, sous forme d'articles scientifiques déjà publiés, dans les deuxième, troisième et quatrième chapitres de la présente thèse. Le deuxième chapitre renferme une revue des données ayant trait aux projections efférentes du pallidum interne chez le singe écureuil, données obtenues principalement suite à l'injection combinée de traceurs rétrogrades dans les cibles potentielles des neurones pallidofuges. Cette technique a permis d'apporter des évidences indirectes quant à la nature hautement collatéralisée des axones des neurones du pallidum interne

ainsi que la présence de

nombreuses projections controlatérales. Le troisième chapitre résume les résultats d'une étude exhaustive par marquage unitaire antérograde des neurones pallidaux, étude qui apporte des évidences directes et définitives quant au fait que ces neurones sont pourvus d'un axone hautement collatéralisé. On y retrouve, en plus, le traçage détaillé du trajet axonal emprunté par les différents types neuronaux rencontrés dans le pallidum interne du singe cynomolgus. Le quatrième chapitre traite du trajet initial des axones pallidofuges. La

réalisation de ce travail s'imposait afin de faire la lumière sur la controverse concernant le trajet tortueux et complexe des axones pallidofuges par rapport à deux faisceaux de fibres majeurs, soit l'anse lenticulaire et le faisceau lenticulaire. Ces données se sont d'ailleurs avérées fort utiles à la compréhension de l'effet des lésions ou stimulations intracérébrales à haute fréquence visant à atténuer les symptômes neurologiques de différentes pathologies liées aux ganglions de la base. Le deuxième volet de mon doctorat à été consacré à l'étude des

afférences

glutamatergiques de la principale structure d'entrée des ganglions de la base, soit le striatum, chez le primate. Les résultats de ces travaux sont à nouveau consignés sous forme d'articles scientifiques, un étant déjà publié et l'autre sous-presse, et constituent les cinquième et sixième chapitres de la présente thèse. Le cinquième chapitre résume une étude détaillée par traçage unitaire des projections corticostriées en provenance du cortex moteur primaire réalisée chez le singe cynomolgus. Ces données révèlent que, contrairement à ce que l'on croyait, il n'y a pas qu'un seul type de projection corticostriée strictement dédiée au striatum chez le singe. Nous avons démontré qu'il existe deux types distincts de neurones moteurs corticaux projetant au striatum, l'un innervant uniquement le striatum et l'autre projetant au striatum par le biais d'une collatérale axonale émise par un axone se dirigeant vers le tronc cérébral. Ces résultats signifient que le cortex moteur primaire des primates influence le striatum de façon directe et indirecte. Le sixième chapitre est formé d'un article dédié à l'organisation neuroanatomique des projections thalamostriées en provenance du complexe mtralaminaire centre médian/parafasciculaire chez le singe écureuil. La visualisation tridimensionnelle des neurones de ce complexe montre clairement que certains d'entre eux sont en mesure d'influencer à la fois le cortex cérébral et le striatum par le biais d'un jeu de collatérale axonale complexe.

Les expérimentations décrites dans cette thèse ont permis d'amasser plusieurs données neuroanatomiques essentielles à la compréhension du fonctionnement des ganglions de la base chez le singe. Ces expériences ont été réalisées à la fois chez des primates de l'Ancien Monde, soit le singe cynomolgus (Macaca fascicularis) et chez des primates du Nouveau Monde, soit le singe écureuil {Saimiri sciureus). Rappelons que, de par sa proximité

phylogénétique avec l'homme, le singe représente un modèle animal très attrayant étant donné que son organisation cérébrale est semblable à celle de l'homme, tout en n'étant pas identique à celle-ci puisque chaque espèce simienne a ses propres spécificités neuromorphologiques. Il existe des différences significatives entre les rongeurs et les primates en ce qui a trait à l'organisation neuranatomique des ganglions de la base. Ces variations seront exposées dans les articles qui forment le cœur de la présente thèse.

1.2

Les ganglions de la base

C'est à Thomas Willis (1621-1675) que l'on doit la première description du « corpus striatum » (corps cannelé ou corps strié). Dans Cerebri anatome (Willis, 1664), Willis note pour la première fois l'existence de structures sous-corticales enfouies dans la partie antérieure des hémisphères cérébraux et dont la morphologie se caractérise par une alternance de stries de matière blanche et de matière grise. Étant donné que ces cannelures semblaient uniques à ces structures, il les engloba sous le nom de corps strié. Il attribua même, et ajuste titre, un rôle moteur à ces structures. Pour Willis et ses contemporains, la notion de corps strié incluait le thalamus (ou corps opto-strié) et c'est Vicq d'Azyr (17481794) en 1786 qui, le premier, réalisa que le striatum et le thalamus n'appartiennent pas à la même entité anatomique. À l'époque, le terme « ganglion basai » désignait un amas de neurones correspondant à l'ébauche du striatum chez les non-mammifères (Déjerine, 1901), « basai » signifiant hémisphère sous-cortical. Le nom anglais « basai ganglia » est une traduction du terme allemand «basai ganglionen » que nous devons à l'Écossais David Ferrier (1843-1928) (Ferrier, 1876). Notons que le terme « ganglions de la base » n'est pas l'équivalence exacte de l'expression «noyaux gris centraux» (Foix et Nicolesco, 1925) puisque cette dernière inclut le thalamus.

Ainsi, le terme « ganglions de la base », tel qu'on l'utilise aujourd'hui, réfère à un ensemble de structures sous-corticales qui jouent un rôle crucial dans le comportement psychomoteur. C'est en étroite collaboration avec le cortex cérébral que les ganglions de la

base exercent leur action sur la motricité. N'ayant aucun accès direct aux motoneurones spinaux, les ganglions de la base influencent le comportement moteur en agissant principalement sur les neurones prémoteurs du thalamus et du tronc cérébral. Ils sont donc reliés en boucle avec le cortex cérébral via un relais dans le thalamus. Chez les primates, l'axe principal de cette boucle est formé d'une série d'éléments dont l'arrangement séquentiel est le suivant: (1) le striatum, comprenant le noyau caudé, le putamen et le noyau accumbens ou striatum ventral, (2) le pallidum ou globus pallidus, comprenant un segment externe, un segment interne (GPi) et une région ventrale, (3) la substance noire (ou locus niger), comprenant une partie compacte (SNc) et une partie réticulée (SNr), et (4) les noyaux ventrolatéraux du thalamus, dont les neurones prémoteurs acheminent l'information ayant été traitée par les ganglions de la base vers le cortex cérébral (Parent, 1990; Parent, 1996) (Fig. 1.1).

L'activité de chacune des composantes des ganglions de la base est modulée par plusieurs structures qui sont situées en marge de l'axe principal et qui fournissent des entrées neurochimiques variées pouvant bloquer ou faciliter le flot de l'information neuronale le long des ganglions de la base. Parmi ces structures ancillaires, notons: (1) le noyau subthalamique (STN), (2) la SNc, (3) le complexe centre médian/parafasciculaire (CM/Pf) du thalamus, (4) le noyau dorsal du raphé, et (5) le noyau pédonculopontin (PPN) (Fig. 1.1). Le striatum constitue la "porte d'entrée" des ganglions de la base.

Ses principales

afférences proviennent de l'ensemble du cortex cérébral, du complexe thalamique CM/Pf et de la SNc. Les projections corticostriées sont massives et imposent au striatum une subdivision fonctionnelle qui, selon certains auteurs (Alexander et al., 1986; Alexander et Crutcher, 1990; Sidibé et al., 1997; Baron et al., 2001), serait maintenue tout au long de l'axe principal des ganglions de la base. Cette hypothèse de traitement de l'information neuronale en parallèle implique que les afférences striatales en provenance du cortex cérébral soit traitées indépendamment, via des circuits parallèles et ségrégés. Quoi qu'il en soit, sur la base de ses afférences corticales, le striatum peut être divisé en territoire associatif, sensorimoteur et limbique (Fig. 1.2). Le territoire associatif comprend principalement la tête du noyau caudé ainsi que le putamen pré-commissural; son

Figure 1.1

Section transversale d'un cerveau humain à un niveau postérieur à la commissure antérieure permettant l'identification de certaines structures composant ou étant étroitement associées aux ganglions de la base. (Nieuwenhuys et al., 1981)

Figure 1.2 Localisation des territoires associatif (as), sensorimoteur (sm) et limbique (li) du striatum chez le primate. Les schémas représentent des sections transversales placées selon un ordre rostro-caudal. Les régions doublement hachurées représentent des zones de chevauchement entre les différents territoires fonctionnels du striatum. (Parent, 1996)

10

B

as

sm

11

li

12 innervation corticale origine surtout des aires corticales de type associatif. Le territoire sensorimoteur comprend la majeure partie du putamen post-commissural, la partie dorsolatérale de la tête du noyau caudé ainsi que la partie latérale du corps du noyau caudé et, comme son nom l'indique, il est essentiellement innervé par les zones corticales motrices et sensorielles. Quant à lui, le territoire limbique comprend le striatum ventral dont la partie la mieux individualisée est le noyau accumbens; il est innervé par les zones corticales limbiques et paralimbiques (Parent, 1990). Puisqu'un chevauchement significatif existe entre les différents territoires fonctionnels décrits plus haut, il serait plus sage de voir ces territoires comme un continuum fonctionnel plutôt que comme des subdivisions comportant des limites strictes. Les neurones striataux situés dans chacun de ces territoires fonctionnels distincts jouent un rôle complémentaire en ce qui a trait à l'acte moteur. Les neurones situés dans le territoire sensori-moteur exercent une action directe sur l'exécution du mouvement, alors que ceux localisés dans le territoire associatif participent à la planification et à l'anticipation de l'acte moteur. Finalement, les neurones striataux situés dans le territoire limbique semblent davantage impliqués dans la gestion des processus motivationnels et émotionnels associés à l'acte moteur (Parent et Hazrati, 1995a).

Les structures de sortie des ganglions de la base sont la SNr et le GPi. Ces structures exercent une influence inhibitrice médiée par l'acide y-aminobutyrique (GABA) sur les neurones thalamocorticaux glutamatergiques situés dans le tiers ventral du thalamus. Elles inhibent aussi certaines structures du tronc cérébral, dont les collicules supérieurs et le PPN.

1.2.1

Le modèle anatomo-fonctionnel des ganglions de la base

Suite à de nombreuses études effectuées à l'aide de modèles animaux et de patients atteints de maladies impliquant des troubles du mouvement, on a proposé, dans les années 1980, un modèle pour expliquer l'organisation anatomique et fonctionnelle des ganglions de la base (Penney et Young, 1983; Alexander et al., 1986; Albin et al., 1989; DeLong, 1990). Ce modèle repose sur la ségrégation de l'information neuronale en provenance du striatum. En

13 effet, on suppose que les neurones qui composent cette structure projettent directement et indirectement aux structures de sortie des ganglions de la base (Fig. 1.3). La projection directe origine d'une population neuronale contenant le neurotransmetteur GABA ainsi que les neuropeptides substance P (SP) et/ou dynorphine (DYN). Dans ce cas, les neurones ciblent de façon monosynaptique le GPi et la SNr. L'information neuronale est ensuite relayée aux neurones prémoteurs situés dans le tiers ventral du thalamus qui projettent au cortex frontal. Quant à elle, la voie indirecte origine d'une population de neurones striataux qui contiennent le GABA et l'enképhaline (ENK). Dans ce cas, l'information est relayée de façon polysynaptique aux structures de sortie des ganglions de la base via (1) un relais dans le segment externe du pallidum et (2) un autre dans le STN. Les efférences du segment externe du pallidum sont inhibitrices alors que celles du STN sont excitatrices.

Suivant ce modèle, la compréhension du fonctionnement des ganglions de la base repose sur le concept de désinhibition (Chevalier et Deniau, 1990) qui implique l'existence d'une chaîne disynaptique formée de deux éléments neuronaux reliés en série et utilisant tous deux le GABA comme neurotransmetteur inhibiteur. C'est par ce mécanisme de désinhibition que le striatum influence les neurones prémoteurs thalamocorticaux. En effet, en utilisant la voie directe, les neurones GABAergiques striataux de projection peuvent inhiber les neurones GABAergiques pallido- et nigro-thalamiques qui cesseront alors d'inhiber les neurones thalamocorticaux. En somme, l'activation de la voie directe désinhibe l'activité thalamique et exerce donc un effet excitateur sur le cortex. Ce processus de désinhibition ne produit pas, en lui-même, une excitation. Il sert simplement à libérer les neurones thalamocorticaux de l'hyperpolarisation tonique qu'exercent sur eux les neurones du GPi et la SNr, permettant ainsi à de nombreuses entrées excitatrices d'exercer un effet significatif. Lorsque les neurones striataux sont silencieux, ce qui est le cas environ 90% du temps, les circuits thalamocorticaux prémoteurs sont maintenus sous une contrainte inhibitrice qui les protège contre tout bruit neuronal incongru. En revanche, lorsque les neurones striataux de projection s'activent, soit lors de la planification d'un acte moteur, les circuits prémoteurs thalamocorticaux deviennent libres de répondre à toutes entrées excitatrices reliées au mouvement devant être exécuté.

Figure 1.3 Diagramme décrivant le modèle des ganglions de la base qui est couramment utilisé dans la littérature afin d'expliquer l'organisation anatomo-fonctionnelle de ces structures souscorticales en condition normale. Les flèches rouges et bleues représentent respectivement la voie directe et indirecte.

14

Voie directe Voie indirecte SNr

15

16

En agissant via la voie indirecte, le striatum peut exercer une influence inverse sur les structures de sortie des ganglions de la base. Dans ce cas, les neurones GABAergiques striataux à l'origine de cette voie inhibent les neurones inhibiteurs du segment externe du pallidum ce qui entraîne la désinhibition des neurones du STN.

Les neurones

glutamatergiques du STN augmentent alors leur effet excitateur sur les neurones GABAergiques du GPi et de la SNr qui, à leur tour, inhiberont davantage les neurones thalamocorticaux prémoteurs. L'activation de la voie indirecte aura donc un effet dépresseur sur l'activité corticale.

Ainsi, selon ce modèle, l'information neuronale traitée dans la boucle cortico-ganglions de la base-thalamo-corticale peut avoir un effet opposé selon que les neurones striataux recevant et intégrant cette information font partie de la voie stnatofuge directe ou indirecte. On présume que le fonctionnement harmonieux des ganglions de la base repose sur un équilibre fragile entre ces deux voies.

1.3 Pathologies associées aux ganglions de la base 1.3.1

La maladie de Parkinson

Le modèle décrit à la section précédente est devenu la référence ultime afin d'interpréter le fonctionnement des ganglions de la base tant en condition normale que pathologique. D'ailleurs, ce modèle est largement utilisé pour expliquer la maladie de Parkinson ainsi que les façons d'intervenir sur ce système afin d'en atténuer les symptômes. Le diagnostique clinique de cette pathologie neurodégénérative repose sur l'identification

d'une

combinaison de symptômes principalement moteurs dont (1) une pauvreté du mouvement (hypokinésie) voire même l'impossibilité d'initier certains mouvements (akinésie), (2) un tremblement au repos, (3) une rigidité musculaire, ainsi que, dans les stades avancés de la maladie, (4) des troubles posturaux de la démarche accompagnés de déficits cognitifs. Bien que les causes primaires de la maladie de Parkinson idiopathique soient encore inconnues, on sait que les principaux symptômes moteurs se manifestent suite à la dégénérescence

17

massive des neurones nigrostriés dopaminergiques localisés dans le tiers ventral de la SNc. Cette mort neuronale provoque une baisse du niveau de dopamine dans le striatum ce qui entraîne un déséquilibre entre la voie directe et indirecte. Il faut mentionner ici que, selon le modèle, la dopamine agit de façon opposée sur les deux populations striatales décrites plus haut. En effet, elle excite faiblement les neurones GABA SP/DYN à l'origine de la voie directe alors qu'elle inhibe fortement les neurones striataux GABA/ENK à l'origine de la voie indirecte. Ce phénomène serait dû à la présence de deux récepteurs dopaminergiques différents, soit les récepteurs Di exprimés par les neurones GABA SP/DYN et les récepteurs D2 exprimés par les neurones GABA/ENK. Dans la maladie de Parkinson, l'inhibition normalement exercée par la dopamine sur les neurones striataux GABA/ENK est perdue. L'augmentation de l'activité de ces derniers leur permet donc d'inhiber davantage les neurones GABAergiques du segment externe du pallidum qui, à leur tour, cessent d'inhiber le STN. Ce dernier devient alors hyperactif et excite fortement les neurones GABAergiques des structures de sortie des ganglions de la base (SNr et GPi). Les neurones du GPi et de la SNr augmentent alors leur contraintes inhibitrices sur les cellules glutamatergiques du thalamus moteur. D'autre part, les neurones GABA SP/DYN à l'origine de la voie directe sont moins excités suite à la diminution de la dopamine dans le striatum. L'inhibition qu'exercent ces neurones striataux sur le GPi et la SNr diminue et ces derniers deviennent hyperactifs. Donc, les neurones GABAergiques du GPi et de la SNr augmentent davantage la contrainte inhibitrice qu'ils exercent sur les neurones thalamocorticaux. La conséquence de cette cascade d'événements est une diminution marquée de l'action excitatrice des neurones thalamocorticaux sur les neurones du cortex moteur impliqués dans l'exécution du mouvement (Fig. 1.4 A). Ceci permet d'expliquer, de façon relativement simple, la bradykinésie observée dans la maladie de Parkinson.

Figure 1.4 Diagramme décrivant le modèle des ganglions de la base qui est couramment utilisé dans la littérature afin d'expliquer l'organisation anatomo-fonctionnelle de ces structures souscorticales en conditions pathologiques : A, la maladie de Parkinson et B, la chorée de Huntington. Les flèches vertes et rouges désignent respectivement les projections excitatrices et inhibitrices. Les voies hyperactives sont représentées par des flèches plus épaisses alors que les voies hypoactives sont indiquées par des lignes pointillées.

18

maladie de Parkinson cortex Glu

;GIU

STR GABA ENK

GABA SP et DYN

GABAI

GABA

thalamus

SNc TGABA

SNr Projections excitatrices

TGlu

GABA,

STN

Projections inhibitrices

maladie de Huntington Glu

GABA

GABA

19

20

1.3.1.1

Traitement de la maladie de Parkinson

Bien qu'il n'existe actuellement aucun traitement curatif, plusieurs traitements palliatifs permettent d'atténuer les symptômes de la maladie de Parkinson. Dès les années 1950, les neurochirurgiens se sont attaqués au tremblement, un symptôme handicapant de la maladie. Utilisant une approche stéréotaxique, les neurochirurgiens ont pu démontrer que la destruction par électrocoagulation de structures nerveuses clés atténuait certains symptômes de la maladie de Parkinson (Hassler et Reichert, 1954) (Fig. 1.5 A). Ces interventions stéréotaxiques ont révélé qu'une destruction partielle ou totale du noyau ventral intermédiaire du thalamus réduisait de façon significative le tremblement. En revanche, l'akinésie et la rigidité musculaire n'étaient pas améliorées par une telle lésion, ni par une destruction

du

GPi.

Quelques

années

plus

tard,

l'avènement

d'une

thérapie

pharmacologique de remplacement sonna le glas de l'approche neurochirurgicale stéréotaxique pour le traitement de la maladie de Parkinson.

C'est au début des années 1960 que jaillit l'idée de suppléer au manque de dopamine cérébrale par l'ajout d'un précurseur métabolique, la lévodopa. Suite à l'administration chronique de fortes doses de lévodopa par voie orale, des résultats convaincants furent obtenus (Cotzias et al., 1967) et la « dopathérapie » s'est imposée comme traitement de choix pour la maladie de Parkinson. Cependant, très tôt, les cliniciens ont noté de nombreux effets secondaires associés à l'administration de ce précurseur, comme les dyskinésies et les fluctuations motrices caractérisées par des périodes dites « on-off » (Cotzias et al., 1969; Barbeau et al., 1971; Barbeau, 1975). Un traitement prolongé à la lévodopa peut engendrer, d'une part, un retour subit à un état rigide et akinétique dans les périodes « off » et, d'autre part, un rétablissement difficile de la motricité normale suite à la prise de lévodopa dans les périodes « on » et ce, à cause de l'amplitude, de la sévérité et de la violence des dyskinésies (Obeso et al., 2000). La nécessité d'un nouveau traitement s'imposait.

La stimulation intracérébrale fut introduite par le chirurgien américain Robert Galbraith Heath (1915-1999) au cours des années 1950 afin de traiter certains troubles psychiatriques

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(Heath, 1963). Cette technique fut ensuite appliquée aux problèmes de douleur chronique. En 1987 deux groupes de cliniciens, l'un à Grenoble (Benabid et al, 1987) et l'autre à Zurich (Siegfried et Pamir, 1987) utilisèrent la stimulation chronique du noyau thalamique ventral intermédiaire pour supprimer de façon permanente le tremblement. En effectuant des lésions électrolytiques du thalamus (thalamotomies), ces neurochirurgiens ont découvert qu'une stimulation électrique à basse fréquence du noyau ventral intermédiaire du thalamus provoquait un tremblement parkinsonien alors qu'une stimulation à haute fréquence de ce même noyau abolissait le tremblement. La méthode de stimulation intracérébrale chronique à haute fréquence a, depuis lors, pris un essor considérable en tant que stratégie alternative aux chirurgies d'ablation.

L'activité anormale de certaines structures nerveuses résultant du manque de dopamine peut être corrigée par une intervention neurochirurgicale visant trois cibles principales, soit le STN, le GPi et le noyau ventral intermédiaire du thalamus. Les symptômes qui répondent le mieux à la stimulation du STN ou du GPi sont le tremblement, la rigidité musculaire, la bradykinésie et les dyskinésies induites par la lévodopa. Dans certains cas, on obtient aussi une réduction appréciable des problèmes posturaux. Cependant, lorsqu'ils sont présents, les symptômes cognitifs ne répondent habituellement pas à la stimulation à haute fréquence et peuvent même s'aggraver. Si la stimulation du GPi permet d'atténuer les dyskinésies, elle améliore relativement peu les fluctuations motrices (phénomène «on-off»). De plus, contrairement à la stimulation du STN, la dopathérapie doit être maintenue suite à la stimulation chronique du GPi. Pour sa part, la stimulation thalamique n'améliore pas l'akinésie et la rigidité musculaire; elle n'est envisagée que dans les cas où le tremblement prévaut sur les autres symptômes (Benabid et al., 1987). D'après les données cliniques actuelles, le STN semble la cible de choix pour traiter la majorité des patients qui souffrent de la maladie de Parkinson (Limousin et al., 1998).

Lors de la descente intracérébrale des microélectrodes, on procède habituellement à des enregistrements électrophysiologiques ainsi qu'à des stimulations afin de bien délimiter la structure dans laquelle la microélectrode sera implantée en permanence. La radiographie et l'imagerie par résonance magnétique permettent de déterminer avec précision la position de

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la microélectrode à l'intérieur du cerveau du patient (Fig. 1.5 B, C). Lorsque l'électrode a atteint la cible visée et que le stimulateur entre en fonction, la rigidité musculaire diminue brusquement et de façon significative. On utilise habituellement une microélectrode à 4 points de contact qui peuvent être de polarité positive ou négative. La stimulation peut être monopolaire ou multipolaire. Les paramètres de stimulation utilisés sont de l'ordre de 130 à 190 Hz et de 2 à 4 V. La microélectrode étant implantée en permanence, le neurochirurgien peut ajuster ces paramètres à tout moment grâce à l'implantation du stimulateur électrique programmable dans la poche sous-cutanée infra-claviculaire du patient (Fig. 1.5 D). L'ajustement des paramètres de stimulation permet de minimiser les effets secondaires et de maximiser les bienfaits sur les symptômes moteurs de la maladie.

Le mécanisme d'action de la stimulation intracérébrale chronique demeure nébuleux. L'hypothèse généralement retenue suppose que la stimulation à haute fréquence produit une inactivation fonctionnelle des neurones situés près de la microélectrode. Cette idée nous vient du fait qu'une lésion produit un effet semblable à celui d'une stimulation à haute fréquence. L'inactivation pourrait être le résultat d'une inhibition neuronale directe, c'est-àdire de la très forte dépolarisation des membranes cellulaires ou encore, d'une stimulation locale des terminaisons synaptiques inhibitrices. Par ailleurs, la stimulation thalamique pourrait abolir ou diminuer le tremblement en brouillant les signaux oscillatoires en provenance des afférences proprioceptives. Il semble que la stimulation intracérébrale chronique n'occasionne aucun dommage tissulaire majeur étant donné la réversibilité immédiate de son effet sur le tremblement. Quoi qu'il en soit, les électrodes de stimulation ainsi implantées affectent non seulement les neurones situés près de l'électrode, mais aussi l'ensemble du réseau neuronal dont font partie les neurones stimulés, y compris les fibres de passage. Des études récentes ont révélé que l'atténuation maximale des symptômes moteurs de la maladie de Parkinson est atteinte lorsque la microélectrode est implantée dans la région antérodorsale du STN (Patel et al., 2003; Yelnik et al., 2003). Ces données suggèrent qu'une telle localisation de la microélectrode permet au courant électrique de diffuser et

Figure 1.5 A, Section transversale d'un cerveau humain montrant la localisation d'une pallidotomie (flèche) effectuée dans la partie caudale du pallidum interne (GPi) chez un patient atteint de la maladie de Parkinson. Radiographies frontale (B) et latérale (C) du crâne d'un patient chez qui des électrodes de stimulations à haute fréquence ont été implantées bilatéralement dans le noyau subthalamique (STN) afin d'atténuer les principaux symptômes moteurs de la maladie de Parkinson. D, Les microélectrodes sont reliées à des stimulateurs électriques programmables logés dans les poches sous-cutanées infra-claviculaires du patient. (Benabid et al., 2000; Lozano et Lang, 2001)

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d'influencer les axones pallidofuges GABAergiques hyperactifs dans la maladie de Parkinson (Saint-Cyr et al., 2002; Voges et al., 2002). Une connaissance détaillée du trajet emprunté par les axones pallidofuges en relation avec les faisceaux de fibres avoisinant le STN est donc essentielle pour comprendre et améliorer le traitement du Parkinson par stimulations à haute fréquence. Ces données seront présentées en détails dans le quatrième chapitre de cette thèse.

La mise en relation des cibles axonales en fonction de la localisation des corps cellulaires au sein du GPi est nécessaire afin d'améliorer le traitement de la maladie de Parkinson par une lésion ablative du GPi (pallidotomie ou ansotomie) ou par stimulations à haute fréquence de cette structure. Le troisième chapitre de cette thèse contient des données démontrant que les corps cellulaires qui possèdent un axone hautement collatéralisé innervant les noyaux moteurs du thalamus sont préférentiellement localisés dans la partie ventrocaudale du GPi. Cette région pallidale est d'ailleurs densément innervée par les projections striatopallidales qui proviennent de la région sensorimotrice du striatum (Parent et Hazrati, 1995a). Une corrélation intéressante peut être faite entre ces données neuroanatomiques et les données cliniques déjà publiées qui démontrent que la réduction la plus marquée des symptômes moteurs de la maladie de Parkinson est obtenues lorsque la pallidotomie affecte principalement la partie ventrocaudale du pallidum interne (Krack et al., 1998; Vitek et al., 1998) (Fig. 1.5A).

1.3.2

La chorée de Huntington

La chorée de Huntington est une maladie neurodégénérative héréditaire causée par une répétition excessive du trinucléotide CAG qui affecte le gène qui code pour la protéine huntingtine (htt). Il s'agit d'un gène à très haute pénétrance et qui se transmet de façon autosomique dominante. La maladie est caractérisée cliniquement par un excès de mouvements (hyperkinésie) et, plus particulièrement, par la présence de mouvements involontaires complexes de type choréiforme. Les patients présentent aussi des troubles

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sévères du comportement et des déficits cognitifs évoluant vers la démence. Le signe pathologique dominant de la maladie est une atrophie progressive du striatum, le noyau caudé étant touché avant et plus sévèrement que le putamen.

Il existe d'ailleurs une

excellente corrélation entre le degré d'atrophie du noyau caudé et la sévérité de la maladie (Vonsattel et al., 1985) (Fig. 1.6). Cette atrophie remarquable du striatum est en grande partie due à la dégénérescence des neurones striataux épineux de projection, les interneurones non épineux étant beaucoup moins affectés dans cette pathologie (Dawbarn et al., 1985; Ferrante et al., 1985).

Les mouvements hyperkinétiques observés dans la maladie de Huntington résultent d'une desinhibition des neurones thalamocorticaux. Cette desinhibition s'explique bien à l'aide du modèle des ganglions de la base présenté à la section 1.2.1. Elle survient suite à la dégénérescence des neurones striataux GABA/ENK qui sont à l'origine de la voie indirecte. La mort de ces neurones de projection permet au segment externe du pallidum d'inhiber davantage le STN. La diminution de l'influence excitatrice glutamatergique en provenance du STN explique la diminution de l'effet des efférences GABAergiques des portes de sorties des ganglions de la base et donc la desinhibition des neurones thalamocorticaux du tiers ventral (Fig. 1.4 B). Ces derniers se mettent donc à répondre de façon anarchique à diverses entrées excitatrices et l'augmentation de leurs influences glutamatergiques sur les neurones moteurs du cortex cérébral engendre une activité motrice excessive et incohérente (Albin et al, 1989; DeLong, 1990). Il faut mentionner ici que les neurones GABA/ENK du striatum seraient affectés plus tôt et plus sévèrement que les neurones GABA SP/DYN à l'origine de la voie directe (Reiner et al., 1988; Albin et al., 1991; Albin et al., 1992). Ceci pourrait expliquer le fait que les patients atteints de la chorée de Huntington sont d'abord hyperkinétiques (choréiques) dans les premiers stades de la maladie et deviennent progressivement akinétiques et rigides dans les stades plus avancés.

Figure 1.6 Sections transversales au niveau de la commissure antérieure montrant l'atrophie progressive du striatum caractéristique de la chorée de Huntington, le noyau caudé (CD) étant touché avant et plus sévèrement que le putamen (PUT). Les cas représentés sont (A) normal, (B) maladie de Huntington grade 1 et (C) maladie de Huntington grade 3. L'atrophie remarquable du striatum est en grande partie due à la dégénérescence des neurones striataux épineux de projection, les interneurones non épineux étant beaucoup moins affectés dans cette pathologie. Barre étalon = 2.5 cm. (Cicchetti et al., 2000)

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Normal

Grade 1

Grade 3

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1.3.2.1 Traitement de la chorée de Huntington La maladie de Huntington est attribuable à une mutation génétique impliquant une répétition de polyglutamine sur le gène htt situé sur le chromosome 4. À l'heure actuelle, nous ne connaissons pas le mécanisme par lequel cette mutation conduit à la mort neuronale. De plus, le fait que les neurones de projection du striatum soient spécifiquement affectés dans cette pathologie demeure inexpliqué. Dans les stades initiaux de la maladie, les mouvements choréiques sont limités au visage et à la partie distale des membres. Ensuite, ces mouvements anormaux se généralisent à l'ensemble du corps et interfèrent grandement avec l'exécution des mouvements volontaires. Ce n'est qu'à ce stade de la maladie, lorsque les mouvements hyperkinétiques handicapent sévèrement le patient, que l'on administre un traitement pharmacologique qui réussit habituellement à atténuer les mouvements hyperkinétiques du patient. Cependant, ces traitements pharmacologiques n'ont aucune influence sur les symptômes cognitifs ainsi que sur la progression de la maladie. La réponse clinique à ces traitements varie énormément d'un patient à l'autre. Les médicaments utilisés, habituellement des antagonistes des récepteurs dopaminergiques, peuvent parfois entraver l'exécution des mouvements volontaires et entraîner plusieurs effets secondaires, comme l'apparition de symptômes parkinsoniens.

D'autres avenues thérapeutiques, pour l'instant moins efficaces, sont présentement envisagées. Ces traitements visent cette fois le ralentissement de la progression de la maladie et le remplacement des neurones perdus. Les thérapies cellulaires dont la greffe de tissu striatal fœtal ainsi que l'administration de facteurs neurotrophiques comptent parmi les stratégies alternatives qui ont un avenir prometteur.

Étant donné que les stimulations à haute fréquence du GPi atténuent efficacement la dystonie induite par la lévodopa chez certains patients parkinsoniens, les neurochirurgiens ont pensé utiliser la même approche pour réduire les mouvement hyperkinétiques chez les patients atteints de la maladie de Huntington (Moro et al., 2004). Des résultats encourageants ont été obtenus chez deux patients, démontrant qu'une stimulation bilatérale du GPi permet d'atténuer les mouvements choréiques et dystoniques sans toutefois

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aggraver la bradykinésie (Moro et al., 2004). Des données électrophysiologiques obtenues lors d'opérations neurochirurgicales ont démontré que les taux de décharge des neurones du GPi enregistrés chez les patients parkinsoniens et ceux atteints de la maladie de Huntignton n'étaient pas statistiquement différents (Tang et al, 2005). Ces données suggèrent que le patron de décharge neuronale, plutôt que le taux de ces décharges, est responsable de l'apparition des symptômes moteurs de ces pathologies. La stimulation intracérébrale à haute fréquence modifierait les patrons de décharge pathologique aidant ainsi à normaliser le comportement moteur des patients.

1.4

L'importance de la collatéralisation axonale au sein des ganglions de la base du primate

L'étude des connexions entre les différentes structures nerveuses peut se faire à plusieurs niveaux. Un premier niveau d'analyse est celui qui conduit à l'élaboration de modèles neuronaux plutôt réducteurs du type boîtes et flèches, comme celui décrit à la section 1.2.1. Certes nécessaire, ce premier niveau d'analyse ne peut évidement pas rendre compte de l'intégration neuronale qui s'opère à l'intérieur des structures et il peut difficilement représenter les éventuelles bifurcations des axones vers plusieurs cibles. Un niveau supérieur d'analyse est celui où l'on tente d'identifier le mode de transfert de l'information neuronale entre un axone et sa cible post-synaptique à l'échelle ultrastructurale. Beaucoup plus sophistiqué que le premier, ce niveau d'étude fournit des éléments essentiels à la compréhension de l'organisation anatomique et fonctionnelle d'un réseau neuronal. Nous avons choisi de situer la présente étude à un niveau intermédiaire, soit au niveau cellulaire ou plus précisément au niveau neuronal unitaire. Cette position permet d'apprécier l'organisation spatiale de l'arborisation axonale entière incluant le degré de collatéralisation axonale présenté par chaque neurone étudié. La visualisation détaillée du domaine somatodendritique et la reconstruction complète de l'arborisation axonale d'un seul neurone permet d'apprécier les capacités que possède ce neurone d'intégrer l'information qui lui arrive de plusieurs sources et d'acheminer une information transformée à d'autres

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neurones souvent situés dans plusieurs structures distinctes ciblées via son axone et ses nombreuses collatérales. Tel que décrit plus haut, le modèle couramment utilisé pour expliquer le fonctionnement des ganglions de la base repose sur une vision plutôt simpliste des connexions reliant les différentes composantes de cet ensemble complexe de structures sous-coricales. Depuis plusieurs années, les études menées dans notre laboratoire ont permis d'apprécier la complexité de l'organisation morphologique des neurones au sein des ganglions de la base et de déceler un niveau de collatéralisation axonale jusqu'alors insoupçonné. Ces données neuroanatomiques remettent en question le schème classique de l'organisation anatomofonctionnelle des ganglions de la base chez le primate qui repose sur la ségrégation de l'information neuronale en provenance du striatum et sur la dualité des voies striatofuges. En effet, la reconstruction d'axones striataux individuels chez le singe a révélé que la majorité des neurones épineux de taille moyenne du striatum projettent aux trois cibles principales de la voie striatofuge, soit les deux segments pallidaux et la SNr (Parent et al., 1995; Parent et al., 2000; Lévesque et Parent, 2005). D'autres études par traçage unitaire, aussi effectuées chez le singe, ont démontré que, bien que la majorité des axones du segment externe du pallidum projettent au STN, aucun ne semble projeter uniquement à cette structure (Sato et al., 2000a). Les efférences du segment externe du pallidum ciblent pratiquement toutes les composantes des ganglions de la base. La projection du pallidum externe vers le GPi et la SNr, démontrée chez le rongeur et le primate (Hazrati et al., 1990; Shink et al., 1996; Bevan et al., 1997; Sato et al., 2000a), nous amène à changer la vision que nous avions de cette structure. Étant donné l'existence de cette projection vers les portes de sortie des ganglions de la base, le pallidum externe semble jouer un rôle intégrateur important entre les structures d'entrée et de sortie des ganglions de la base plutôt qu'un rôle de simple relais de la voie indirecte. Suite à l'étude par traçage unitaire des projections efférentes du STN chez le singe macaque (Sato et al., 2000b), il a été démontré que le STN est composé de plusieurs types de neurones dont certains possèdent un axone hautement collatéralisé. Plusieurs neurones du STN sont en mesure d'influencer, non seulement le GPi et la SNr, les deux portes de sortie des ganglions de la base, mais aussi le pallidum externe avec lequel ils entretiennent une relation réciproque. Ces

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importantes données neuroanatomiques sont évidemment difficiles à concilier avec le schème actuel des ganglions de la base. Le modèle des ganglions de la base présenté plus haut possède une valeur heuristique indéniable. En plus de stimuler la recherche, il a servi de cadre de référence à de nombreuses études anatomiques, physiologiques et cliniques sur les ganglions de la base. Cependant, les nouvelles données obtenues par traçage unitaire nous laissent entrevoir une image plus complexe de l'organisation anatomo-fonctionnelle des ganglions de la base. Nous devrons donc tenir compte de ces nouveaux résultats si nous espérons en arriver à une meilleure compréhension du rôle de ces structures sous-corticales dans le contrôle du comportement moteur en conditions normale et pathologique.

1.5 Le pallidum interne 1.5.1

Anatomîe

Le segment interne du globus pallidus (GPi) appartient au complexe pallidal qui lui-même forme la partie interne du noyau lenticulaire. La lame médullaire externe constitue une bande de substance blanche qui sépare le putamen du complexe pallidal. Ce dernier est bordé dorsomédialement par le bras postérieur de la capsule interne. C'est la lame médullaire interne qui divise le complexe pallidal en segments interne et externe (Fig. 1.1). D'autres auteurs préfèrent parler de segments médian et latéral pour décrire le pallidum interne et externe respectivement. Lorsqu'on observe attentivement le GPi, on peut voir une autre lame médullaire appelée lame médullaire accessoire, beaucoup plus discrète que les autres lames médullaires du noyau lenticulaire. Chez le primate, la lame médullaire accessoire permet de diviser le GPi en parties médiane et latérale.

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1.5.2

Cytologie

Plusieurs axones myélinisés traversent le pallidum ce qui lui confère une couleur pâle comparativement au striatum. Une étude morphométrique réalisée sur du tissus postmortem humain indique que le segment externe du pallidum représente 70% du volume du complexe pallidal et qu'il dispose d'une densité cellulaire plus élevée que celle mesurée dans le GPi (Schroder et al., 1975). En moyenne, le volume des corps cellulaires des neurones appartenant au GPi est cependant 10% supérieur à celui des neurones du segment externe. On estime que le nombre total de neurones contenus dans le GPi varie entre 143 000 et 171 000. Les neurones de projection du GPi ont un corps cellulaire volumineux (3550 /xm de diamètre), de forme ovoïde ou polygonal, ainsi que des dendrites pouvant atteindre plus de 1 000 /xm de longueur (Fox et al., 1974; Percheron et al., 1984a; Percheron et al., 1984b) (Fig. 1.7). L'organisation somatodendritique de ces neurones se présente sous la forme de disques plats de très grande dimension orientés parallèlement les uns aux autres ainsi qu'au bord latéral de chaque segment pallidal (Percheron et al., 1984a; Percheron et al., 1984b; Yelnik et al., 1984; Parent et Hazrati, 1995a) (Fig. 1.8). L'orientation du domaine somatodendritique de ces neurones, perpendiculaire aux axones striatofuges, implique qu'un neurone du GPi est en mesure de recevoir un très grand nombre d'afférences en provenance de différentes régions fonctionnelles du striatum suggérant ainsi le rôle intégrateur important joué par les neurones GPi. On a aussi décrit l'existence de neurones cholinergiques au sein du GPi. Ces neurones sont en fait une extension du noyau basalis de Meynert qui repose ventralement au globus pallidus et dont quelques-uns des éléments envahissent la lame médullaire interne (Parent, 1979).

Figure 1.7 A, Dessins de neurones du pallidum interne chez le macaque effectués à partir de matériel imprégné par la méthode de Golgi. Les flèches indiquent le segment initial de l'axone. B, Photomicrographie d'un neurone du pallidum interne à partir d'une préparation de Golgi chez le macaque. (Fox et al., 1974)

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Figure 1.8 Shéma montrant une vue frontale de l'organisation en disque de l'arborisation dendntique des neurones du pallidum chez le singe macaque. (Percheron et al, 1984a)

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MACAQUE

Pallidal dendritic arborizat ion

FRONTAL

Med Inf

Substantia n i g ra

1mm

1.2

3.4

37

5.6

N.caudatus

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1.5.3

Hodologie 1.5.3.1 Afferences pallidales

Les principales afferences du GPi proviennent du striatum, du STN et du pallidum externe (Cowan et Powell, 1966; Szabo, 1967; Nauta et Cole, 1978; Parent et al., 1991). Le fait que les afferences en provenance des différents territoires fonctionnels du striatum occupent des régions pallidales plus ou moins distinctes suggère l'existence d'une certaine ségrégation en ce qui a trait au traitement des informations neuronales sensorimotrices, associatives et limbiques au sein du pallidum. Certains auteurs ont donc été tentés de diviser le pallidum en trois territoires fonctionnels distincts sur la base des projections striatopallidales (Hedreen et DeLong, 1991; Shink et al., 1997; Sidibé et al., 1997; Baron et al., 2001). Selon ces auteurs, le territoire sensorimoteur du GPi correspondrait à la partie postcommissurale du pallidum ventrolatéral puisque cette région reçoit d'importantes afferences en provenance du putamen, où est centré le territoire sensorimoteur du striatum. De même, les territoires associatif et limbique occuperaient respectivement la portion dorsolatérale et la pointe médiane du GPi. Cette division territoriale du GPi découle de l'hypothèse d'un traitement de l'information neuronale en parallèle suggérant que cette dernière soit transmise à travers les ganglions de la base selon des circuits fonctionnellement ségrégés (Alexander et al., 1986; Alexander et Crutcher, 1990). Les études ayant mené à l'élaboration de cette hypothèse ont été réalisées souvent à partir d'injections massives de traceurs antérogrades, une méthode qui ne permet pas une distinction claire entre les fibres de passage et les terminaisons axonales (Hedreen et DeLong, 1991). L'utilisation d'une technique beaucoup plus sensible nous a permis de démontrer l'existence de neurones hautement collatéralisés au sein du GPi, ce qui va à rencontre d'un traitement strictement ségrégé et canalisé de l'information neuronale à travers toute la boucle cortico-ganglions de la base-thalamo-corticale. Par ailleurs, des études antérieures ont démontré que les fibres striatopallidales s'arborisent sous forme de bandes parallèles aux lames médullaires (Percheron et al., 1984b; Parent et Hazrati, 1995a). Considérant ce patron de terminaison des fibres striatofuges ainsi que la dimension et l'orientation du domaine somatodendritique des neurones du GPi, ces derniers semblent

39

être en mesure d'intégrer les informations neuronales convergentes provenant de plus d'un territoire fonctionnel du striatum. Il serait donc hasardeux d'inférer des territoires fonctionnels au GPi uniquement sur la base de l'origine des projections striatopallidales. Il s'avère primordial d'étudier en détail les projections efférentes des neurones du GPi avant de pouvoir supputer sur leur rôle fonctionnel dans l'organisation des ganglions de la base. Des études antérieures ont démontré que les afférences en provenance du STN et du STR convergent sur les mêmes neurones du GPi. Les fibres striatales s'enroulent autour des longues dendrites pallidales alors que les axones en provenance du STN démontrent des terminaisons périsomatiques (Hazrati et Parent, 1992). Contrairement aux afférences striatales qui utilisent le neurotransmetteur inhibiteur GABA, les afférences en provenance du STN se servent du glutamate comme neurotransmetteur et sont donc excitatrices. Puisque ces deux afférences convergent sur les mêmes neurones du GPi, les décharges neuronales de ces derniers sont le résultat d'une interaction complexe de ces deux principales afférences. À ces projections s'ajoute une afférence dopaminergique en provenance d'axones collatéralisés de la SNc innervant aussi le striatum (Cossette et al., 1999; Gauthier et al., 1999; Prensa et al., 2000). Cette projection permet à la dopamine d'influencer directement les structures de sortie des ganglions de la base et pourrait avoir une implication importante dans l'apparition des taux de décharge pathologiques enregistrés chez les patients parkinsoniens. Le GPi reçoit de plus une projection sérotoninergique du noyau dorsal du raphé (Lavoie et Parent, 1990) ainsi qu'une projection cholinergique du PPN (Lavoie et Parent, 1994a), tous deux situés dans le tegmentum mésencéphalique.

1.5.3.2 Efférences pallidales L'article de Nauta et Mehler publié en 1966 décrit de façon remarquable les efférences du GPi (Nauta et Mehler, 1966). Cette étude autoradiographique a permis d'identifier les principales cibles du GPi soit les noyaux ventral antérieur (VA) et ventral latéral (VL) du thalamus, le CM/Pf, le PPN et l'habénula latérale. De plus, ces auteurs ont suggéré que les projections pallidofuges se terminant dans le CM puissent être des collatérales d'axones

40

innervant les noyaux du tiers ventral du thalamus. La vision d'une organisation collatéralisée des efférences du GPi a été appuyée par des études électrophysiologiques d'invasions antidromiques (Filion et Harnois, 1978; Harnois et Filion, 1980; Harnois et Filion, 1982) ainsi que par des études utilisant des traceurs rétrogrades (Parent et De Bellefeuille, 1982; Parent et De Bellefeuille, 1983). Ces travaux, résumés dans le deuxième chapitre de cette thèse, suggèrent que la majorité des neurones du GPi projettent à la fois aux noyaux VA/VL et au noyau PPN, aux noyaux VA/VL et au CM ou encore, à ces trois structures à la fois. Au contraire, il semble que la projection vers l'habénula latérale provienne d'un groupe neuronal distinct du GPi. Les neurones ciblés par les axones du GPi appartiennent à différents systèmes neuronaux : (1) les neurones prémoteurs, situés dans les noyaux de relais du thalamus (VA/VL) et dans le tegmentum mésencéphalique (PPN), ainsi que (2) les neurones limbiques de l'habénula latérale. Recevant d'importantes projections de l'aire hypothalamique latérale, de la substantia innominata et de l'aire sep taie, l'habénula latérale est une structure de relais importante du système limbique. Des études anatomiques (Herkenham et Nauta, 1977) et électrophysiologiques (Garland et Mogenson, 1983) ont suggéré que la projection pallidohabénulaire puisse agir à titre d'interface fonctionnelle entre les systèmes moteur et limbique.

La projection du GPi qui descend vers le tronc cérébral pour s'arboriser principalement au niveau du PPN est souvent négligée dans le schème actuel de l'organisation anatomofonctionnelle des ganglions de la base. Pourtant, le PPN du tegmentum mésencéphalique reçoit une projection importante non seulement du GPi mais aussi de la SNr. Il projette en retour à ces mêmes structures ainsi qu'au STN et au complexe CM/Pf (Mehler et Nauta, 1974; Lavoie et Parent, 1994a; Lavoie et Parent, 1994b). Certaines études ont démontré que les ganglions de la base pouvaient influencer la motricité et ce, en l'absence du cortex moteur chez le chat et le rat (Harnois et Filion, 1980; Harnois et Filion, 1982). Les projections descendantes des ganglions de la base vers la moelle épinière via un relais dans le PPN pourraient expliquer ces observations. Le PPN permettrait donc à l'information neuronale de sortir de la boucle cortico-ganglions de la base-thalamo-corticale afin d'atteindre les motoneurones spinaux. La projection pallidotegmentaire pourrait aussi jouer un rôle important dans l'akinésie observée dans la maladie de Parkinson.

41

Les axones pallidofuges émergent et cheminent le long de deux voies principales: (1) l'anse lenticulaire et (2) le faisceau lenticulaire. Les fibres de l'anse lenticulaire longent la partie ventrale du pallidum pour passer ventralement, médialement et antérieurement autour du bras postérieur de la capsule interne et ensuite se diriger postérieurement afin d'entrer dans le champ H de Forel (ou aire prérubrale). Les fibres qui composent le faisceau lenticulaire émergent de la partie dorsomédiane du pallidum, un peu plus caudalement que celles de l'anse lenticulaire, traversent la partie ventrale de la capsule interne et cheminent ventralement sous la zona incerta. Bien que la plupart des fibres qui composent le faisceau lenticulaire demeurent dans la partie rostrale du STN, certaines d'entre-elles longent la partie dorsale du STN. Les axones composant le faisceau lenticulaire, aussi nommé champ H2 de Forel, rejoignent ceux de l'anse lenticulaire dans le champ H de Forel. Ce faisceau de fibres ainsi constitué entre dans le faisceau thalamique (champ Hl de Forel) situé dorsalement à la zona incerta. Selon les données de la littérature, les axones de l'anse lenticulaire proviennent de neurones dont le corps cellulaire est situé dans la partie latérale du GPi alors que les fibres du faisceau lenticulaire proviennent de la partie médiane du GPi (Kuo et Carpenter, 1973; Carpenter, 1976). Nous avons jugé bon de suivre en détail le trajet initial des axones marqués par injection microiontophorétique afin de clarifier ce schème organisationnel présent dans la littérature. La technique utilisée étant tout à fait adaptée à cette problématique, nous avons obtenu des données nouvelles qui nous forcent à réviser le schème couramment admis en ce qui a trait aux relations entre l'origine cellulaire et la trajectoire des axones pallidofuges dans l'anse et le faisceau lenticulaire. Ces données ont fait l'objet d'un article qui sera présenté dans le quatrième chapitre de cette thèse.

1.6 Le cortex moteur primaire 1.6.1

Anatomie et organisation fonctionnelle

L'aire 4 de Brodmann, communément appelé l'aire motrice primaire, est située sur la paroi antérieure du sulcus central ainsi que sur le gyrus précentral adjacent. Elle s'étend davantage à la limite supérieure de l'hémisphère alors qu'elle se restreint à la paroi

42

antérieure du sulcus central près du gyrus frontal inférieur. Sur la surface médiane de l'hémisphère, l'aire motrice primaire s'étend jusqu'à la portion antérieure du lobule paracentral (Parent, 1996). Le cortex moteur primaire est très épais; il est de l'ordre de 3,5 à 4,5 mm chez l'homme et de 2,5 à 3,0 mm chez le singe cynomolgus. L'idée d'une organisation somatotopique au sein du cortex moteur a été soulevée pour la première fois au cours des années 1860 par l'Anglais John Hughlings Jackson (1835-1911). Ce dernier a formulé cette hypothèse pour expliquer le fait que les convulsions motrices focalisées qui apparaissent chez certains patients atteints d'épilepsie Bravais-Jacksonnienne progressent le long des différents segments du corps suivant un patron hautement prévisible (Jackson, 1863). Ce concept fut ensuite démontré expérimentalement par les Allemands Gustav Fristsch (1838-1927) et Eduard Hitzig (1838-1907) suite à des stimulations électriques chez le chien (Walshe, 1948). Le fameux homonculus moteur dessiné par Wilder Penfield (1891-1976) illustre de façon très frappante l'organisation somatotopique du cortex moteur chez l'homme (Penfield et Rasmussen, 1950).

La vision classique de l'organisation somatotopique du cortex moteur primaire implique une représentation continue et déformée des différentes parties corporelles à la surface de ce dernier. Ce schème organisationnel nous vient d'études au cours desquelles des mouvements discrets et isolés ont été produits dans l'hemicorps controlateral suite à des stimulations effectuées en surface du cortex et ce, chez plusieurs espèces animales ainsi que chez l'humain. L'organisation somatotopique suggère que les régions corticales participant au contrôle des muscles de la face, des bras, et des jambes soient ségréguées. Jusqu'au milieu du 20ème siècle, on pensait que cette ségrégation pouvait aller jusqu'à la représentation ordonnée, en un site cortical unique, de chacune des parties corporelles comprises dans ces régions. Par exemple, on croyait que la région du cortex moteur primaire impliquée dans le contrôle du bras était composée de la représentation séquentielle des muscles du pouce, de l'index, du majeur, de l'annulaire, de l'auriculaire, du poignet et de l'épaule. Depuis les 25 dernières années, plusieurs données expérimentales nuancent l'idée selon laquelle il existerait une organisation somatotopique stricte au sein du cortex moteur primaire. Bien que la ségrégation fonctionnelle des grandes régions corporelles

43

comme la face, le bras et la jambe soit généralement observée, de nombreuse données neurophysiologiques obtenues par microstimulations intracorticales nous indiquent qu'un muscle peut être recruté suite à la stimulation de plusieurs sites corticaux (Merzenich et al, 1978; Strick et Preston, 1978; Donoghue et al., 1992; Schneider et al., 2001). Ces sites de recrutement multiples sont non contigus et entremêlés de sites corticaux permettant le recrutement d'autres muscles appartenant à la même région corporelle. Cette mosaïque fonctionnelle fait en sorte que les territoires du cortex moteur primaire impliqués dans le contrôle de certains muscles se chevauchent ce qui pourrait faciliter les interactions biomécaniques locales à travers des territoires corticaux représentant différentes synergies musculaires (Schieber, 2001).

1.6.2

Cytologie

Bien que le cortex cérébral contienne une quantité impressionnante de neurones, le nombre de types cellulaires est, quant à lui, relativement restreint. Les types neuronaux principaux qu'on y retrouve peuvent être regroupés en deux grandes catégories soit (1) les cellules excitatrices pyramidales (Golgi type I) utilisant le glutamate comme neurotransmetteur, et (2) les cellules inhibitrices stellaires ou granulaires (Golgi type II) synthétisant le GABA (Ribak, 1978; Houser et al., 1983; DeFelipe et Farinas, 1992). Les neurones pyramidaux, incluant les cellules fusiformes, sont les neurones de projections principaux du cortex cérébral alors que les cellules granulaires agissent à titre d'interneurones. Des classifications basées sur la morphologie somatodendritique ont permis de distinguer plusieurs types d'interneurones dont (1) les cellules en panier, (2) les cellules en chandelier, (3) les cellules en double bouquet, (4) les cellules de Martinotti, (5) les cellules de forme neurogliale ainsi que (6) les cellules en griffes (Ramôn y Cajal, 1909, 1911; Jones, 1975a; Feldman et Peters, 1978). Comme leur nom l'indique, les neurones pyramidaux sont caractérisés par un soma de forme triangulaire. Ils possèdent une dendrite apicale ainsi que plusieurs dendrites basales. L'axone émerge de la base du corps cellulaire pour se terminer dans les couches profondes du cortex ou encore pour entrer dans la substance blanche souscorticale et former ainsi des neurones d'association ou de projection. La hauteur du corps

44

cellulaire varie entre 10 et 50(im. Les cellules géantes de Betz peuvent avoir un corps cellulaire atteignant une hauteur de 120um.

Les différents types neuronaux décrits plus haut se retrouvent en couches cellulaires au sein du cortex cérébral. Ces couches cellulaires sont organisées séquentiellement de la superficie vers la substance blanche sous-corticale selon l'ordre suivant : (I) la couche moléculaire, (II) la couche granulaire externe, (III) la couche pyramidale externe, (IV) la couche granulaire interne, (V) la couche pyramidale interne et finalement (VI) la couche multiforme. Le cortex moteur primaire est qualifié d'agranulaire puisque la couche granulaire interne, la couche IV, est difficile à mettre en évidence étant donné que les cellules granulaires normalement contenues dans cette couche sont pratiquement absentes. Notons que c'est dans la couche IV où l'on retrouve la majorité des terminaisons thalamocorticales. Cette couche cellulaire atteint son épaisseur maximale dans le cortex somatosensoriel. Le cortex moteur primaire, dit « cortex de type 1 » selon von Economo (von Economo, 1929), se distingue donc par son épaisseur ainsi que par la faible quantité de cellules granulaires. Les neurones pyramidaux des couches III et V y sont davantage développés et de plus grande taille que ceux retrouvés dans les autres aires corticales. Même les petits neurones dont le corps cellulaire est situé dans les couches II et IV sont principalement de forme pyramidale. Ceci fait en sorte qu'il peut être difficile de distinguer les couches cellulaires individuelles du cortex moteur primaire.

1.6.3

Hodologie 1.6.3.1 Afférences du cortex moteur primaire

Les axones afférents du cortex cérébral comprennent principalement (1) les projections thalamocorticales, (2) les projections associatives provenant d'autres aires corticales homolatérales, ainsi que (3) les projections commissurales originant de l'hémisphère controlatéral. Les projections thalamocorticales provenant des noyaux ventraux du thalamus forment de denses plexus terminaux dans la couche IV qui parfois s'étendent dans la couche III (Jones, 1985). Le système thalamocortical non spécifique joue un rôle crucial

dans la modulation des états de vigilance (Jones, 1998; Steriade, 2000; Jones, 2001). Les axones appartenant à ce système émergent des noyaux intralaminaires du thalamus et projettent dans une vaste région du cortex cérébral (Jones et Leavitt, 1974). Ces axones s'arborisent principalement dans les couches V et VI ainsi que, de façon moins importante, dans la couche I (Berendse et Groenewegen, 1991). Ce sujet sera traité de façon plus approfondie dans la section 1.7 ainsi que dans le sixième chapitre. Les axones d'association proviennent de corps cellulaire situés dans la partie superficielle des couches II et III. Les terminaisons axonales de ces neurones sont distribuées principalement dans les couches III et IV mais peuvent s'étendre dans les couches corticales profondes (Jones et Powell, 1968). Les projections commissurales sont issues de gros corps cellulaires situés dans la partie profonde de la couche III. Ces projections sont distribuées à travers toutes les couches corticales (Szentagothai, 1978). À ces principales afférences s'ajoutent plusieurs projections monoaminergiques. La projection noradrénergique provient des neurones du locus coeruléus situé dans la partie dorsomédiane du tegmentum pontin alors que la projection sérotoninergique provient des noyaux dorsal et médian du raphé situés dans la portion médiane du tegmentum mésencéphalique caudal (Parent, 1996). La projection sérotoninergique est plus profuse que la projection noradrénergique et elle cible toutes les couches corticales. La projection dopaminergique quant à elle, origine de la SNc et de l'aire tegmentaire ventrale toutes deux situées à la base du mésencéphale. Chez les primates, il semble que le cortex moteur primaire soit davantage innervé que les autres aires corticales par la dopamine (Morrison et Hof, 1992; Lewis et al., 2001). La distribution régionale et laminaire de l'innervation dopaminergique suggèrent que les cellules pyramidales donnant origine aux projections corticofuges

et corticocorticales sont les cibles préférentielles

des terminaisons

dopaminergiques (Lewis et al., 1986; Lewis et al., 1987). Dans l'ensemble, les projections monoaminergiques ascendantes constituent des systèmes très divergents ce qui signifie qu'un seul neurone est en mesure d'innerver un vaste territoire cortical (Lindvall et Bjôrklund, 1984; Foote et Morrison, 1987; McCormick, 1992). Il existe une afférence corticale cholinergique substantielle provenant des neurones situés dans le noyau basalis de Meynert (groupe cholinergique Ch4) ainsi que, de façon moins

46

importante, du bras vertical de bande diagonale de Broca située dans l'aire septale (groupe, cholinergique Ch2) (Parent, 1986; Mesulam et Geula, 1988). Tout comme les projections noradrénergiques et sérotoninergiques, les axones cholinergiques innervent pratiquement l'ensemble du cortex cérébral, la couche I étant la plus densément innervée, suivie des couches II et III.

1.6.3.2 Efférences du cortex moteur primaire Les neurones pyramidaux peuvent être groupés en sous-types sur la base de leur projection axonale (Jones, 1984). Les axones corticofuges proviennent de corps cellulaires principalement situés dans les couches corticales profondes et peuvent être regroupés selon les catégories suivantes : les axones (1) corticospinaux, (2) corticobulbaires, (3) corticoréticulaires, (4) corticopontins, (5) corticothalamiques, (6) corticostriés et (7) corticonucléaires.

Ce

corticosubthalamiques,

dernier

regroupement

corticorubrales,

corticotectales

comprend ainsi

que

les des

projections projections

corticocérébelleuses. Les corps cellulaires dont l'axone contribue aux différentes projections corticofuges ont des distributions laminaires spécifiques. Chez les primates, on pense que les neurones corticospinaux sont majoritairement contenus dans la partie profonde de la couche V (Vb) alors que les neurones corticostriés originent principalement d'un groupe de cellules pyramidales de plus petite taille localisé dans la partie superficielle de la couche V (Va) (Jones et al., 1977; Arikuni et Kubota, 1986; Saint-Cyr et al., 1990). Les projections corticopontines, corticobulbaires et corticorubrales originent principalement de la partie centrale de la couche V. La vaste majorité des neurones corticothalamiques se retrouvent dans la couche VI, bien que certains d'entre eux soient situés dans la partie profonde de la couche V. Le soma des neurones d'association se trouve dans les couches superficielles. Les projections corticocorticales homolatérales proviennent des couches II et III alors que les projections commissurales originent de la couche III. Ces données ont été obtenues principalement par l'injection de traceurs rétrogrades (Jones, 1984). Elles nous révèlent l'organisation laminaire des neurones qui composent le cortex moteur primaire et dont l'axone constitue les différents systèmes corticofuges.

47

Le claustrum est une structure ne faisant pas partie des ganglions de la base au sens strict. Cette structure est cependant réciproquement liée à pratiquement toutes les aires corticales et peut être divisée en zones somesthésique, visuelle, auditive et limbique (Riche et Lanoir, 1978; Pearson et al., 1982; Sherk, 1986; Minciacchi et al., 1991; Tanne-Gariepy et al., 2002). Des projections provenant du cortex précentral et qui innervent à la fois le striatum et le claustrum ont été décrites chez plusieurs espèces (Kemp et Powell, 1970; Kiinzle et al., 1976; Jurgens, 1984; Selemon et Goldman-Rakic, 1988; Stanton et al., 1988; Lévesque et Parent, 1998).

1.6.3.2.1 La projection corticostriée L'ensemble du cortex cérébral projette massivement au striatum et, tel que mentionné plus haut, le cortex moteur primaire innerve principalement la partie dorsolatérale du putamen correspondant au territoire sensorimoteur du striatum (Parent, 1990). La première interprétation de l'organisation des projections corticostriées fut celle de Kemp et Powell (Kemp et Powell, 1970) qui ont proposé que les aires corticales se projettent d'une manière topographique stricte sur le striatum de sorte que chaque territoire striatal reçoit une projection provenant de la région corticale la plus proche. Ces auteurs ont suggéré qu'une zone restreinte du cortex cérébral projette à une région striatale bien circonscrite avec peu de chevauchement. Utilisant la méthode de Golgi, Ramôn y Cajal a démontré que les axones corticostriés s'arborisaient selon un patron axodendritique cruciforme (Ramôn y Cajal, 1909, 1911). Il faisait alors référence aux fibres corticostriées qui présentent une arborisation longitudinale croisant ainsi plusieurs dendrites appartenant aux neurones striataux et faisant synapse en passant avec elles. Donc, plutôt que de transmettre une information neuronale spécifique a un petit groupe de neurones striataux tel que proposé par Kemp et Powell, le système corticostrié pourrait influencer un vaste groupe de neurones au sein d'un territoire striatal donné. Il semble tout de même exister une représentation somatotopique de la jambe, du bras et de la face sous forme de bandes obliques dans le striatum sensorimoteur (Kûnzle, 1975; Liles etUpdyke, 1985; Flaherty et Graybiel, 1993).

48 Le striatum est une structure nerveuse hétérogène pouvant être divisée en deux compartiments majeurs, soit les striosomes et la matrice.

Ces compartiments ont été

identifiés suite à l'étude de la distribution topographique de différentes protéines dans le striatum (Gerfen, 1984; Flaherty et Graybiel, 1991; Berendse et al., 1992; Brown, 1992; Parthasarathy et al., 1992; Flaherty et Graybiel, 1993). On sait maintenant que la matrice est innervée principalement par le cortex sensorimoteur alors que les striosomes sont la cible préférentielle des neurones du cortex limbique et préfrontal (Flaherty et Graybiel, 1993; Kincaid et Wilson, 1996; Lévesque et Parent, 1998). Cette hétérogénéité pourrait constituer un moyen de rassembler les signaux corticaux fonctionnellement reliés au sein du striatum afin qu'ils soient intégrés de façon plus efficace (Parent et Hazrati, 1995a). Chez les primates, les striosomes peuvent être facilement discernés dans le territoire associatif et limbique du striatum. Cependant, ils sont beaucoup plus difficiles à visualiser dans le territoire sensorimoteur (Côté et Parent, 1992; Desjardins et Parent, 1992; Sadikot et al., 1992a). Cette observation soulève des doutes quant à la validité du concept striosomes/matrice pour l'ensemble du striatum chez les primates. Depuis le début du 20ème siècle, on a tenté de savoir si la projection corticostriée était indépendante des autres projections corticofuges. À cette époque, l'analyse de sections du prosencéphale de rongeurs imprégnées par la méthode de Golgi a conduit Ramôn y Cajal à conclure que la projection corticostriée dérive de collatérales axonales émises par des axones corticofuges en route vers le tronc cérébral (Ramôn y Cajal, 1909, 1911). Cette observation a été supportée par des études anatomiques et électrophysiologiques chez le rat (Webster, 1961; Donoghue et Kitai, 1981; Cowan et Wilson, 1994). Chez les rongeurs, on pense maintenant que la projection corticostriée est composée principalement de collatérales d'axones projetant vers le tronc cérébral ou la moelle épinière mais aussi, de façon moins importante, d'axones corticostriés dédiés principalement au striatum mais ciblant souvent d'autres noyaux télencéphaliques (Donoghue et Kitai, 1981; Landry et al., 1984; Wilson, 1987; Cowan et Wilson, 1994; Lévesque et al., 1996a; Lévesque et al., 1996b; Lévesque et Parent, 1998; Zheng et Wilson, 2002; Reiner et al., 2003).

49 Chez les primates, la première représentation précise de l'organisation corticostriée nous vient d'études autoradiographiques effectuées chez le singe cynomolgus par Heinz Kûnzle (Kûnzle, 1975). Ces études ont permis de déterminer l'organisation topographique des champs terminaux corticostriés mais n'ont fourni que peu d'information en ce qui a trait au trajet axonal ainsi qu'aux patrons d'arborisations terminales difficiles à distinguer sur du matériel autoradiographique. Malgré ces difficultés techniques, Kûnzle suggéra que, plutôt que d'être formées de collatérales d'axones projetant vers le tronc cérébral ou la moelle épinière, les projections corticostriées font partie d'un système indépendant dédié uniquement au striatum (Kûnzle, 1975). Cette proposition a par la suite été appuyée par des études utilisant des traceurs rétrogrades (Jones et Wise, 1977) ainsi que par des investigations électrophysiologiques (Bauswein et al., 1989; Turner et DeLong, 2000) conduites chez le singe suggérant que le putamen reçoit un message en provenance du cortex cérébral qui est différent de celui transmis au tronc cérébral et à la moelle épinière.

Afin d'en arriver à une meilleure compréhension de l'organisation de la projection corticostriée chez les primates, nous avons effectué une analyse quantitative de l'organisation anatomique au niveau neuronal unitaire de ce système. Les résultats de cette étude sont colligés dans le cinquième chapitre de cette thèse; ils démontrent, contrairement à toutes attentes, l'existence de deux populations neuronales corticostriées distinctes au sein du cortex moteur primaire du primate. La première cible de façon directe le putamen dorsolatéral alors que la deuxième innerve le même territoire striatal, mais par le biais de collatérales émises par des axones en route vers le tronc cérébral.

1.7

Le complexe thalamique centre médian/parafasciculaire 1.7.1

Anatomie topographique

Les noyaux intralaminaires sont formés de différents groupes cellulaires localisés à l'intérieur de la lame médullaire interne du thalamus. Cette lame médullaire sépare la

50

masse thalamique médiane de la masse thalamique latérale. Chez les primates, les noyaux intralaminaires sont regroupés en une division rostrale comprenant les noyaux paracentral, central latéral et central médial ainsi qu'en une division caudale formée des noyaux centre médian (CM), parafasciculaire (Pf) et subparafasciculaire (Parent, 1996). Le noyau CM est situé entre le noyau dorsomédian et le noyau ventral postérieur du thalamus. Il est pratiquement entièrement entouré de fibres appartenant à la lame médullaire interne, à l'exception de sa partie médiane, adjacente au noyau Pf. Ce dernier entoure la partie dorsomédiane du faisceau habénulo-interpédonculaire ou faisceau rétroflexe de Meynert d'où il tire son nom. Les noyaux intralaminaires du groupe caudal appartiennent à une région thalamique qui semble avoir subi de nombreux changements au cours de l'évolution des espèces. Ainsi, bien que le noyau Pf ait été identifié chez tous les mammifères, le CM, quant à lui, semble être apparu plus tard au cours de l'évolution phylogénétique, parallèlement au développement du putamen (Mehler, 1966; Mehler, 1981).

Le neuropsychiatre français Jules Bernard Luys (1828-1897) est le premier à avoir réalisé que le thalamus est une masse hétérogène constituée de plusieurs groupements nucléaires qu'il dénommait « centre ». C'est lui qui, à partir de coupes de cerveaux humains durcies à l'acide chromique et colorées au carmin, a pour la première fois identifié le CM. Les magnifiques schémas en trois dimensions que l'on retrouve dans l'atlas accompagnant son traité de 1865 illustrent de façon remarquable l'organisation du thalamus (Luys, 1865) (Fig. 1.9). Bien qu'ils soient attenants l'un à l'autre, les noyaux CM et Pf n'ont pas été décrits en totalité par le même auteur. En effet, plus de quarante années séparent la découverte du CM par Luys de celle du Pf par C. Vogt puis par M. Friedemann chez le cercopithèque. Le noyau Pf fut d'abord nommé champ parafasciculaire par Vogt (Vogt, 1909) et ensuite nucleus parafascicularis par Friedemann (Friedemann, 1911). Ce n'est que récemment que le CM et le Pf ont été regroupés en un seul complexe (Jones, 1985). Ce regroupement peut être justifié par le fait que le complexe CM/Pf est séparé du reste du thalamus par une lame thalamique ainsi que par la similitude qui existe entre les efférences et les afférences du CM et du Pf.

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En se basant sur certains critères cytologiques et hodologiques, des auteurs ont proposé une division tripartite du complexe CM/Pf chez le primate (Aronson et Papez, 1934; François et al, 1988; François et al., 1991; Percheron et Filion, 1991; Sidibé et al, 1997). Dans l'article présenté au sixième chapitre de la thèse, nous avons plutôt préconisé une division bipartite de ce complexe telle que décrite dans l'atlas de Emmers et Akert (Emmers et Akert, 1963). Le choix de cette division se justifie par le fait que la coloration utilisée dans nos expériences ne nous a pas permis de visualiser les trois parties du complexe, telles que décrites par Percheron et collaborateurs, soit la pars lateralis, la pars média et la pars parafascicularis. En revanche, la coloration permettait une distinction nette entre le CM et le Pf. Mentionnons aussi que nos études n'impliquaient pas d'injections stéréotaxiques dans le noyau subparafasciculaire, une structure située entre le complexe CM/Pf et le tegmentum mésencéphalique (Aronson et Papez, 1934).

1.7.2

Cytologie

II existe peu d'études portant sur l'organisation somatodendritique des neurones du CM/Pf chez le primate. Deux types neuronaux ont été identifiés chez le singe dans le noyau Pf selon la présence ou non d'épines dendritiques et un seul dans le CM (Hazlett et al., 1976; Pearson et al., 1984). Une étude effectuée à partir de matériel imprégné selon la méthode de Golgi a démontré que les dendrites des neurones du Pf sont longues, épaisses, lisses et peu branchées alors que celles du CM ont un branchement plus important avec de nombreux prolongements axoniformes (Fénelon et al, 1994) (Fig. 1.10). Ces types neuronaux se distinguent clairement des autres neurones thalamocorticaux appartenant aux noyaux de relais du thalamus. Ces deniers sont appelés neurones en buisson puisqu'ils présentent un branchement dendritique beaucoup plus important.

Figure 1.9

Le thalamus selon Jules Bernard Luys. A, Schéma tridimensionnel montrant, entre autres, les connexions entre les quatre centres thalamiques (A, B, C, D) et certaines régions spécifiques du cortex cérébral. B, Schéma d'une section transversale du thalamus humain montrant, entre autres, deux des centres thalamiques définis par Luys, soit le centre moyen (9, 9') et le centre médian (10, 10'). (Luys, 1865)

52

A

4 :c

49

B

Figure 1.10 Dessins de neurones du complexe thalamique centre médian/parafasciculaire chez le macaque effectués à partir de matériel imprégné par la méthode de Golgi. A, B, Neurones du noyau centre médian (pars paralateralis) muni de dendrites ramifiées. La partie distale des dendrites se courbe parfois vers le corps cellulaire d'origine (astérisque) et présente des appendices axoniformes (têtes de flèche). C-F, Neurones du noyau parafasciculaire (C, D : pars média et E, F : pars parafascicularis) caractérisés par une arborisation dendritique moins ramifiée que celle présentée par les neurones du noyau centre médian. (Fénelon et al., 1994)

54

55

56

L'analyse tridimensionnelle du domaine somatodendritique des neurones du CM et du Pf. présentée au sixième chapitre de la thèse révèle l'existence de variations morphologiques importantes entre les neurones de ces deux noyaux thalamiques. Les corps cellulaires des neurones du CM marqués lors de nos expériences sont de taille moyenne et dotés de longues dendrites fortement branchées. Tel que décrit précédemment par Fénelon et collaborateurs, les dendrites sont souvent caractérisées par des appendices axoniformes pouvant accroître significativement leur capacité intégrative. La taille des corps cellulaires des neurones du Pf est plus importante que celle des neurones du CM. De plus, les neurones du Pf présentent un nombre moindre de dendrites primaires et la plupart d'entre eux sont dépourvues d'appendices axoniformes. La reconstruction tridimensionnelle du domaine somatodendritique des neurones du CM nous a permis de décrire une organisation trois fois plus étendue sur le plan sagittal que frontal permettant ainsi à un neurone du CM d'intégrer une multitude d'afférences distribuées à travers un vaste territoire du CM.

1.7.3

Hodologie 1.7.3.1 Afférences du complexe centre médian/parafasciculaire

Le Pf reçoit une projection importante en provenance du cortex prémoteur ainsi que des noyaux cérébelleux profonds (Mehler, 1971; Stanton, 1980; Asanuma et al., 1983). Le CM quant à lui reçoit des afférences du cortex moteur et sensoriel, de la moelle épinière, du noyau caudal du nerf trijumeau, du noyau parabrachial, du noyau cunéiforme, du prétectum ainsi que du noyau dorsal du raphé (Jones, 1985). Le complexe CM/Pf fait partie du système activateur ascendant puisqu'il reçoit une projection cholinergique du noyau PPN et participe à la modulation des états de vigilance de par ses projections diffuses au cortex cérébral (Groenewegen et Berendse, 1994). D'autre part, le noyau Pf semble impliqué dans l'occulomotricité puisqu'il reçoit une projection des collicules supérieurs et du cortex occulomoteur (Benevento et Fallon, 1975; Partlow et al., 1977; Harting et al., 1980).

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Le complexe thalamique CM/Pf reçoit aussi d'importantes afférences en provenance des « portes de sortie » des ganglions de la base, soit le GPi et la SNr (Beckstead et al, 1979; Ilinsky et al, 1985; Sidibé et al., 1997). Les axones du CM/Pf projettent à leur tour à la principale porte d'entrée des ganglions de la base soit le striatum. Ces projections font partie d'une boucle trisynaptique impliquant (1) les projections striatopallidales qui originent des neurones situés dans le territoire sensorimoteur du striatum et qui projettent au GPi, (2) la projection pallidothalamique qui provient de neurones du GPi et qui projettent au complexe thalamique CM/Pf, et (3) la projection thalamostriée qui origine du CM et dont les axones innervent le territoire sensorimoteur du striatum (Nauta et Mehler, 1966; Hazrati et Parent, 1992; Sadikot et al., 1992a; Parent et al., 2001). Cette boucle, appelée « boucle Nauta-Mehler », constitue un mécanisme neuronal susceptible de produire un effet de rétroaction positif puissant sur l'entrée de l'information neuronale au sein des ganglions de la base. Comme nous le verrons dans le troisième chapitre de la thèse, tous les neurones du GPi étudiés qui projettent au CM/Pf ciblent à la fois les noyaux moteurs du tiers ventral du thalamus ainsi que le noyau PPN par le biais de collatérales axonales. Une proportion importante des neurones du CM/Pf innervent à la fois le striatum et le cortex cérébral. L'ensemble de ces données suggère que, plutôt que de constituer une boucle fermée et liée en parallèle, la boucle Nauta-Mehler est ouverte et entremêlée à la boucle cortico-ganglions de la base-thalamo-corticale qu'emprunte l'information neuronale à travers les ganglions de la base. De plus, ces données indiquent que la projection du GPi vers le CM joue un rôle important en ce qui a trait à la modulation de l'effet excitateur qu'exercent les axones thalamostriés sur les neurones de taille moyenne du striatum. De par cette projection, les neurones du GPi, qui composent la principale porte de sortie des ganglions de la base chez le primate, participent à la modulation de l'entrée synaptique au sein du striatum, la porte d'entrée des ganglions de la base.

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1.7.3.2 Efférences du complexe centre médian/parafasciculaire

Les études de dégénérescence cellulaire rétrogrades conduites par Cowan et Powell ont mis en évidence une projection striatale dense en provenance des noyaux intralaminaires chez le singe (Cowan et Powell, 1956). On sait aujourd'hui que le complexe CM/Pf constitue la principale source de projections thalamostnees et que ces projections s'arborisent de façon complémentaire au sein du striatum (Kalil, 1978; Parent et al., 1983; Smith et Parent, 1986; Sadikot et al., 1990; Sadikot et al., 1992a), le CM innervant le territoire sensorimoteur du striatum et le Pf, le territoire associatif. De par ses afférences pallido- et nigro-thalamiques ainsi que ses efférences striatales, le complexe thalamique CM/Pf est étroitement lié aux ganglions de la base. Cette affirmation est supportée par la dégénérescence de certains neurones du complexe CM/Pf dans la maladie de Parkinson (Henderson et al., 2000).

On sait que les axones thalamostriés et corticostriés ciblent principalement les neurones épineux de taille moyenne situés dans la matrice striatale (Sadikot et al., 1992a; Sadikot et al., 1992b; Flaherty et Graybiel, 1993). Des études en microscopie électronique ont révélé que la plupart des contacts synaptiques d'origine thalamique s'établissent sur les dendrites alors que ceux provenant du cortex cérébral sont retrouvés principalement sur la tête des épines dendritiques (Frotscher et al., 1981; Somogyi et al., 1981; Sadikot et al., 1992b; Smith et al., 1994). Cet arrangement ultrastructural indique que les synapses asymétriques thalamostnees pourraient moduler de façon précise, l'activité des neurones épineux de taille moyenne du striatum recevant une afférence corticale (Fig. 1.11). Plusieurs évidences suggèrent une interaction fonctionnelle entre les terminaisons glutamatergiques et dopaminergiques au sein du striatum. On sait que la dopamine libérée par les terminaisons axonales des neurones de la SNc peut moduler directement l'activité des neurones épineux de taille moyenne du striatum et ce, en faisant synapse principalement sur les dendrites de ces derniers (Smith et al., 1994). Une étude récente a démontré de façon convaincante que la dopamine libérée dans la fente synaptique pouvait diffuser afin d'exercer une action inhibitrice sur la libération de glutamate en agissant sur les récepteurs D2 localisés sur la membrane présynaptique des terminaisons corticostriées (Bamford et al., 2004). Il semble

59

que cette inhibition hétérosynaptique soit sélective, agissant principalement sur un sousgroupe de terminaisons glutamatergiques les moins actives. La dopamine pourrait donc filtrer les informations neuronales qui entrent dans les ganglions de la base permettant de renforcer un sous-groupe spécifique de contacts corticostriés augmentant ainsi le contraste entre les signaux les plus significatifs et ceux qui le sont moins. On peut émettre l'hypothèse que cette interaction fonctionnelle entre la libération de dopamine et de glutamate puisse aussi s'appliquer aux axones thalamostriés. Jusqu'à ce jour, aucune étude convaincante n'en a fait la démonstration.

Faisant partie des noyaux intralaminaires, le complexe CM/Pf est reconnu pour projeter de manière diffuse au cortex cérébral (Jones et Leavitt, 1974). On croit que cette projection thalamocorticale joue un rôle crucial dans les mécanismes d'attention et d'éveil (Jones, 1998; Steriade, 2000; Jones, 2001). Le CM projette principalement au cortex moteur (Strick, 1975; Kûnzle, 1976; Akert et Hartmann-von Monakow, 1980) alors que le Pf projette au cortex cingulaire antérieur, préfrontal et prémoteur (Kievit et Kuypers, 1977; Vogt et al., 1987). Notre étude par traçage unitaire a démontré que les axones thalamocorticaux en provenance du CM/Pf s'arborisent principalement dans la couche V, suivie, en ordre décroissant, des couches VI et I.

Le complexe thalamique CM/Pf est donc très bien positionné pour moduler l'activité globale du cerveau ainsi que pour contrôler le comportement moteur en agissant à la fois sur le cortex cérébral ainsi que sur le striatum. Peu de données neuroanatomiques permettent de savoir si l'influence sur le cortex cérébral et le striatum est exercée par deux populations neuronales distinctes ou par des neurones innervant à la fois les deux structures. Une étude réalisée suite à l'injection de traceurs rétrogrades a suggéré que les neurones du CM qui projettent au cortex cérébral forment une population distincte de ceux innervant le striatum (Sadikot et al., 1992a). En revanche, une étude par traçage unitaire chez'le rat a démontré que pratiquement tous les neurones du Pf projettent à la fois au cortex cérébral ainsi qu'au striatum (Deschênes et al., 1996).

Figure 1.11 Organisation ultrastructurale des principales afférences qui convergent sur les neurones épineux de taille moyenne

du striatum.

L'encadré

montre

que

les

synapses

dopaminergiques (DA) sont retrouvées principalement sur le cou des épines dendntiques alors que les synapses glutamatergiques (GLU) en provenance du thalamus (THAL) et du cortex cérébral (CTX) se retrouvent principalement sur les dendrites et sur la tête des épines dendritiques, respectivement. (Parent, 1996)

60

o CD

CORTEX

1 LU

O CO

CE

Large Interneurons Medium-sized Interneurons

61

62

Plusieurs études anatomiques (Jones, 1975b; Cornwall et Phillipson, 1988; Kolmac et Mitrofanis, 1997; Tsumori et al., 2000) ont démontré que le noyau réticulaire du thalamus, dont les neurones utilisent le neurotransmetteur inhibiteur GABA (Oertel et Mugnaini, 1984), est lié réciproquement au complexe CM/Pf dont les neurones utilisent le neurotransmetteur excitateur glutamate (Streit, 1980; Christie et al., 1987). L'étude par traçage unitaire des projections efférentes du CM/Pf présentée au sixième chapitre de cette thèse démontre que la plupart des axones émergeant du CM/Pf émettent quelques collatérales au noyau réticulaire avant de quitter le thalamus. Ces projections pourraient faire partie d'un circuit intrathalamique de rétroaction négative capable de moduler l'effet synaptique exercé par les axones du CM/Pf sur le striatum ou le cortex cérébral. On peut émettre l'hypothèse que ce circuit constitue un substrat morphologique permettant une inhibition latérale pouvant atténuer l'effet des efférences des neurones du CM/Pf adjacents aux neurones du CM/Pf actifs. Ce filtre augmenterait ainsi la précision spatiale et temporelle de l'information neuronale envoyée au striatum et/ou au cortex cérébral.

1.8 Problématique de recherche Beaucoup de données sur les connexions des ganglions de la base ont été amassées au cours des dernières années suite à l'utilisation de différentes techniques telles que l'étude des terminaisons nerveuses en dégénérescence, l'injection de traceurs rétrogrades ou encore l'injection massive de traceurs antérogrades. Ces études ont permis de déceler les principales relations entre les différentes composantes de ganglions de la base. Peu d'études concernant la microcircuiterie fine sont cependant disponibles chez le primate. Ces études procurent une vision détaillée de l'organisation neuroanatomique unitaire des neurones composant les ganglions de la base. Elles permettent d'évaluer de façon juste et précise le degré de collatéralisation axonale des neurones étudiés en relation avec la localisation du corps cellulaire d'origine et l'organisation somatodendritique.

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1.9 Objectifs de recherche 1.9.1

Objectifs généraux

Mon programme de recherche au doctorat avait pour but de mieux comprendre l'organisation anatomique et fonctionnelle des ganglions de la base chez le primate. Les objectifs généraux de mon projet étaient: (1) de faire une analyse détaillée de la microcircuiterie fine au sein de certaines composantes des ganglions de la base, (2) d'établir l'importance et le rôle de la collateralisation axonale au niveau de ces mêmes structures, et (3) d'intégrer le phénomène de la collateralisation axonale en une façon nouvelle de voir l'organisation anatomo-fonctionnelle des ganglions de la base.

1.9.2

Objectifs spécifiques

Les objectifs spécifiques de mon projet de doctorat étaient de reconstruire, par traçage unitaire, l'arborisation axonale et dendritique complète des neurones de projection du GPi, du cortex moteur primaire et du complexe thalamique CM/Pf. Ces reconstructions neuronales unitaires devaient me permettre de (1) caractériser chaque type neuronal rencontré sur la base de ses projections efférentes, (2) déceler l'importance relative de chacun de ces types neuronaux, (3) déterminer de façon précise le trajet emprunté par les axones reconstruits, (4) déterminer le degré de collateralisation axonale de chacun des types neuronaux tout en apportant une attention particulière aux projections controlatérales, (5) déterminer le mode d'innervation axonale dans chacune des cibles, (6) déterminer la force de l'entrée synaptique de chaque collatérale axonale en dénombrant précisément les varicosités axonales émises dans chaque structure cible, et (7) déterminer la localisation des corps cellulaires de chaque type neuronal au sein de la structure étudiée afin de révéler une éventuelle organisation topographique basée sur les efférences de la structure.

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1.10 Approche méthodologique On retrouve parfois une divergence importante entre les résultats tirés des différentes études qui traitent des efférences d'une structure donnée. Cette disparité pourrait refléter une diversité interspécifique des systèmes neuronaux étudiés, mais elle pourrait aussi résulter des limitations imposées par les techniques utilisées. Tel que mentionné précédemment, l'injection de traceurs rétrogrades a permis d'amasser une quantité impressionnante de données sur les relations qu'entretiennent les composantes des ganglions de la base entre elles. Une des limitations majeures de la technique d'injection de double traceurs rétrogrades est la possibilité de voir apparaître des faux-négatifs. Ce phénomène peut survenir s'il existe une différence entre les deux traceurs utilisés en ce qui a trait au taux de capture par les terminaisons axonales ou encore, si les deux traceurs sont injectés dans des régions qui ne concordent pas parfaitement avec les sites de terminaisons axonales du neurone étudié. Cette limitation est d'autant plus importante lorsque l'emplacement exact des champs terminaux de l'axone n'est pas connu. Elle s'applique aussi, et pour les mêmes raisons, aux études électrophysiologiques faisant usage d'invasions antidromiques. L'injection massive de traceurs antérogrades est une autre technique couramment utilisée en hodologie. Cependant, tout comme c'est le cas pour la double injection de traceurs rétrogrades, la possibilité de marquer des fibres de passage a souvent créé une discordance entre les données retrouvées dans la littérature. La limitation principale de l'injection massive de traceurs antérogrades réside dans la trop grande quantité d'axones marqués ne permettant pas l'étude de la microciruiterie au niveau neuronal unitaire. Ce problème est particulièremet

sérieux lorsqu'une

structure contient des populations

neuronales

hétérogènes, comme c'est souvent le cas dans les ganglions de la base.

Les travaux de recherche décrits dans cette thèse ont permis d'aborder l'importante question de la coUatéralisation axonale au sein des ganglions de la base chez le primate en utilisant cette fois une approche beaucoup plus directe et puissante. Cette dernière consiste en l'injection stéréotaxique d'un traceur antérograde par microiontophorèse ce qui permet de marquer un petit groupe de neurones et de reconstruire complètement et

65 individuellement leurs arborisations axonale et dendritique. De plus, cette technique de pointe permet d'enregistrer l'activité électrophysiologique des neurones rencontrés lors de la descente de la micropipette de verre qui sert à la fois aux enregistrements et à l'injection. Puisque les neurones composant les structures étudiées possèdent des patrons de décharge caractéristiques, nous sommes en mesure de confirmer l'emplacement de la micropipette déterminé à l'aide des coordonnées stéréotaxiques et de l'atlas stéréotaxique (Emmers et Akert, 1963; Szabo et Cowan, 1984).

Ces études ont été réalisées chez le singe écureuil (Saimiri sciureus) et chez le singe macaque (Macaca fascicularis). Les animaux ont été maintenus en captivité au sein de colonies composées de plusieurs individus hébergés dans une salle commune munie de cordes et de perchoirs sur lesquels ils pouvaient se déplacer et se reposer. L'ensemble du travail méthodologique a été réalisé conformément au manuel canadien sur les soins et l'utilisation des animaux d'expérimentation. Les procédures chirurgicales ainsi que les soins prodigués aux animaux ont été approuvés par le comité de protection des animaux de l'Université Laval. Étant donné l'importance de la variation interindividuelle des dimensions de l'encéphale chez les primates, il est important d'effectuer une ventriculographie préalablement à l'injection du traceur. Pour ce faire, les animaux sont anesthésiés avec un mélange de kétamine et de xylazine et leur tête est maintenue dans un appareil stéréotaxique spécialement conçu pour les primates. Suite à la trépanation, une solution radio-opaque est injectée par pression à l'aide d'une microseringue insérée dans le ventricule latéral droit. Quelques minutes après l'injection, des radiographies latérales et frontales du système ventriculaire sont prises afin de localiser précisément la commissure antérieure et postérieure de chaque animal (Percheron, 1975) (Fig. 1.12).

Figure 1.12 Radiographies frontale (A) et latérale (B) du crâne d'un singe écureuil {Saimiri sciureus) prises lors d'une ventriculographie. B, L'injection d'un liquide radio-opaque dans le ventricule latéral droit nous permet de localiser précisément la commissure antérieure (1) et postérieure (2). Avec ces mesures, il est possible d'ajuster les coordonnées stéréotaxiques de l'atlas afin de pallier les différences interindividuelles des dimensions de l'encéphale. (Percheron, 1975)

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67

Une à trois semaines après la ventriculographie, les animaux sont anesthésiés à nouveau et placés dans le même appareil stéréotaxique. Nous utilisons alors les coordonnées stéréotaxiques retrouvées dans les atlas (Emmers et Akert, 1963; Szabo et Cowan, 1984) corrigées par les données obtenues lors de la ventriculographie. L'injection d'un traceur antérograde soit la biotine dextran aminé (BDA, Molecular Probes, Eugène, OR) s'effectue par microiontophorèse à l'aide d'une micropipette de verre dont le diamètre à la pointe est de 2-3/xm. Suite à l'injection, il faut laisser vivre l'animal pendant une période de 7 à 10 jours afin de permettre la migration complète du traceur neuronal jusqu'aux terminaisons axonales. Par la suite, une perfusion intracardiaque est pratiquée afin d'effectuer une première fixation du cerveau. Subséquemment, le cerveau est post-fixe et coupé en tranches de 70 /xm à l'aide d'un microtome à congélation. Le traceur neuronal injecté est ensuite révélé par immunohistochimie. En utilisant la technique d'injection juxtacellulaire développée dans notre laboratoire (Pinault, 1996), il est possible de marquer un seul neurone à la fois. L'ensemble des expérimentations présenté dans cette thèse a été réalisé chez le singe. Nous avons donc préconisé le marquage de 5-10 neurones par site d'injection afin de s'assurer d'obtenir suffisamment de neurones complètement marqués et reconstruits pour définir les différents types neuronaux existant dans la structure étudiée.

Habituellement, la BDA est injectée sur deux sites différents de chaque côté du cerveau de l'animal. Il faut noter que l'injection d'une faible quantité de BDA permet d'éviter un trop grand entremêlement des axones marqués. Ceci facilite la reconstruction individuelle entière des neurones. L'arborisation axonale peut être très longue. Dans le cas des neurones du GPi, la longueur axonale totale peut atteindre 27 cm chez le singe macaque. L'axone présente habituellement un trajet tortueux et est donc sectionné à plusieurs reprises par les plans de coupe d'une épaisseur de 70 um. Les reconstructions axonales sont donc réalisées en alignant et en mettant bout à bout les segments axonaux retrouvés sur les différentes sections. L'arborisation axonale est d'abord dessinée et reconstruite à partir de coupes sériées et à l'aide d'un microscope optique et d'une caméra lucida afin d'obtenir une visualisation intégrale plane. L'utilisation d'un microscope muni d'une platine motorisée et reliée à un ordinateur nous permet de reconstruire, dans les trois plans de l'espace, l'arborisation axonale et dendritique des neurones marqués. Pour ce faire, chaque segment

69 axonal est tracé à l'écran et transformé en un ensemble de points dont les coordonnées tridimensionnelles sont enregistrées par l'ordinateur. L'utilisation d'un logiciel d'analyse neuroanatomique (Neurolucida, MicroBrightField, Inc., Colchester, VT) nous permet d'obtenir des données précises concernant (1) les paramètres topologiques, comme l'étendue dans l'espace de l'arborisation axonale et dendritique, ainsi que (2) les paramètres métriques, comme la longueur et le diamètre de l'axone et des dendrites ainsi que le nombre de varicosités axonales émis par un neurone dans ses différentes structures cibles. Puisque les varicosités axonales correspondent aux sites présumés de libération du neurotransmetteur, cette dernière analyse permet d'évaluer la force relative de l'entrée synaptique de chaque collatérale axonale. Dans l'ensemble, cette approche méthodologique constitue un outil puissant permettant d'obtenir une vision juste, précise et détaillée de l'organisation unitaire des neurones composant les ganglions de la base. Les résultats obtenus avec cette technique modifient considérablement la vision que nous avions de l'organisation anatomo-fonctionnelle des ganglions de la base chez les primates.

CHAPITRE 2 THE PALLIDOFUGAL PROJECTION SYSTEM IN PRIMATES: EVIDENCE FOR NEURONS BRANCHING IPSILATERALLY AND CONTRALATERALLY TO THE THALAMUS AND BRAINSTEM

CHAPITRE 2

THE PALLIDOFUGAL PROJECTION SYSTEM IN PRIMATES: EVIDENCE FOR NEURONS BRANCHING IPSILATERALLY AND CONTRALATERALLY TO THE THALAMUS AND BRAINSTEM

Martin Parent, Martin Lévesque and André Parent

Laboratoire de Neurobiologie Centre de recherche Université Laval Robert-Giffard 2601, de la Canardière, Beauport, Québec Canada Gl J 2G3

The Journal of Chemical Neuroanatomy (1999) 16:153-165

2.1 RESUME Cet article fait la synthèse des résultats neuroanatomiques portant sur les projections pallidofuges du singe écureuil (Saimiri sciureus) accumulés durant ces dernières années dans notre laboratoire. Des données obtenues par injections de traceurs antérogrades, par double injections de traceurs rétrogrades fluorescents ainsi que par traçage unitaire y sont présentées. L'injections de traceurs antérogrades dans le pallidum interne a permis de marquer des axones qui entrent dans plusieurs noyaux thalamiques dont le centre médian, l'habénula latérale, les noyaux du tiers ventral du thalamus ainsi que dans le noyau pédonculopontin du tegmentum mésencéphalique. Les projections pallidofuges semblent être composées d'axones qui projettent à la fois au tiers ventral du thalamus et au noyau pédonculopontin ainsi que de neurones qui s'arborisent à la fois dans les noyaux du tiers ventral et dans le noyau intralaminaire centre médian du thalamus. L'utilisation de la technique de double marquage rétrograde ainsi que la reconstruction axonale unitaire confirment ce haut degré de collatéralisation. L'injection massive de traceurs antérogrades a permis de décrire des axones pallidofuges qui croisent la ligne médiane et s'arborisent dans les principales cibles pallidales controlatérales. L'injection combinée de traceurs rétrogrades fluorescents appuie l'existence de ces projections controlatérales. Les axones pallidofuges projetant homolaterallement ainsi que controlaterallement au thalamus et au tronc cérébral décrits dans cet article sont susceptibles de jouer un rôle crucial dans l'organisation fonctionnelle des ganglions de la base du primate.

2.2 ABSTRACT

This paper summarizes the results of some of our previous neuroanatomical studies on the pallidofugal projections in squirrel monkeys (Saimiri sciureus) and also reports more récent data obtained with double rétrograde and single-axon tracing methods.

Injections of

anterograde tracers in the internai pallidum label axons that reach the ventral tier, centromedian and latéral habenular thalamic nuclei, as well as the pedunculopontine tegmental nucleus. The pallidofugal projections are composed of axons that branch to the ventral tier and pedunculopontine nuclei, and to ventral tier and centromedian nuclei. Double rétrograde labeling with fluorescent tracers and single-axon tracing confirm this high degree of collateralization.

Furthermore, some pallidal labeled axons cross the

midline and arborize contralaterally in the major pallidal targets. fluorescent labeling experiments support thèse findings.

Double rétrograde

Pallidal axons that branch

ipsilaterally as well as contralaterally to the thalamus and brainstem could play a crucial rôle in the functional organization of primate basai ganglia.

74

2.3

INTRODUCTION

The internai segment of the globus pallidus (GPi) in primates, the presumed homologue of the entopeduncular nucleus (EN) in non-primates, is a major output nucleus of the basai ganglia (Carpenter, 1981; Parent and Hazrati, 1995a). The GPi in primates is known to project to several thalamic nuclei, principally the ventral anterior (VA)/ventral latéral (VL) nuclei (the so-called ventral tier nuclei), the centromedian (CM)/parafascicular (Pf) complex, the latéral habenular nucleus (HL), and it also sends input to the pedunculopontine tegmental nucleus (PPN) of the midbrain-pontine tegmentum (Nauta and Mehler, 1966; Kuo and Carpenter, 1973; Kim et al., 1976; DeVito and Anderson, 1982; Parent and De Bellefeuille, 1982; Parent and De Bellefeuille, 1983; Parent and Hazrati, 1995a). The pioneering axonal degeneration study of Nauta and Mehler (1966) has led to the suggestion that the pallidal projections to the thalamus and brainstem arise mostly from axon collaterals of the same GPi neurons. This view received support

from

electrophysiological (antidromic invasion) experiments (Filion and Harnois, 1978; Harnois and Filion, 1980; Harnois and Filion, 1982) and double rétrograde cell labeling studies (Parent and De Bellefeuille, 1982; Parent and De Bellefeuille, 1983). In view of the rôle that the GPi plays in the overall organization of the basai ganglia, we thought it useful to briefly review our knowledge of the anatomical organization of the pallidofugal projections in primates.

The présent paper stems from a talk that was given at a symposium entitled 'Thalamic Intégration of Basai Ganglia Signais' as part of the 1998 Forum of European Neuroscience held in Berlin. The aim of the paper is to provide a personal view of the organization of the pallidofugal projection System in primates. The présent contribution should by no means be considered a detailed review on the subject, and we apologize to ail the investigators whose relevant work has not been cited hère because of space limitation. The first part of the paper consists of a brief summary of the results of our previous studies on the pallidofugal projections undertaken with both rétrograde and anterograde labeling methods in the squirrel monkeys. This sets the scène for the second part of the paper which deals with

75 more récent data obtained with double rétrograde cell labeling procédures and with singleaxon tracing methods in both squirrel and cynomolgus monkeys. In this paper, emphasis will be placed on the contralateral pallidothalamic projections, which may play a crucial rôle in the functional organization of the primate basai ganglia.

2.4 2.4.1

MATERIALS AND METHODS Fluorescence double rétrograde labeling experiments

Twenty maie, adult (body weight, 900-1100 g) squirrel monkeys (Saimiri sciureus) were used for fluorescence double rétrograde labeling experiments. We employed a procédure based on the rétrograde transport of two or more substances that fluoresce maximally at différent wavelengths and which is particularly suitable for the study of neuronal Systems having multiple axonal processes terminating in différent brain areas (van der Kooy et al., 1978). Ail animais were anesthetized with ketamine hydrochloride (Ketaset, 40 mg/kg, i.m.) before receiving fluorescent tracer injections. The anesthesia and surgical procédures were performed according to the guidelines of the Canadian Council on Animal Care and the Laval University Committee on Animal Research approved our expérimental protocol. We used three différent combinations of fluorescent tracers: (1) Evans Blue (EB) and DapiPrimuline (DP); (2) Fast Blue (FB) and Nuclear Yellow (NY); and (3) True Blue (TB) and NY. The tracers were injected with 1-ul Hamilton syringes in quantities ranging from 0.2 to 0.4 ul, over 2-3 microsyringe pénétrations. The stereotaxic coordinates were chosen according to the atlas of Emmers and Akert (1963). The injections were made on the same side according to the following combinations: (1) VA/VL and HL (three animais); (2) VA/VL and CM/Pf (four animais); (3) VA/VL and PPN (six animais); (4) CM/Pf and PPN (three animais); (5) substantia nigra (SN) alone (two animais); and (6) VA/VL/CM and PPN (two animais). The thalamic injections were made with a vertical approach, whereas a latéral approach was used to reach the PPN and the SN in order to avoid spillage of the tracer in the mesencephalic tegmentum and the superior colliculus. After the appropriate

76

survival period, the animais were administered an overdose of pentobarbital and perfused with 10% sucrose in phosphate buffer, followed by 1 liter of a fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer, followed by a solution of 4% paraformaldehyde. The brain was stereotaxically eut into transverse slabs and placed in phosphate buffer containing 30% sucrose for 12 h at 4°C. The brain slabs were then sectioned on a freezing micro tome at 40 um in the transverse plane. The sections (one out of three) were serially collected in distilled water, mounted on slides without coverslip and air-dried. They were examined with a Leitz microscope equipped with an epifluorescence condenser and appropriate excitation/émission filters. Some sections were also stained with cresyl violet in order to ensure a proper identification of brain structures.

2.4.2

Anterograde labeling expérimenta

Two other squirrel monkeys were used for axon tracing experiments. The animais were anesthetized with ketamine hydrochloride (Ketaset, 40 mg/kg, i.m.) and received unilatéral iontophoretic injections of the anterograde tracer Phaseolus vu/gam-leucoagglutinin (PHA-L) into the GPi over two needle pénétrations. A 2.5% solution of PHA-L (Vector Labs, Burlingame, CA) was prepared by dissolving 5 mg of PHA-L in 200 (il of phosphate buffer (0.01 M, pH 8.0). This solution was loaded in a glass micropipette with a tip diameter of 25-30 um and was iontophoretically injected with a 7-10 uA positive (cathodal) current delivered in 7-s puises every 14 s over a 20-30 min period using a constant current generator (Midgard Electronics). The micropipette was attached to a micromanipulator that was stereotaxically driven and the stereotaxic coordinates were chosen according to the atlas of Emmers and Akert (1963). Following a survival period of 12 days, the animal was deeply anesthetized with an overdose of pentobarbital and perfused transcardially with 250 ml of phosphate buffer (0.1 M, pH 7.4) containing 10% sucrose. The perfusion was pursued by 600 ml of a fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4), and finally with 400 ml of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) at 4°C. Following perfusion, the head of the animal was placed in the stereotaxic apparatus and,

77

after removal of the cranial vault, the brain was sectioned stereotaxically in 5-10 mm-thick transverse slabs. Thèse slabs were postfixed for 1 h in 4% paraformaldehyde fixative, and placed for 18-24 h in a phosphate buffer (0.1 M, pH 7.4) containing 30% sucrose at 4°C. The brain slabs were then sectioned on a freezing microtome at 40 um in the transverse plane. The sections were serially collected, kept in 0.1 M Tris-buffered saline (TBS, pH 7.4) at 4°C, and then processed for the immunohistochemical localization of PHA-L. In addition, some alternate sections taken at forebrain and brainstem levels were stained with cresyl violet.

The présence of PHA-L was revealed immunohistochemically by pre-incubating the sections for 1 h in TBS containing 0.1% Triton X-100 and 2% normal rabbit sérum (NRS). They were then incubated overnight at 4°C with a goat anti-PHA-L (Vector labs) diluted 1:2000 in TBS containing 0.1% Triton X-100 and 1% NRS. After this primary incubation, the sections were rinsed three times (10 min each) in TBS, incubated for 1 h at room température with biotinylated rabbit anti-goat IgG (Vector Labs) diluted 1:200 in a TBS solution containing 0.1% Triton X-100 and 1% NRS. They were then rinsed three times (for 20 min) in TBS and incubated for 1 h at room température in a solution containing the avidin-biotin-peroxidase complex (ABC; Vector Labs, 1:100 dilution). After a 30-min rinse in TBS, the bound peroxidase was revealed by placing the sections in a solution containing 3,3'-diaminobenzidine tetrahydrochloride (DAB, 0.05%) and hydrogen peroxide (H2O2, 0.005%) in TBS buffer (0.05 M, pH 7.6) for 15-20 min at room température. The immunostained sections were then rinsed in TBS (5x5 min) and mounted onto gelatincoated slides with Permount to be examined with a light microscope under both bright- and darkfield illumination.

2.4.3

Single-axon tracing experiments

Two maie, adult (body weight of 3-3.5 kg), cynomolgus monkeys (Macaca fascicularis) were used for single-axon tracing experiments. The animais were anesthetized as above and microiontophoretic injections of biocytin (Sigma, St Louis, MO) or biotin dextran

78 aminé (BDA; Molecular Probes, Eugène, OR) were made in différent portions of the GPi bilaterally using the stereotaxic atlas of Szabo and Cowan (1984). Microiontophoretic labeling was carried out with glass micropipettes (tip diameter 3-4 um) filled with a solution of potassium acétate (0.5 M) plus 2% biocytin or 2% BDA. Thèse électrodes had an impédance of 15-25 MQ and were used to monitor the typical spontaneous activity of GPi neurons. Once in the GPi, the pipette was connected to a high compliance iontophoresis device (Neuro Data) and the tracer was ejected by passing positive current puises of 300-400 nA (1 s on/1 s off) for 30 min. After a survival period of 24 h (biocytin) or 48 h (BDA), the animais received an overdose of sodium pentobarbital. They were then perfused with 500 ml of phosphate buffered saline (PBS, 0.1 M, pH 7.4) containing 0.5 ml heparin, followed by 1 liter of a fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4). After a final wash with 400 ml of a 10% sucrose solution in phosphate buffer, the brains were dissected out and placed in a cryoprotective solution (30% sucrose in phosphate buffer) until immersion. The brains were then sectioned in the sagittal plane at 50 um on a freezing microtome and the sections processed for the visualization of biocytin or BDA according to the ABC histochemical protocol

(Lévesque

et

al.,

1996b).

Nickel-intensified

3,3'-diaminobenzidine

tetrahydrochlonde (NiDAB) was used as the chromogen to reveal either biocytin or BDA (dark purple precipitate). Sections were mounted on gelatin-coated slides and covered with Permount. Labeled neurons were drawn with a caméra lucida using *20

an

d

x

40

objectives. Their axonal fields were also mapped at low magnification to refer their position to corresponding planes in the atlas of Szabo and Cowan (1984).

79

2.5 RESULTS 2.5.1

Rétrograde tracer experiments: ipsilateral cell labeling

2.5.1.1

Thalamus (VA/VL) and habenula (HL) injections

In animais that received injections of one fluorescent tracer into the VA/VL nuclei and the complementary tracer into the HL on the same side of the brain, a large number of neurons retrogradely labeled with the tracer injected into the thalamus occurred in the central twothirds of the ipsilateral GPi (Fig. 2.1 A). In contrast, pallidal neurons containing the tracer injected into the habenula were much less numerous and mostly confïned to the periphery of the GPi. Thèse pallidohabenular neurons merged with a large population of similarly labeled neurons in the latéral hypothalamus. Very few doubly labeled neurons could be found at pallidal level after thalamus-habenula injections.

2.5.1.2

Thalamus (VA/VL) and intralaminar nuclei (CM/PJ) injections

In this group of animais, numerous neurons labeled with the tracer injected in the VA/VL thalamic nuclei were présent in the center of GPi, compared to a much smaller number of neurons projecting to the CM/Pf thalamic complex (Fig. 2.1 B). We estimated that the number of pallidointralaminar neurons amounted approximately to 30-35% of the number of pallidothalamic neurons. The pallidointralaminar neurons formed two interrelated clusters, one located dorsolaterally in the GPi (Fig. 2.1 B,D) and the other ventromedially along the accessory medullary lamina (Fig. 2.1 B). As much as 80% of the neurons in thèse two clusters were doubly labeled.

80

2.5.1.3

Thalamus (VA/VL) and brainstem (PPN) injections

A multitude of doubly labeled neurons occurred in the GPi after thalamus/brainstem injections (Fig. 2.1 C). It was estimated that the number of doubly labeled neurons amounted approximately to 70% of ail the labeled GPi neurons. A significant number of neurons projecting to the thalamus and brainstem were also présent in the contralateral GPi.

2.5.1.4

Intralaminar nuclei (CM/PJ) and brainstem (PPN) injections

The distribution of GPi cells projecting to the CM/Pf complex and to the PPN in this expérimental group was overall similar to that reported for groups 2 and 3, respectively (Fig. 2.1 D). However, at variance with the results obtained after thalamus/intralaminar nuclei injections, only approximately 15-20% of the neurons forming the two clusters were doubly labeled after intralaminar nuclei/brainstem injections.

2.5.1.5

Substantia nigra (SN) injections

In the monkeys of this expérimental group, the SN injections were rather massive and involve both the pars compacta (SNc) and reticulata (SNr) of the structure, but spared the subthalamic nucleus (STN). After such injections a moderate number of labeled cells occurred in the GPe, whereas no rétrograde cell labeling was found in the GPi. The latter structure was nevertheless closely surrounded by numerous retrogradely labeled neurons belonging to the nucleus basalis of Meynert (Fig. 2.1 E).

2.5.2

Rétrograde tracer experiments: bilatéral cell labeling

As mentioned above, some retrogradely labeled cells were also encountered in the contralateral GPi after injections in either the thalamus or the brainstem. The degree of

81

bilatéral labeling was studied in détails in the two monkeys of this sixth expérimental group, which received massive injections of NY at the thalamic level and of FB in the PPN. The injection sites in the thalamus involved the dorsal two-thirds of the VA/VL and the central portion of the CM (Fig. 2 C-E). Thèse injection loci did not encroach upon the adjacent reticular nucleus and did not spread into the contralateral side. The PPN injection site covered most of the rostrocaudal extent of the nucleus. The tip of cannula was centered upon the brachium conjunctivum, but the tracer had diffused within most of the dorsoventral extent of the PPN (Fig. 2.2 F). Retrogradely labeled cells were encountered bilaterally in the GPi, the SNr, the reticular thalamic nucleus, the GPe and the zona incerta (Fig. 2.2). In the ipsilateral GPi, the majority (70-80%) of labeled cells contained both fluorescent tracers and thèse doubly labeled cells were distributed throughout the core of the structure (Fig. 2.3 A). Singly labeled pallidothalamic cells were found to be particularly abundant dorsolaterally, whereas singly labeled pallidotegmental cells appeared more numerous ventromedially in the GPi (Fig. 2.2 A-D; Fig. 2.3). In the contralateral GPi, the number of retrogradely labeled cells was approximately 10-20% that found in the ipsilateral GPi. The pallidotegmental cells were more numerous than the pallidothalamic cells, and some doubly labeled neurons also occurred in the contralateral GPi (Fig. 2.2 BE). Thèse doubly labeled neurons represent approximately 35% of the total number of retrogradely labeled cells in the contralateral GPi. The retrogradely labeled cells in the reticular nucleus of thalamus contained NY only and were présent bilaterally throughout most of the rostrocaudal and dorsoventral extent of the structure (Fig. 2.2). In the zona incerta, NY-labeled cells abounded rostrally, whereas FB-labeled cells predominated caudally. The bilatéral labeling in the SN was confmed to the SNr, and the majority of the labeled cells contained the tracer FB injected into the PPN (Fig. 2.2 E). In the SN the number of labeled cells was much greater ipsilaterally than contralaterally. In the reticular nucleus, the retrogradely labeled cells were also more numerous ipsilaterally although the number of contralateral NY-labeled cells was impressive (Fig. 2.2 B-E).

82 2.5.3

Anterograde tracer experiments: ipsilateral axonal Iabeling

In the monkeys of this expérimental group, two major PHA-L injection sites in continuity with one another occurred in the GPi. The first one had its maximal extent at a level corresponding to the stereotaxic plane A 11.5, according to the atlas of Emmers and Akert (1963). This injection site covered most of the central core of the GPi (Fig. 2.4 A). The second injection site had its maximal extent at stereotaxic level A 10 and was about the same size as the first injection site (Fig. 2.4 B).

Numerous anterogradely labeled axons emerged from the injection sites in the GPi and coursed along the ventral and dorsal surfaces of the pallidal complex, that is either along the ansa lenticularis rostrally and the lenticular fasciculus caudally, en route to the thalamus. Thèse iïbers met at the medioventral tip of the GPi and continued medially at the basis of the internai capsule. Rostrally, the fïbers reached the thalamus mostly by coursing along the ansa lenticularis and arborized principally within the rostral portion of the VA nucleus (Fig. 2.4 A). More caudally, they formed several fascicles that pierced the internai capsule and accumulated into the lenticular fasciculus, which appeared as a rather compact bundle (Fig. 2.4 B). This bundle broke-off into two différent fiber Systems. The first one curved laterodorsally and swept rostrally along the thalamic fasciculus to reach the caudal portion of the VA nucleus and the oral part of the VL nucleus (VLo). The second one was composed of several fascicles that separated from the lenticular fasciculus in a straight caudal and dorsomedial course to reach the CM, the VL and, less abundantly, the ventralmost portion of the lateroposterior (LP) thalamic nuclei (Fig. 2.4 C, D).

Caudal to the lenticular fasciculus, numerous labeled fïbers accumulated within the Forel's field H, and from there a large portion of the labeled fïbers could be followed up to the caudal portions of CM and VL (Fig. 2.4 D). At the level of the caudal pôle of CM, a smaller contingent of fïbers ran dorsocaudally close to the midline and reached the habenula (Fig. 2.4 E). Other labeled fïbers left the area of ForePs fields, swept caudally, and descended within the central portion of the midbrain tegmentum (Fig. 2.4 E). Thèse fïbers could be followed down to the PPN where most of them appeared to terminate.

83 Additionally, some labeled fibers ran into the cérébral peduncle and contributed to the innervation of the PPN.

2.5.4

Anterograde tracer experiments: axonal arborization

The pallidothalamic fibers displayed différent patterns of arborization within each target structure. They appeared as typical glomerule-like plexuses in the VA, VLo, LP and VL nuclei (Fig. 2.5 A). Thèse glomeruli occurred throughout the rostrocaudal extent of the VA, but abounded particularly in the dorsolateral sector of the nucleus. Similar plexuses were encountered in the dorsolateral région of the VLo and in much of the remaining portion of the VL, except for an area near the medullary lamina, which was devoid of such plexuses but contained numerous thin and rather unbranched fibers heading for the dorsolateral portion of the VA/VL. Only a few glomerule-like terminal fields were noted in the LP, and most of them occurred at the rather fuzzy junction zone between LP and VL.

In the CM most labeled fibers were long, varicose and directed mostly mediolaterally. Thèse long and coarse fibers were intermingled with shorter and thinner fibers that gave rise to a rather dense terminal field covering a large portion of the CM (Fig. 2.5 B). By companson, the Pf was virtually devoid of labeled fibers and terminais. In the dorsal portion of CM, several long and coarse fibers were seen to continue their course laterally beyond the limits of CM to reach the VL nucleus. In the habenula, the labeling appeared as a small but dense terminal field within the latéral portion of HL (Fig. 2.5 C). By companson to the numerous and highly ramified fibers that occurred in the thalamus after GPi injections, the labeled fibers in the PPN were fewer and poorly branched. Most of thèse fibers exhibited a rather short and sinuous course.

84 2.5.5

Anterograde tracer experiments: contralateral axonal labeling

Numerous labeled axons were seen crossing the midline at both thalamic and brainstem levels. The majority of thèse decussating axons traversed the midline at the rostral pôle of the CM and in the supramammillary decussation (Fig. 2.4 C). Other fibers decussated more caudally in the core of the midbrain tegmentum and in the posterior commissure. Several fibers crossing at the CM level terminated within the contralateral CM, where their pattern of arborization was similar to that seen in the ipsilateral CM (Fig. 2.4 D). It consisted of thin, long and varicose axons directed mediolaterally and giving rise to a dense terminal field composed of shorter axon collaterals and axon terminais that occurred in the CM, but completely avoid the Pf. Some coarse fibers were also noted among the thinner axons in the dorsal portion of the CM. Thèse fibers continued their course laterodorsally to reach the VA (Fig. 2.4 B), the VLo and the remaining portion of the VL (Fig. 2.4 C, D). They formed glomerule-like plexuses identical to those found ipsilaterally in the thalamus. At brainstem levels some relatively short, sinuous and nonvaricose labeled axons were found in the contralateral PPN; no labeling was detected in the habenula.

2.5.6

Single-axon tracing experiments

Microiontophoretic injections of biocytin or BDA in the GPi led to the labeling of single cells within différent portions of the structure. Thèse neurons had a large and elongated perikarya that gave rise to several long, thick and slightly varicose dendrites (Fig. 2.6 B). The axon of thèse neurons emerged either from the perikarya or a proximal dendrite and could be traced along its entire trajectory. Thèse axons were seen to branch within the GPi itself. They gave rise first to a long branch that coursed caudally to the mesopontine brainstem tegmentum (Fig. 2.6). This branch passed beneath the STN and did not émit collaterals along its way. Other branches were seen to émerge from the main axon of the pallidal neurons in the GPi. Thèse branches also headed towards the brainstem, but gave rise to at least one major collatéral which, at the STN level, ascended dorsally within the ventral tier thalamic nuclei (Fig. 2.6). This major collatéral branched into about 10-15 smaller collaterals that ran throughout large terri tories of the ventral tier thalamic nuclei.

85

Thèse collaterals, however, developed varicosities only at their terminal tips and only in very spécifie sectors of the VA/VL thalamic nuclei. At thèse levels, numerous axonal varicosities and terminal boutons were seen to closely surround the unstained neurons of the ventral tier thalamic nuclei (Fig. 2.6 A).

2.6 DISCUSSION 2.6.1

Origin and collateralization of the pallidofugal projections

Our rétrograde cell labeling studies hâve revealed that neurons projecting to the VA/VL nuclei occurred in the core of the GPi along its entire rostrocaudal extent, whereas those projecting to the CM formed two more or less continuous clusters lying in the dorsolateral and ventromedial portions of the GPi. Pallidal neurons projecting to the PPN were also confined to the core of the GPi, whereas those projecting to the habenula were located more peripherally and abounded particularly at the ill-defmed junction between the GPi and the latéral hypothalamus. Based on its connections, the latter area is considered as the 'limbic' territory of the pallidum (Parent, 1990). Data from our rétrograde cell labeling studies reveal that the limbic territory of the GPi and the adjoining latéral hypothalamus represent major sources of afférents to HL in primates.

In agreement with the results of antidromic invasion studies (Harnois and Filion, 1980; Harnois and Filion, 1982), fluorescence rétrograde double-labeling investigations (Parent and De Bellefeuille, 1982; Parent and De Bellefeuille, 1983) hâve revealed that GPi neurons in primates display a high degree of axonal collateralization. In fact, the majority of pallidal neurons were found to send axon collaterals to the VA/VL and the PPN, the VA/VL and the CM, or to the three of them. In contrast, the pallidohabenular projection was found to arise predominantly from a distinct neuronal population. In rats and cats the pallidohabenular projection was reported to arise from pallidal neurons other than those projecting to the VA/VL, the CM and the PPN (Filion and Harnois, 1978; van der Kooy and Carter, 1981), but this projection in non-primates appeared much more prominent than that in primates (Nauta, 1974; van der Kooy and Carter, 1981). As in primates, however,

86

the pallidal neurons in the entopeduncular nucleus (EN) of rats and cats that project to the VA/VL, the CM or the PPN were highly collateralized (Filion and Harnois, 1978; van der Kooy and Carter, 1981). The data obtained with the rétrograde double-labeling technique confirm the high degree of collateralization of the pallidothalamic and pallidotegmental projections in primates. Our single-axon traeing studies hâve provided the first direct évidence of the high degree of collateralization of the pallidofugal projections and hâve also contributed further insights into the highly complex organization of this neural System. Our data hâve revealed that the main axon of the GPi neurons bifurcate within the pallidum itself giving rise to a least two major branches heading towards the brainstem. One of the branches courses straight to the mesopontine tegmentum, whereas the other gives off one major collatéral that ascends to the thalamus before reaching the brainstem. The major collatéral that reaches the thalamus branches frequently before terminating in the form of small clusters in spécifie sectors of the VA/VL nuclei. Thèse new findings reveal that, although thalamic terminal fields of pallidal axons are wide and highly branched, they in fact anse from a thicker branch that descends towards the mesopontine tegmentum and arborize poorly in the PPN. Pallidal axons directed only at the thalamus could not be detected in the présent study. However, the sampling of labeled axons examined hère is too small to completely rule out this possibility. Furthermore, the fact that GPi neurons labeled only with the tracer injected in the thalamus were encountered in significant number in double-retrograde labeling experiments involving thalamic and brainstem injections favors the existence of pallidal neurons projecting only to the thalamus.

2.6.2

Contralateral pallidofugal projections

Haring Nauta made the first allusion to a crossed pallidothalamic projection in his autoradiographic traeing studies of the pallidal projections in the cat (Nauta, 1974; Nauta, 1979). This investigator reported a very sparse contralateral projection attributable to a few EN labeled axons, which crossed the midline in the supramammillary decussation and to a

87

lesser extent in the massa intermedia, to sparsely innervate the contralateral CM and habenula. Thèse fmdings hâve been supported by a rétrograde cell labeling study, in which retrogradely labeled cells in the contralateral EN hâve been detected after injections of horseradish peroxidase (HRP) into either the VA/VL or the CM of the cat (Nakano et al, 1983). In primates, the existence of a contralateral pallido-centromedian projection was categorically denied in a HRP study of the CM afférents from the GPi in macaque monkeys (Fénelon et al., 1990). Likewise, previous anterograde labeling studies of the pallidothalamic projections in monkeys made no mention of a contralateral contribution (Nauta and Mehler, 1966; Kuo and Carpenter, 1973; Kim et al., 1976), except one investigation in which the occurrence of a sparse anterograde autoradiographic labeling in the contralateral habenula was noted (DeVito and Anderson, 1982).

Our PHA-L anterograde labeling experiments (Hazrati and Parent, 1991) hâve provided évidence for the existence of prominent crossed pallidothalamic and pallidotegmental projections in primates. The contralateral pallidothalamic projection was found to be much more profusely arborized than the pallidotegmental projection. However, the patterns of termination of thèse pallidofugal fibers in the VA/VL, the CM and the PPN were strikingly similar to those observed ipsilaterally. The existence of such crossed pallidothalamic and pallidotegmental projections was confirmed by rétrograde double-labeling experiments, which revealed the présence in the GPi of neurons projecting contralaterally to the thalamus (VA/VL-CM) or the brainstem (PPN), and even to both thalamus and brainstem via axon collaterals.

In contrast to the profuse contralateral labeling found in the VA/VL and the CM, no anterogradely PHA-L-labeled fibers or terminais were noted in the contralateral habenula and this projection has not been investigated with rétrograde tracer. However, it must be recalled that in our experiments, the PHA-L injection sites were partial and covered mostly the central portion of the GPi. Since the majority of pallidal neurons projecting to the HL are located peripherally, it is likely that only a small portion of pallidohabenular neurons

hâve taken up the PHA-L in thèse experiments, which would explain the sparse labeling observed in ipsilateral HL and the absence of labeling in contralateral HL. This point needs further investigation with rétrograde labeling and more extensive injections of anterograde tracers. Thèse future studies should investigate the possibility that the contralateral pallidohabenular projection may originate from a distinct cell population in the GPi. Interestingly, our rétrograde labeling experiments hâve revealed that the number of labeled cells in the contralateral GPi amounted only to 10-20% that in the ipsilateral GPi. In the contralateral GPi only one-third of ail retrogradely labeled neurons contained the two tracers and the number of neurons containing the tracer delivered in the PPN were more numerous than those labeled with the tracer injected in the thalamus. Thèse data confirmed the existence of bilatéral pallidotegmental projections, as demonstrated in a previous study (Parent and De Bellefeuille, 1982). The results also provide rétrograde cell labeling évidence for a contralateral pallidothalamic projection in primates, and establish the existence of GPi neurons projecting contralaterally to both the thalamus and the brainstem. The results of our anterograde and rétrograde labeling experiments indicate that the contralateral pallidothalamic projection involves relatively few GPi neurons, but that thèse neurons arborize extensively in their contralateral thalamic targets.

Although the exact rôle of the contralateral pallidofugal projections in the normal functioning of the basai ganglia is not known, it is worth noting that the other major output structure of the basai ganglia, the SNr, also gives rise to a significant contralateral thalamic input (Gerfen et al., 1982). Thèse contralateral projections could play a very important compensatory rôle in cases involving unilatéral basai ganglia lésions, such as in hemiparkinsonism. Indeed, some changes in GABA receptors and glucose metabolism hâve been noted in some contralateral basai ganglia components in monkeys rendered hemiparkinsonian following unilatéral intracarotid injections of the neurotoxin l-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) (Mitchell et al., 1989; Robertson et al., 1991). Thèse contralateral changes hâve been tentatively explained by complex loop Systems involving the basai ganglia-thalamo-cortical circuit, and particularly by the bilatéral corticostriatal projections. However, we believe that the more direct bilatéral

89 pallidothalamic and/or pallidotegmental projections should be taken into account in the interprétation of thèse contralateral changes.

2.6.3

The reticular thalamic nucleus

Besides cell labeling in the GPi, injections of rétrograde tracers in the thalamus also led to cell labeling in the ipsilateral and contralateral reticular nucleus of the thalamus. This finding indicates that reticular thalamic neurons might influence the activity of the thalamic nuclei receiving a projection from the ipsilateral GPi. The reticular nucleus projections to the dorsal thalamus hâve long been thought to be strictly ipsilateral. However, rétrograde cell labeling studies undertaken in rats and cats more than a décade ago hâve suggested the existence of reticular nucleus commissural connections, notably with the ventromedial thalamic nucleus and the adjacent ventrolateral thalamic nucleus (VL) (Herkenham, 1979; Rinvik, 1984). Several récent reports hâve confirmed the existence of commissural reticular nucleus projections to distinct dorsal thalamic nuclei in several species (Velayos et al., 1989; Hazrati and Parent, 1991; Chen et al., 1992; Paré and Steriade, 1993; Raos and Bentivoglio, 1993; Hazrati et al., 1995). The primate CM/Pf complex or the rodent Pf nucleus are involved in gênerai cortical activation and play an important rôle in attention to visual, auditory and somatosensory stimuli (Kaufman and Rosenquist, 1985). Thus, the projection of the contralateral reticular nucleus to the caudal intralaminar nuclei, which are among the main targets of the reticular nucleus (Steriade et al., 1984), could be important for the bilatéral synchronization of cortical rhythmic activities between the two hémisphères (Paré and Steriade, 1993).

It is worth noting, however, that in contrast to the fibers that form the massive corticothalamic projection System, the GPi axons that arborize within the ventral tier thalamic nuclei do not provide collaterals to the thalamic reticular nucleus.

90 2.6.4.

Concluding remarks

The séries of experiments reviewed in the présent paper hâve revealed some of the major organizational features of the pallidofugal projection system in primates. One of thèse features is the highly collateralized nature of the axons that constitute this important output system of the basai ganglia. Indeed, the majority of the GPi axons branch to the thalamus and brainstem. Unfortunately, the pallidotegmental projection has been largely ignored in the current model of the basai ganglia organization, at the expense of the pallidothalamic projection (Parent and Cicchetti, 1998). Yet, GPi axons descending towards the brainstem could be involved in various motor déficits that are part of the typical syndromes resulting from basai ganglia disorders, including dystonia for which there is at présent no adéquate explanation. The other major feature of the organization of the pallidofugal projection system is the existence of fibers that arborize contralaterally, either in the thalamus, the brainstem, or both. Thèse contralateral pallidothalamic

and pallidotegmental or

pallido/thalamo/tegmental fibers may play a major rôle in the functional organization of the basai ganglia. Such decussating fibers may explain, for example, the bilatéral effects often seen after various unilatéral neurosurgical interventions for parkinsonism (Parent and Cicchetti, 1998). The existence of such contralateral pallidofugal fibers should be taken into account if one hopes to reach a more global understanding of the functional organization of the primate basai ganglia.

Figure 2.1

Semischematic drawings of transverse half-sections through the globus pallidus and adjacent areas in the squirrel monkey to illustrate the distribution and relative proportion of retrogradely labeled neurons in a représentative case of each of the five rétrograde doublelabeling experiments (A-E). The various combinations of double injections are indicated in the upper right portion of each drawing. The blue and red circles represent the two types of singly labeled neurons, whereas the asterisks represent doubly labeled neurons.

91

VA/VL - HL () ()

VA/VL - CM/Pf PUT

SI

CM/Pf-PPN

VA/VL - PPN

r- LH P H $ CA

SI lmm

92

III

Figure 2.2

Schematic drawings of transverse sections taken through the rostrocaudal extent of the forebrain and upper brainstem of a squirrel monkey that received injections of NY in the thalamus (VA/VL and CM) (red areas in C-E) and FB in the upper brainstem (PPN) (blue area in F). The red circles indicate cells rétrogradely labeled with NY, the blue circles represent cells retrogradely labeled with FB, and the asterisks indicate doubly labeled (NY/FB) cells.

93

A

C

94

Figure 2.3

Darkfield photomicrographs showing examples of singly and doubly labeled neurons disclosed after the injection of NY in the thalamus (VA/VL and CM) and FB in the upper brainstem (PPN) on the same side. A, Doubly labeled cells in the GPi ipsilateral to the injections. B, Doubly labeled cells in the GPi contralateral to the injections. C, Singly (FB) labeled in the contralateral GPi.

95

96

Figure 2.4

Séries of drawings of transverse sections through the rostrocaudal extent forebrain and upper brainstem of a squirrel monkey that received PHA-L injections into the GPi (red areas in A, B). The anterogradely labeled axons are illustrated as sinuous red lines, whereas the small dots represent isolated axon terminais.

97

A

lmm

Figure 2.5

Darkfield photomicrographs showing examples of anterogradely labeled axons and terminal arborization observed ipsilaterally after PHA-L injection into the GPi. A, a terminal fîeld displaying glomerule-like features in the ventrolateral thalamic nucleus. B, numerous thins fibers forming a dense terminal field in the centromedian thalamic nucleus. C, typical terminal field in the latéral habenula.

99

100

Figure 2.6 Diagram illustrating the course of a single axon of a GPi neuron that was microiontophoretically injected with BDA. The trajectory of the axon has been reconstructed with the help of caméra lucida drawings of several sagittal sections that were superimposed upon each other. A, This insert shows a high magnification drawmg of one of the many small terminal flelds that characterize pallidothalamic axons. B, Photomicrograph of the labeled GPi neuron whose exact location is indicated by an arrow in the main diagram.

101

Towards brainstem

102

CHAPITRE 3

MOTOR AND LIMBIC NEURONS IN THE INTERNAL PALLIDUM OF PRIMATES: SINGLE-AXON TRACING AND THREEDIMENSIONAL RECONSTRUCTION

CHAPITRE 3

MOTOR AND LIMBIC NEURONS IN THE INTERNAL PALLIDUM OF PRIMATES: SINGLE-AXON TRACING AND THREEDIMENSIONAL RECONSTRUCTION

Martin Parent, Martin Lévesque and André Parent

Laboratoire de Neurobiologie Centre de recherche Université Laval Robert-Giffard, 2601, Chemin de la Canardière, Local F-6500 Beauport, Québec, Canada, G1J 2G3

The Journal of Comparative Neurology (2001) 439:162-175

3.1

RESUME

Les projections efférentes du pallidum interne (GPi) chez le singe cynomolgus {Macaca fascicularis) ont été étudiées suite à l'injection microiontophorétique d'un traceur antérograde, la biotine dextran aminé, qui permet le marquage de quelques neurones par site d'injection. À l'aide d'un microscope optique et d'une caméra lucida, 52 axones ont été complètement reconstruits. Deux types de neurones qui composent le GPi ont été identifiés sur la base de leurs projections axonales. La branche axonale principale des neurones du type moteur descend vers le tronc cérébral et s'arborise discrètement dans le noyau pédonculopontin. L'axone principal de ces neurones émet des collatérales qui montent vers le thalamus et se subdivisent en 10-15 collatérales secondaires, couvrant ainsi un large secteur des noyaux thalamiques moteurs. Les segments terminaux de ces fines collatérales forment plusieurs bouquets de terminaisons axonales souvent retrouvées en étroite apposition avec les perikarya des neurones thalamiques. Environ la moitié des neurones moteurs émettent des collatérales qui s'arborisent dans les deux composantes du complexe centre médian/parafasciculaire du thalamus. La longueur totale des axones moteurs varie de 5.86 à 27.22 cm. Les axones limbiques grimpent le long du pôle rostral du thalamus, empruntent la strie médullaire thalamique pour s'arboriser profusément dans l'habénula latérale, qui s'avère être la cible pallidale la plus densément innervée. Certains axones limbiques émettent une collatérale vers les noyaux antérieurs du thalamus. Une faible proportion des neurones moteurs et limbiques émettent une collatérale qui croise la ligne médiane afin de s'arboriser dans les cibles controlatérales du GPi. La reconstruction tridimensionnelle révèle un aspect frappant de l'arborisation axonale des neurones moteurs, soit son expansion dans le plan sagittal. Cette étude par traçage unitaire nous apporte une évidence directe du haut degré de collatéralisation axonale des neurones moteurs du GPi chez le primate, leur permettant ainsi d'exercer une influence variée sur l'ensemble du réseau neuronal. Les neurones limbiques, quant à eux, agissent d'une manière beaucoup plus focalisée sur l'habénula latérale.

3.2

ABSTRACT

The axonal projections of the internai pallidum (GPi) in cynomolgus monkeys (Macaca fascicularis) were studied by labeling small pools of neurons with biotinylated dextran aminé. A total of 52 axons were entirely reconstructed from sériai sections with a caméra lucida. Two types of GPi neurons, termed motor and limbic, were identified on the basis of their target sites. Motor axons gave rise to a long branch that descended directly to the pedunculopontine tegmental nucleus, where it arborized discretely. Other branches ascended to the thalamus and broke into 10-15 smaller collaterals that ran through most of the ventral tier thalamic nuclei, where they terminate as typical plexuses. About half of the motor axons gave rise to collaterals that arborized in both components of the centre médian/parafascicular thalamic complex. The total length of motor axons ranged from 5.86 to 27.22 cm. Limbic axons climbed along the rostral thalamic pôle, coursed along the stria medullaris and arborized profusely within the latéral habenular nucleus, which stands out as the most densely innervated pallidal target. Some limbic axons also provided collaterals to the anterior thalamic nuclei. A small proportion of motor and limbic axons had branches that crossed the midline to arborize in contralateral GPi target structures. Three-dimension reconstruction revealed the unique aspect of the motor neuron axonal arborization, which was strikingly expanded along the sagittal plane. Thèse data suggest that motor GPi neurons exert a multifaceted influence through a widely distributed axonal network, whereas limbic GPi neurons act in a much more focused manner upon the habenula.

107

3.3

INTRODUCTION

The internai segment of the globus pallidus (GPi) in primates is one of the major output structures of the basai ganglia, the other one being the substantia nigra pars reticulata (SNr). The pioneering axon degeneration study of Nauta and Mehler (1966) has clearly identified the major GPi targets, which are: (1) the ventral tier motor nuclei of the thalamus, corresponding to various parts of ventral anterior nucleus (VA) (Ilinsky and Kultas-Ilinsky, 1987); (2) the centre médian thalamic nucleus (CM); (3) the latéral habenular nucleus (HL), and (4) the brainstem pedunculopontine tegmental nucleus (PPN). This study further suggested that the GPi projections that terminate in the CM might arise from collaterals of axons that arborize in the ventral tier thalamic nuclei (Nauta and Mehler, 1966).

The identity of the GPi récipient structures was confirmed by a multitude of rétrograde and anterograde labeling studies (Kuo and Carpenter, 1973; Kim et al., 1976; DeVito and Anderson, 1982; Hazrati and Parent, 1991; Shink et al., 1997; Sidibé et al., 1997; Parent et al., 1999; Baron et al., 2001). Moreover, the suggestion that pallidothalamic projections might be collateralized was supported and further extended by electrophysiological (antidromic invasion) experiments (Filion and Harnois, 1978; Harnois and Filion, 1980; Harnois and Filion, 1982) and rétrograde double-cell labeling investigations (Parent and De Bellefeuille, 1982; Parent and De Bellefeuille, 1983). The latter studies showed that, except for the peripherally located pallidohabenular neurons that appear to form a distinct neural population, up to 80% of ail retrogradely labeled neurons in the core of the primate GPi were double labeled after injections of complementary rétrograde tracers in VA and PPN (Parent and De Bellefeuille, 1982), compared to approximately 30-40% after injections in VA and CM (Parent and De Bellefeuille, 1983).

More direct évidence for axonal collateralization was obtained recently by Percheron and his colleagues who traced a few single anterogradely labeled fibers following bulk injections of biocytin in macaque GPi (Arecchi-Bouchhioua et al., 1996; Arecchi-

108

Bouchhioua et al., 1997). Although the proximal portion of thèse axons was not described. in détail, the tracing of their distal segment provided: (1) an overall view of the terminal patterns of single pallidothalamic fibers in the ventral tier thalamic nuclei (ArecchiBouchhioua et al., 1996), and (2) clear évidence for collaterals departing from the pallidothalamic branches to innervate the CM (Arecchi-Bouchhioua et al., 1997). In addition to being highly collateralized, évidence gathered in several species suggests that the pallidofugal System is, at least in part, bilaterally distributed (Parent et al., 1999).

In the hope to draw a more complète picture of this widely distributed neuronal System in primates, we used a procédure that allows microiontophoretic injections of anterograde tracers in very small subsets of electrophysiologically identified neurons scattered throughout the GPi in cynomolgus monkeys. Single labeled pallidofugal axons were then entirely reconstructed from sériai sections and a three-dimensional rendering of the entire network was obtained with the help of a computerized image-analysis System. This study has yielded novel fïndings that should further our understanding of the anatomical and functional organization of primate basai ganglia.

3.4 3.4.1

MATERIALS AND METHODS Préparation of the animais

A total of twelve adult cynomolgus monkeys (Macaca fascicularis) of both sexes, with a body weight that ranged from 3-4 kg, were used in the présent study. Ail surgical and animal care procédures adhered to the guidelines for the use and care of expérimental animais of the Canadian Council of Animal Care. The Animal Care Committee of Laval University also approved our expérimental protocol. The animais were first anesthetized with ketamine (75 mg/kg) plus xylazine (5 mg/kg) and their head placed in a specifically designed stereotaxic apparatus. After trépanation, a radiopaque solution (Omnipac or Iohexol, 0.8 ml of a 65% solution, Nicomed Imaging, Brandford, Ontario, Canada) was injected through a microsyringe into the right latéral ventricle. A few minutes after the injection, latéral and frontal X-ray pictures of the ventricular System were taken to precisely

109 localize the baseline formed by the anterior and posterior commissures in each animal. (Percheron, 1975).

3.4.2

Anterograde labeling

3.4.2.1

Injection procédures

Eleven monkeys were used for the anterograde labeling studies. One to three weeks after ventriculography, the animais were anesthetized as above and placed in the same stereotaxic apparatus. They were then maintained under propofol (10 mg/ml, i.v.) anesthesia while microiontophoretic injections of biotin dextran aminé (BDA, Molecular probes, Eugène, Or) were being made in différent portions of the GPi. Three monkeys were injected unilaterally to verify the possibility of contralateral projections. The remaining animais received bilatéral injections, but in ail cases only two injections were made in each GPi. The target was aimed at by using the stereotaxic coordinates of the atlas of Szabo and Cowan

(1984),

as

modified

by

the

data

collected

from

ventriculography.

Microiontophoretic labeling was carried out with glass micropipettes (tip diameter 2-3 /xm) filled with a solution of potassium acétate (0.5 M) plus 2% BDA. Thèse électrodes had impédance ranging between 10-15 MCI and were used to monitor the extracellular activity of the neuronal populations encountered during the pénétration of the micropipette. The external segment of the pallidum (GPe) was easily recognizable by the characteristic spontaneous firing pattern of its neurons, which includes high frequency discharges with pauses (DeLong, 1971; Filion, 1979). The GPi neurons also discharged at high frequency (70-120 spikes/second), but their tonic firing pattern was not interrupted by long periods of silence (Hutchison, 1998) (Fig. 3.1 A). Once in the chosen target, the micropipette was connected to a high compliance iontophoresis device (NeuroData) and the tracer was injected by passing positive current puises of 300-400 nA (ls on/ ls off) for 25 min.

110 3.4.2.2

Tracer visualization and cytochrome oxidase staining

After a survival period of 8-10 days, the animais were deeply anesthetized with sodium pentobarbital and perfused transcardially with 1 liter of saline solution (0,9%) followed by 2 liters of a fixative solution containing 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4) and 1 liter of 10% sucrose solution in PB. The brains were dissected out and placed in a cryoprotective solution composed of 1/3 paraformaldehyde (4% solution in PB) and 2/3 sucrose (30% solution in PB) for 24h at 4°C. The brains were then eut along the sagittal (7 monkeys) or frontal plane (3 monkeys) at 70 [xm using a freezing microtome. The sections were collected serially in phosphate buffer saline (PBS, 0.1 M, pH 7.4) and processed for the visualization of BDA according to the avidine-biotin-peroxydase method (ABC, Vector Labs, Burlingame, CA). In brief, the sections were incubated overnight at 4°C in a solution containing ABC diluted 1:100 in PBS (0.1M, pH 7.4), plus 1% normal rabbit sérum and 1% Triton X-100. They were then rinsed twice in PBS and once in Tris buffer. The bound peroxidase was revealed by incubating the sections in a solution containing 0.025% 3,3'diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO), 0.3% nickel-ammonium sulfate, 0.008% cobalt chloride and 0.008% hydrogen peroxide in 0.05M Tris buffer, pH 7.6, for 10-15 minutes at room température. The reaction was terminated by a rinse in Tris buffer followed by two rinses in PBS.

To help identifying nuclei and structures that harbored labeled neurons and axons, most sections were counterstained for cytochrome oxidase, according to the histochemical protocol of Wong-Riley (Wong-Riley, 1979). The counterstaining was performed before BDA révélation, and nickel-cobalt-intensified DAB (dark blue reaction) and unintensifïed DAB (diffuse brown precipitate) were used to reveal BDA and cytochrome oxidase, respectively.

111 3.4.2.3

Calbindin immunohistochemistry

One monkey was used to compare the distribution of pallidal axons with that of the calcium-binding protein calbindin D-28K (CB) at thalamic level. In this case, the brain was eut with a freezing microtome into 50 /zm-thick frontal sections that were collected in cold PBS. The tracer was revealed using nickel-cobalt-intensified DAB as the chromogen (as described above) and the sections were then processed for the visualization of CB immunoreactivity. In brief, after three rinses of 10 minutes each in PBS, the sections were incubated overnight at 4°C in a solution containing 2% of normal horse sérum, 0.1% Triton X-100 and the CB primary antibody. The CB antibody was a mouse monoclonal antibody highly spécifie for this cytoplasmic protein (Sigma, St. Louis, MO, dilution 1:2500). After three rinses in PBS, the sections were incubated for 1 hour at room température in biotinylated horse IgG. After three more rinses in PBS, the sections were reincubated for 1 hour at room température in 2% ABC. The sections were then washed twice in PBS and once in 0.05M Tris buffer (pH 7.6), and the bound peroxidase was revealed by using DAB as the chromogen.

3.4.2.4

Material analysis

Ail sections were mounted on gelatin-coated slides, dehydrated in graded alcohols, cleared in toluène, and coverslipped with Permount. They were examined under a Nikon light microscope equipped with a caméra lucida. Détails of single BDA-labeled neurons were drawn at 200X and 400X magnifications. The terminal fields and cell body of the labeled neurons were mapped at lower magnifications to détermine their topographie location according to the atlas of Szabo and Cowan (1984). The atlas of the macaque thalamus by Olszewski (Olszewski, 1952) and spécifie descriptions of thalamic motor nuclei in macaques (Ilinsky and Kultas-Ilinsky, 1987; Percheron et al., 1996) were also used for the detailed mapping of the pallidothalamic projections. The photomicrographs were digitally captured (AGFA Studiocam, Woburn, NA) and handled with the Adobe Photoshop software (version 5.5, Adobe, San José, CA). Most axons were entirely reconstructed along either the sagittal or frontal planes with the help of a caméra lucida. Furthermore, a

112

computerized image-analysis System (Neurolucida, MicroBrightField, Inc., Colchester, VT) was used to reconstruct in three dimensions the entire axonal trajectory and terminal arborization of three typical GPi neurons. This computerized System was also employed to gather quantitative estimâtes of the number of terminal boutons and total axonal length of the same three neurons.

3.4.3

Rétrograde labeling

The fluorescent tracer Fast Blue (FB, 2% solution, Sigma, St. Louis, MO) was injected into the habenula in one cynomolgus monkey. A total volume of 0.5 /xl of FB was injected over two needle pénétrations by means of 1 /xl Hamilton microsyringe. Three days later, the animal was perfused transcardially, as described above, and its brain was eut at 40 jiim in the frontal plane with a freezing microtome. The sections were serially collected in distilled water, mounted on gelatin-coated slides, air-dried, and coverslipped with DPX (Sigma, St Louis, MO). The sections were examined with a Leitz Ploemopack fluorescence microscope equipped with filter mirror System A (360 nm), which allows the visualization of FB. The distribution of the rétrograde pallidohabenular labeled cells was mapped out on caméra lucida drawings of six frontal sections taken at regular intervais throughout the rostro-caudal extent of the GPi.

3.5 3.5.1

RESULTS General labeling features

The injection procédure used in the présent study produced very small injection sites involving no more than 3 to 6 GPi neurons per site. Most of the injection loci had a dense core of BDA precipitate surrounded by several neurons labeled in a Golgi-like manner (Fig. 3.1 B). Thèse singly labeled GPi neurons had a spindle-shaped, ovoid or triangular cell

113 body emitting 2-5 long and poorly ramified primary dendrites (Fig. 3.1 C). The dendrites were characteristically thick, smooth or sparsely spined and occasionally varicose. Most GPi neurons displayed a typical discoid-like dendritic field that extended maximally along the dorsoventral and sagittal planes. However, the most medially located GPi neurons had dendrites that tended to ramify in ail directions. The dendrites of GPi neurons were rather long and some extended as far as 1000 jiim along the rostrocaudal and/or dorsoventral planes. This typical feature of GPi neurons explains the fact that labeled neurons were occasionally found at some distance from the core of the injection sites. Intensely labeled axons could be seen to émerge from either the core of the injection sites or from individually labeled neurons located peripherally. In the latter case, the axons emerged from either the cell body or a primary dendrite. Thèse axons could easily be followed individually throughout each section and were thus entirely reconstructed along the sagittal or frontal planes with a caméra lucida. Because labeled axons traveled in parallel and did not intermingle, there was very little possibility of confusing one axon with another.

In the présent study, 52 axons were traced entirely from the injection site to their terminal fields and half of them were connected to their parent cell body in the GPi. The remaining axons could not followed up to their cell body of origin because their proximal segment merged within the dense core of BDA precipitate at the injection site. Thèse axons had nevertheless branching patterns similar to those that were connected to their cell body of origin and were thus taken into account in the présent study. Detailed analysis of the material ensured that thèse axons émerge form a cell body located in the GPi and not from fibers of passage that could hâve taken up the tracer. Ail the main structures that project to the GPi, including the striatum, the GPe and the subthalamic nucleus (STN), and whose axons could hâve taken up the tracer were carefully scanned for the présence of retrogradely labeled neurons. However, no such retrogradely labeled neurons were encountered, probably because of the very small amount of BDA delivered in each GPi sites.

114

3.5.2

Axonal trajectory

3.5.2.1

General features

Based on their target sites and axonal branching patterns, the primate GPi appears to be essentially composed of two types of neurons. The type I neurons are referred to as "motor neurons" because their axons project profusely to premotor neurons located in the ventral tier thalamic nuclei and PPN. The motor neurons can be further subdivided into two subtypes: type la neurons that project only to ventral tier nuclei and PPN, and type Ib neurons that arborize more profusely than type la and, in addition to collaterals to the ventral tier nuclei and PPN, also innervate the centre médian/parafascicular thalamic complex (CM/Pf). The type II neurons are hère termed "limbic neurons" because their axon arborizes principally within the HL, a major relay in the limbic System circuitry. The limbic neurons can be further subdivided into two subtypes: type Ha neurons that project only but densely to the HL, and type Ilb neurons that innervate profusely the HL, but also provide collaterals to the anterior thalamic nuclei.

It is worth noting that the notion of motor and limbic neurons, as used in the présent study, has nothing to do with the functional subdivision the globus pallidus into the so-called "associative", "sensorimotor" and "limbic" territories, as proposed by Smith and collaborators (Shink et al., 1997; Sidibé et al., 1997; Baron et al, 2001). The latter concept implies that the striatal organization is directly transposed at pallidal level. Thus, by virtue of the topography of the striatopallidal projections, the dorsomedial third, the ventrolateral two-thirds and the rostroventromedial pôle of the GPi would correspond respectively to the associative, sensorimotor and limbic territories of the GPi (Shink et al., 1997; Sidibé et al., 1997; Baron et al., 2001). Hère, GPi neurons hâve been defined following a detailed study of their efferent projections and not on the basis of an a priori location in any putative pallidal territories. Thus, the motor neurons of the présent study may réside anywhere within the GPi, including the so-called associative or sensorimotor territories. The same applies to limbic neurons, which do not hâve to be confmed to the presumed limbic GPi territory.

115 Occasionally, the axon of motor or limbic neurons was seen to émit 1 or 2 thin collaterals that arborized poorly within, and sometimes beyond, the somatodendritic domain of their parent neuron. Thèse local collaterals exhibited some varicosities of various sizes reminiscent of boutons-en-passant or boutons terminaux. Besides local collaterals, the main axon of motor neurons branched either within the GPi or just after leaving it. In contrast, the axons of limbic neurons remained unbranched, at least until they reached the thalamus. Ail limbic axons exited the GPi through the lenticular fasciculus (LF, Forel's field H2), whereas motor axons emerged either through the ansa lenticularis (AL) or the LF, irrespective of the position of their parent cell body in the GPi (Fig. 3.2). Some axons emerged at approximately the same rostrocaudal level than that of their cell body, whereas others coursed along a considérable distance rostrally or caudally within the GPi before exiting the nucleus via either the LF or the AL. One of the labeled axons that bifurcated within the GPi had a major collatéral that exited the GPi through the LF caudally, at about the same level as that of its parent cell body, whereas the other major collatéral coursed for about 3 mm rostrally and ventrally and emerged from the GPi via the AL (Fig. 3.2).

3.5.2.2

Type la neurons

The axons of 15 neurons of this subtype were entirely reconstructed and a typical example is illustrated in Figure 3.3. The cell body of this particular neuron was located ventrally in the medial portion of the GPi at mid-pallidal level (Fig. 3.7 C). Its main axon exited through the AL, emitted two short and varicose collaterals in the rostral part of Forel's field H (FH), or prerubral field, and then bifurcated more caudally in the same area. One of the main branches ascended directly to the ventral tier thalamic nuclei via the thalamic fasciculus (Forel field Hl), while the other descended without further branching toward the brainstem tegmentum (PPN) (Fig. 3.3). The main ascending branch passed through the ventromedial thalamic nucleus (VM), which corresponds to the medial part of the ventral latéral nucleus of Olszewski (1952) (Ilinsky and Kultas-Ilinsky, 1987), without emitting any collaterals or terminais at this level. It remained unbranched until it reached the densicellular part of the VA nucleus (VAdc), which corresponds closely to the oral part of

116

the ventral latéral nucleus of Olszewski (1952) (Ilinsky and Kultas-Ilinsky, 1987). There it broke out into several short terminal collaterals (Fig. 3.3). The main branch continued more dorsally and caudally to arborize within the parvicellular part of the VA nucleus of Olszewski (1952) (VApc). Thus, this single branch spread throughout a large portion of the pallidal territory of the ventral tier nuclei, an area that is globally referred to as the nucleus lateralis oralis (LO) by Percheron and colleagues (Percheron et al., 1996). The terminal arborization of this single axon at thalamic level derived from one major branch that departed from the main axon within the FH. In other cases, as much as 2-5 branches were seen to départ from the main axon to innervate the thalamus. Irrespective of their number, thèse initial branches subdivided successively into 10-15 long and thin branches that covered most of the pallidal thalamic territory. As they approached the end of their trajectory, thèse long branches broke out into numerous shorter terminal collaterals that formed typical dense cluster-like terminal fields composed of a multitude of very thin, highly varicose and closely intermingled terminal axon collaterals (Fig. 3.5 A). Some terminal branches endowed with few varicosities also ended in the vicinity of the clusters, which had a mean rostrocaudal extent of about 100 /xm. The number of thèse terminal clusters ranged from 3 to 10 per axon and, occasionally, collaterals from 2 distinct axons were seen to contribute to the formation of a single cluster.

The terminal field in the PPN was markedly différent than the one at thalamic level. It consisted of a small number of varicose terminal branches that emitted a few collaterals oriented at right angles from the main axonal branch. Thèse short terminal collaterals displayed numerous varicosities, including some at their very end portions (Fig. 3.5 C). Most of thèse varicosities were larger than those at thalamic level. One collatéral did traverse the caudal limit of the PPN, but after a short trajectory in the central portion of the pontine tegmentum, it turned back and reentered the PPN (Fig. 3.3 A).

Numerous terminal varicosities were also noted at the level of FH and the zona incerta (Zi). Thèse terminais were part of short collaterals provided en passant by the main axons that coursed through this area before heading to the thalamus or the brainstem. As judged by the

117 number of axonal branches and varicosities, the density of the innervation was much higher at thalamic (468 terminal boutons) than brainstem (41 terminal boutons) level. This type la axon also provided a significant innervation to the FH (142 axonal varicosities). A count of the total length of ail the various axonal segments encountered in the différent structures, as well as along the main axonal trajectory, revealed that this particular type la axon was 5.86 cm long. Two other type la neurons emitted a collatéral at the PPN level that crossed the midline by passing dorsally to the decussation of the superior cerebellar peduncle (SCP), near the medial longitudinal fasciculus. Thèse decussating fibers were weakly labeled and thus difficult to follow further on the contralateral side in this particular case. However, observations of labeled axons in the contralateral PPN in monkeys that were injected unilaterally revealed that the pattern of arborization at PPN level was similar on both sides, although the number of fibers and terminais was much greater ipsilaterally than contralaterally.

3.5.2.3

Type Ib neurons

The axons of 16 neurons of this subtype were entirely reconstmcted and an example is illustrated in Figure 3.4. The cell body of this particular neuron was located centrally in the medial half of the GPi at its caudal third level (Fig. 3.7 E). The main axon of this neuron traveled for a certain distance within the GPi before leaving the structure. However, it did not émit any collaterals along its course within the GPi. This axon did not run through the AL or the LF, but exited directly through the medial pôle of the GPi. It gave rise to three major collaterals within the FH, and each of thèse collaterals emitted several, thin and varicose collaterals within the FH itself. The three major collaterals of this axon were drawn in différent colors in figure 4 to facilitate the visualization of their individual arborization patterns. The most ventrally located collatéral (green) descended toward the PPN, but yielded one short collatéral that ascended back to the FH and a much longer collatéral that reached the thalamus by coursing along the Hl field (Fig. 3.4 A). The most

118 dorsally located collatéral (blue) bifurcated within the FH, where it provided two short and varicose collaterals before emitting two major collaterals that ascended toward the VAdc/VApc thalamic subnuclei via the Hl field. Thèse major collaterals did pass through the VM nucleus, but did not provide any collatéral or terminal at this level. The main axonal branch of this dorsal collatéral continued its caudal course for some distance before it bifurcated and gave rise to two collaterals that both reached the CM/Pf from its caudal part. The middle collatéral (red) divided itself into two branches within the FH. One of thèse two branches emitted three collaterals that arborized in the FH and Zi before it ascended dorsally and caudally to reach the CM/Pf, where it arborized profusely. The other branch descended toward the retrorubral area (RRA), but remained poorly ramified at this level (Fig. 3.4 A).

The thalamic innervation from this particular type Ib neuron derived from three main axonal branches that arborized profusely within large sectors of the VApc/VAdc subnuclei and three other axon collaterals that densely innervated the CM/Pf complex (Fig. 3.4). Although they occupied a vast territory within the ventral tier thalamic nuclei, the axonal branches that reached this région of the thalamus did not arborize very densely, except at their very end portion, where they formed typical cluster-like plexuses, as described above. The axon collaterals did not form dense plexuses in the CM/Pf as they did in the VApc/VAdc subnuclei. Instead, the fibers became thin and varicose well before terminating and formed typical loops oriented along the longest extent of CM/Pf. They emitted several short and varicose collaterals that ran at right angle to the main axonal trunk and thèse thin varicose terminal fibers were often seen in close contact to unlabeled cell bodies in the CM/Pf (Fig. 3.5 B). Most of thèse fibers arborized within the medial part of the CM and the latéral part of the Pf, which correspond respectively to the pars média and the latéral sector of pars parafascicularis of the central complex, as defined by Percheron and collaborators (Fénelon et al., 1994; Percheron et al., 1996). The VA subnuclei were found to contain only 237 axonal varicosities in comparison to 540 terminal varicosities for the entire CM/Pf, which appeared as the most densely innervated target of this particular type Ib axon.

19 The only branch that reached the PPN did so after an extremely long but rather straightforward course through the midbrain tegmentum. Along most of its proximal course, this rather thick labeled fiber was bordered by the red nucleus dorsomedially and the substantia nigra pars compacta (SNc) ventrolaterally. The fiber swept ventrally in the core of the midbrain tegmentum and then coursed immediately latéral to the SCP. The fiber finally arborized in the form of several short and varicose collaterals uniformly scattered along the dorsal and ventral aspects of the SCP, within the so-called subnucleus diffusus (or pars dissipata) of the PPN (Olszewski and Baxter, 1954). A few isolated varicose collaterals, some with large size varicosities in close contact with cell bodies, were also seen in the subnucleus compactus (or pars compacta) of the PPN, more laterally. Many of thèse short terminal collaterals were oriented dorsoventrally at right angle from the main fiber (Fig. 3.5 C). At least one fiber collatéral coursed caudal to the PPN. This fiber could be followed for a certain distance as it traveled dorsolaterally along the latéral aspect of the nucleus raphe pontis. However, the fiber became progressively too faintly labeled and was lost at the pontomedullary junction.

In ail, the fiber collaterals at the PPN level yielded a total of 57 axonal varicosities. The axon collatéral that reached the RJRA divided into three short and varicose collaterals and the total numbers of axonal varicosities at this level was 12, in comparison to 132 in the FH. This Ib axon was thus much more profusely arborized than the la axon examined above and its total length was estimated at 27.22 cm. The three-dimensional reconstruction of this particular neuron revealed a striking feature that appeared to be shared by ail motor (type I) neurons, which was that the widest extent of their axonal arborization occurred along the sagittal plane. It was along that plane that the entire axonal arborization was, by far, the most spread out (Fig. 3.4 A). In contrast, the axonal arborization of the same neurons had a narrower and more compacted appearance when viewed along the frontal (Fig. 3.4 B) or horizontal (Fig. 3.4 C) planes. The total axonal arborization of this particular neuron extended over a surface of about 140 mm2 along the sagittal plane, compared to 30 mm2 along the frontal or horizontal planes. The axonal arborization of the type Ib neurons was thus 4.6 times more spread out along the sagittal plane than along the frontal or horizontal planes.

120

Other type Ib axons were seen to emit collaterals that crossed the midline at the level of the CM/Pf, as well as within the supramammillary decussation. The fibers that decussated at the CM/Pf level innervated the same structures contralaterally. The pattem of innervation of the CM/Pf was similar on both sides, except that the labeled fibers were much less abundant contralaterally than ipsilaterally. One of the fibers that crossed the midline within the supramammillary decussation could be followed for a considérable distance within the contralateral

thalamus. After

its decussation the fiber

ascended

close to the

mammilothalamic tract, traversed the VM nucleus and then climbed along the latéral border of the paracentral intralaminar nucleus. It then swept laterally to penetrate the VAdc thalamic nucleus. The fiber remained unbranched throughout its long journey through the thalamus. Unfortunately, it became too faintly labeled to be followed further within the contralateral VAdc nucleus. Another type Ib neuron emitted axon collaterals that crossed the midline by coursing along the dorsal surface of the SCP and innervated the contralateral PPN. The pattern of arborization of the PPN was similar on both sides, except for the number of labeled axon collaterals, which was much smaller contralaterally than ipsilaterally. Several other type Ib axons were seen to emit a few collaterals that coursed more caudally within the brainstem than the PPN level. One of thèse weakly labeled collaterals was followed up to central gray matter near the dorsal raphe nucleus, whereas others could be traced down as far as the parabrachial nuclei where they vanished.

In sections immunostained for CB, virtually ail thalamic labeled terminal fibers were confined to the CB-rich sectors of the VApc/VAdc subnuclei or the LO nucleus (Fig. 3.4 D). However, in contrast to the ventral tier nuclei, the CM/Pf was completely free of CB immunostaining in our double stained préparations.

3.5.2.4

Type lia neurons

The axons of 17 neurons of this subtype were entirely reconstructed and an example is illustrated in Figure 3.6. The cell body of this particular neuron was located ventrally in the

121 rostral pôle of the GPi (Fig. 3.7 A). The axon exited through the LF, traversed obliquely the latéral hypothalamic area by coursing along the inferior thalamic peduncle, ascended along the rostral pôle of the thalamus to enter directly within the stria medullaris. From there the fiber followed a rather straight course until it approached the habenula. After a small caudal détour, the labeled axon entered directly into the habenula and immediately branched into three major collaterals that arborized within most of the HL, leaving the medial habenular nucleus completely devoid of labeled axonal varicosities. Of the three main collaterals that penetrated the HL, the first one remained rather poorly branched, the second one arborized abundantly (Fig. 3.6 B), and the third one became extremely thin and could not be followed entirely. The pattern of arborization within the HL consisted of a multitude of very fine fibers that displayed innumerable axonal varicosities of very small and uniform size (Fig. 3.6 C). Thèse highly varicose fibers formed typical pericellular arrangements around the small, round and closely packed cell bodies in the HL. This particular axon was found to yield 647 terminal varicosities in the HL, but this number is obviously an underestimation because it was not possible to count the axon terminais of one of the three major collaterals that innervated the HL because of its faint labeling.

Although the high density of innervation has complicated the tracing of individual axonal branches in the HL, many type II axons emitted one major axonal branch that crossed the midline within the interhabenular commissure (Fig. 3.6 C, inset) and innervated the contralateral HL. In cases of unilatéral GPi injections, the pattern of pallidal innervation of the contralateral HL was found to be largely similar to that seen ipsilaterally, except that the density of innervation was significantly less contralaterally than ipsilaterally.

Instead of climbing immediately within the stria medullaris, other type lia axons ascended along the reticular thalamic nucleus and entered within the stria terminalis that arched along the medial border of the caudate nucleus near its junction with the thalamus. As we moved caudally, thèse labeled fibers progressively left the stria terminalis to course medially within the stratum zonale until they progressively joined the stria medullaris and the HL.

122 3.5.2.5

Type Ilb murons

The trajectory and branching pattern of this type of neurons (n = 4) were very similar to those described above for the type Ha axons, except that they provided one to three major collaterals to the anterior thalamic nuclei on their way to the HL. Thèse collaterals arborized principally in the anterior médian and anterior ventral thalamic nuclei. Overall, the pallidal innervation of the anterior nuclei was much less dense than that of the HL. The pattern of terminal arborization at the level of the anterior nuclei consisted essentially of a few short, thin and varicose axon collaterals that appeared to make contact en passant with the cell bodies of the anterior thalamic nuclei.

In one particular case, the axon of a type Ilb neuron was found to travel for a considérable distance within the VApc subnucleus before reaching the stria medullaris and subsequently the HL. This singular axon provided only a few axonal varicosities as it coursed through the VApc nucleus. It also emitted small varicose collatéral that reached the anterior ventral thalamic nucleus. The terminal field of this axon within the HL was similar to that of the other type II neurons described above.

3.5.3

Topographical distribution

Despite the fact that pallidal projection neurons of type I and II were not distinguishable from one another on the basis of their somatodendritic morphology, they were found to occupy a very distinct sector of the internai pallidum. The plotting of each type of GPi projection neurons, the axons of which could be connected to their parent cell bodies, on a séries of equally spaced (850 lira apart) parasagittal sections (Fig. 3.7) has revealed that type I neurons abounded preferentially within the large central portion of the GPi, whereas type II neurons were largely confined to the periphery of the nucleus. Furthermore, the most profusely arborized type I neurons (type Ib motor neurons) tended to be more abundant ventrally in the caudalmost portion of the GPi. The peripheral distribution of type II neurons, as delineated after single-cell labeling experiments with BDA, was confirmed in the animal that received a FB injection in the habenula. Indeed, following a FB injection

123 that was confmed to the dorsal two thirds of the habenula (Fig. 3.8 B, inset), the retrogradely labeled pallidal neurons were mostly confmed to the periphery of the GPi, as well as along the accessory meduUary lamina and the inner border of the internai meduUary lamina (Fig. 3.8 A). A rostrocaudal-decreasing gradient of the FB labelling was also noted in that particular animal; the pallidohabenular neurons were scattered throughout the rostral pôle of the GPi, but were progressively displaced at the periphery of the nucleus in its remaining caudal portion. Thèse pallidohabenular neurons merged medially with a large population of similarly labeled neurons in the latéral hypothalamus (not shown). The retrogradely labeled neurons in the GPi had a typical triangular or oval cell body with two or three primary dendrites oriented parallel to the internai meduUary lamina (Fig. 3.8 B). A rough quantitative estimate revealed that the type II retrogradely labeled neurons in this experiment represented approximately 10% of the total number of neurons in the GPi, as visualized by cytochrome oxidase or cresyl violet counterstaining. However, this might be an underestimate because the FB injections did not involve the ventral third of the HL.

3.6

DISCUSSION

The présent study has provided the first detailed account of the axonal branching patterns of the GPi neurons in primates. Our investigation has yielded the first direct évidence for the existence of numerous GPi neurons endowed with a widely distributed axonal arborization that allows them to influence structures as broadly dispersed as the motor thalamic nuclei and the mesopontine brainstem tegmentum. Besides thèse wide reaching motor neurons, the primate GPi was also found to arbor a smaller number of neurons with a much more focused terminal arborization. Thèse limbic neurons target principally the HL, which stands out as the most densely innervated target structures of the GPi in primates. The significance of thèse and other results will now be discussed in the light of relevant data gathered in various species.

124

3.6.1

The ansa lenticularis or the lenticular fasciculus

Since the original description of the AL (Lisenkernschinge) by von Monakow (von Monakow, 1895), the organization of the pallidofugal fibers located around and within the GPi has been a matter of controversy (Nauta and Mehler, 1966; Grofova, 1970). Following the démonstration by Ranson and Ranson (Ranson and Ranson, 1939) that the GPi was the main and probably the sole contributor to the AL, Carpenter and co-workers hâve proposed a simple scheme of organization based on the results of their own tract-tracing studies (Kuo and Carpenter, 1973; Kim et al., 1976; Carpenter, 1981). The concept was that fibers in the AL anse predominantly from the outer part of the GPi and pass ventromedially through portions of the medial part of the GPi to their site of émergence. Fibers of the LF were said to arise mainly from the inner part of the GPi and project dorsomedially through the internai capsule. This attractive scheme was challenged recently by the results of an anterograde labeling studies in squirrel monkeys that involved large injections of BDA, mainly in the posterior aspects of the GPi (Baron et al., 2001). This investigation led to the suggestions that pallidofugal fibers from the caudal half of the GPi exit from the medial border of the nucleus without traveling to any significant extent in the rostrocaudal plane, and that the AL is largely composed of axons that originate from the rostral half of the GPi.

In the présent single-axon labeling study, ail limbic neurons exited via the LF, whereas motor axons emerged trough either the AL or the LF, irrespective of the location of their cell bodies in the GPi. Some motor axons also emerged directly along the medial aspect of the GPi, at various distances between the LF dorsally and the AL ventrally. Furthermore, two neurons, one located on each side of the accessory medullary lamina, had their respective axon coursing in the AL, a finding that does not support Carpenter's concept about the composition of the AL and LF. One motor neuron with an axon that bifurcated within the GPi had one branch that emerged through the LF at about the same anteroposterior level than that of its parent cell body, as predicted by the results of Baron et al. (2001). The other branch, however, coursed for more than 3 mm within the GPi before emerging via the AL rostrally. Thèse findings reveal that pallidal axons and collaterals often follow complex and tortuous courses within the GPi so that the location of the cell

125 body within the nucleus is not a good predictor of where the pallidal axon (or its multiple branches) might émerge.

3.6.2

Pallidothalamic projection

The pallidothalamic axons traced in the présent study arborized in the same thalamic territory as the one defined by previous anterograde tracing studies in primates (see reviews by Ilinsky and Kultas-Ilinsky, 1987; Percheron et al., 1996). However, the VM nucleus (or the medial part of the ventral latéral nucleus of Olszewski, 1952), which is often included in the pallidal thalamic territory (Kim et al., 1976; Carpenter, 1981; DeVito and Anderson, 1982), was found to contain several labeled fîbers but very few axon terminais. Thus, in agreement with a previous suggestion made by Ilinsky and Kultas-Ilinsky (1987), we believe that the VM nucleus is principally a zone of passage for the pallidothalamic fîbers, not a zone of termination. The terminal arborization of the pallidothalamic fîbers was largely confîned to the CB-rich portion of the ventral tier nuclei, as suggested previously by Percheron et al. (1996). This calcium-binding protein can thus be used as a reliable marker for the pallidal territory in the ventral tier thalamic nuclei, but not in the CM/Pf because this complex is largely devoid of CB.

The fact that each labeled axon expanded over a large portion of the pallidal territory, irrespective of the location of their parent cell body in the GPi, argues against the idea of a direct transposition at thalamic level of the pallidal functional territories (associative, sensorimotor and limbic) (Sidibé et al., 1997). Perhaps the major organizational feature of the pallidothalamic system is the fact that each of its axon gives rise to 5-10 cluster-like terminal plexuses that are widely distributed in the ventral tier nuclei. This mode of organization allows single pallidothalamic axons to transmit the same neural information to small groups of about 20 thalamocortical cells (Ilinsky and Kultas-Ilinsky, 1987; ArecchiBouchhioua et al., 1996; Ilinsky et al., 1997) dispersed throughout the pallidal territory. The neural information can then be processed in parallel by each of thèse small cell clusters before being conveyed to the cérébral cortex. This mode of organization is the opposite of

126

the sériai type of organization favoured by most classical somatosensory Systems. In such a case, each axon carried a spécifie type of information that is transmitted to a single thalamic unit, which in turn projects to a spécifie cortical domain. Another important organizational feature of the pallidothalamie system relates to the fact that it is part of a much wider neuronal network. Virtually ail single pallidothalamie fibers that we hâve traced were collaterals of single axons that also innervated the PPN (type la neurons) and, in about 50% of the cases, the CM/Pf as well (type Ib neurons). This form of organization implies that single pallidal neurons can send efferent copies of the same neural information to the small cell clusters in the ventral tier thalamic nuclei, and to more diffuse cell populations in the PPN and CM/Pf.

The results of the présent study agrée with those of our previous fluorescent double rétrograde labeling investigation, which showed that the vast majority (about 80%) of the rétrogradely labeled neurons in the GPi were double labeled following the injection of complementary tracers into the ventral tier thalamic nuclei and the PPN of squirrel monkeys (Parent and De Bellefeuille, 1982). A small number of GPi neurons labeled only with the tracer injected into the ventral tier thalamic nuclei (or in the PPN) were encountered in this double-labeling study. However, we believe that the absence of one of the two tracers in thèse neurons could hâve resulted from the fact that the two injections were not in perfect register, that is, the tracers did not reach in equal amount the two terminal fields of the same axon at brainstem and thalamic levels. In accord with the data of the présent single-axon tracing study, we propose that virtually ail pallidofugal axon that projects to the ventral tier thalamic nuclei also projects to the PPN.

3.6.3

Pallidointralaminar projection

The anterograde degeneration study of Nauta and Mehler (1966) was the first to clearly establish the existence of a projection from GPi to CM in primates. That projection was confirmed by numerous anterograde tract-tracing and rétrograde cell-labeling studies in

127 monkeys (Sadikot et al., 1992a). A similar projection was documented in rats and cats from the entopeduncular nucleus (EN), the presumed homologue of the GPi in non-primates (Nauta, 1974; Carter and Fibiger, 1978; Hendry et al., 1979; Larsen and McBride, 1979). However, the présent study is the first to demonstrate that the pallidointralaminar projection anses from collaterals of single axons that project also to the ventral tier thalamic nuclei and PPN. This fmdings is in agreement with the original suggestion made by Nauta and Mehler (1966) that the CM/Pf pallidal innervation dérives principally from collaterals of fibers that arborize within the ventral tier thalamic nuclei, as confirmed by double rétrograde fluorescent studies (Parent and De Bellefeuille, 1983) and single-axon tracing studies (Arecchi-Bouchhioua et al., 1997).

Our data reveal that about half of the axons that bifurcate to the thalamus and brainstem provide collaterals to the CM/Pf. This number is slightly higher than the proportion of double labeled neurons (30-40%) encountered in the GPi following injections of fluorescent tracers in the ventral tier nuclei and CM/Pf (Parent and De Bellefeuille, 1982), and much higher than the proportion of double labeled pallidal neurons (15-20%) observed after CM/Pf and PPN injections (Parent and De Bsllefeuille, 1983). The discrepancy between thèse numbers might reflect some bias towards CM/Pf-projecting axons in the sampling of the présent study or some false négative problems in our previous double labeling investigations due to the diffïculty of injecting the two corresponding terminai fïelds of the same axon. It must also be mentioned that no attempt has yet been made to triple-label pallidal neurons by injecting their three major termination sites with différent rétrograde tracers. Such a study would yield numbers more directly comparable to those of the présent study.

The pallidofugal axons that provide collaterals to the CM/Pf (motor type Ib) are by far the most widely arborized axons and their cell bodies of origin are slightly more abundant in the caudalventral portion of the GPi than elsewhere in the nucleus. The latter fînding is congruent with the results of a previous rétrograde cell labeling study in monkeys, which has revealed that pallidal neurons projecting to the "central complex" (the term used by Percheron and colleagues to refer to the CM/Pf) are particularly abundant in the caudal and

128 ventral sector of the GPi, a région crossed by the fibers of the putamen sensorimotor territory (Fénelon et al., 1990). This finding is interesting in view of the fact that this part of the GPi is a good target for placing radiofrequency lésions (Vitek et al., 1998) or electrical stimulators (Krack et al., 1998) to alleviate hypo- and hyperkinetic movement disorders.

The pallidointralaminar fibers traced hère terminated massively in the CM, but also provided a significant input to the latéral part of the Pf, as predicted from previous studies in cats (Nauta, 1979) and monkeys (DeVito and Anderson, 1982; Arecchi-Bouchhioua et al., 1997; Sidibé et al., 1997). The présent study has further shown that most single-labeled fibers innervate both the CM and Pf nuclei via long and varicose collaterals that form loops between the two structures.

3.6.4

Pallidotegmental projection

The pallidotegmental projection differs in many respects from the pallidothalamic and pallidointralaminar projections. In most cases, it was composed of a single fiber that emerged rather close from the cell body and remained poorly branched and uniformly thick throughout its long trajectory toward the PPN. It is only when it reached the PPN that the fiber broke out into numerous collaterals that arborized within both the pars diffusa and compacta of the PPN. The fact that the number of varicosities is about 5-10 times less numerous in the PPN than in thalamus does not indicate that the pallidotegmental branch is functionally less important than its pallidothalamic branch. First, this différence in the number of terminais reflects, at least in part, the enormous size variation that exists between the small PPN and the voluminous latéral thalamic mass. Second, the pallidotegmental terminal fïeld is much more focused than the widespread thalamic innervation and it comprises terminal collaterals with large varicosities that closely surround PPN neurons. Third, the fiber that descends to the PPN remains largely unbranched, whereas the pallidothalamic innervation dérives from multiple, long and thin collaterals that branch frequently. Since nerve conduction velocity is directly proportional to the axon diameter and inversely proportional to the degree of axonal branching, it may

129

be presumed that PPN neurons will receive GPi information well before thalamic neurons. This spatiotemporal mode of organization of single pallidofugal axons is important because the PPN is known to project back to many basai ganglia structures, principally the SNc, the STN and the GPi himself (Lavoie and Parent, 1994b). This feedback projection, which is excitatory and mediated through acetylcholine and glutamate, or both (Di Loreto et al., 1992; Lavoie and Parent, 1994a; Charara et al., 1996), could play an important rôle in the functional organization of the basai ganglia.

3.6.5

Pallidohabenular projection

The pioneering anterograde degeneration studies (Wilson, 1914; Ranson et al., 1941; Nauta and Mehler, 1966) provided the first évidence of a pallidohabenular projection, but thèse investigations were fraught with the problem of fibers of passage. Further évidence for such a projection was later provided by an autoradiographic anterograde tracing study in the cat, which showed that neurons of the EN could reach the HL through at least five différent routes (Nauta, 1974). The existence of a pallidohabenular projection was then confirmed by a multitude of rétrograde cell labeling studies and anterograde tract tracing investigations in rats, cats and monkeys (Parent et al., 1999).

The présent single-axon tracing study has revealed that the pallidohabenular or type II neurons: (1) form a distinct population in primate GPi; (2) are distributed at the periphery of the GPi and also along the accessory medullary lamina; (3) represent at least 10% of the total neuronal population of the GPi; (4) can be divided into neurons that project only to HL (Ha) and those that project to the HL and the anterior thalamic nuclei (Ilb); and (5) project to the HL and the anterior thalamic nuclei by coursing principally along the stria medullaris.

Some of thèse findings agrée with the data of our previous double rétrograde fluorescent labeling studies, which hâve shown that the pallidohabenular neurons form a ring around the pallidal neurons that project, according to various combinations, to the three other target

130 structures of the GPi (Parent and De Bellefeuille, 1982). A similar study in the rat has revealed that the pallidohabenular neurons occupy the rostral two-thirds of the EN, whereas neurons that project to the ventrolateral thalamus, the Pf and the PPN are ail confined to the caudal third of the EN (van der Kooy and Carter, 1981). However, a radically différent view of the organization of the EN has been advocated in a récent single-axon tracing investigation of the pallidothalamic projections in the rat (Kha et al., 2000). In this study, two populations of pallidothalamic neurons hâve been identified: those that project only to the motor thalamus and those that project to the motor thalamus and other nuclei, including the habenula (Kha et al., 2000). This view is difficult to reconcile with the current concept that pallidal neurons projecting to the HL are distinct from those projecting to the motor thalamus. However, it is possible that the few axons that were traced to the motor thalamus and HL by Kha et al. (2000) correspond to the single axon that was found to follow an aberrant course through the ventrolateral thalamic région before terminating in the HL in the présent study. Obviously, more detailed single-axon tracing studies of the rat EN are needed before a more complète picture of the organization of the rodent pallidofugal System émerges.

The double-labeling study of the pallidofugal System undertaken in rats (van der Kooy and Carter, 1981) and monkeys (Parent and De Bellefeuille, 1982) led to the belief that the pallidohabenular projection is much more prominent in non-primates than in primates. However, the data of the présent study reveal that the HL is, in both absolute and relative terms, the most densely innervated of ail GPi target sites in monkeys. The limbic nature of thèse pallidohabenular neurons in primate GPi is reinforced by the fact that some of them provide axon collaterals to the limbic anterior thalamic nuclei along their way to the HL. The anterior thalamic nuclei, which receive fibers of the mammillothalamic tract and, in turn, project to the cingulate cortex, are considered as key relay station within the limbic circuitry (Jones, 1985).

Altogether, thèse data reveal that the pallidohabenular projection is a highly prominent component of the pallidal outflow in primates, a component that has been unfortunately largely neglected in our récent attempts to redefine the functional organization of the basai

131

ganglia. The existence of a pallidohabenular projection suggests that the basai ganglia may be able to influence neural mechanisms other than the one strictly related to somatic musculature and movement. In that perspective the HL could be viewed as a functional interface between the basai ganglia and limbic System (Spooren et al., 1995). However, because the HL is a highly compartmentalized nucleus (Herkenham and Nauta, 1977), it is possible that the basai ganglia and limbic outflows remain as well segregated at the HL level than they are at the GPi level.

3.6.6

Contracterai pallidofugal projections

Haring Nauta was the first to allude to contralateral pallidofugal projections in his autoradiographic studies of the efferent projections of the féline EN (Nauta, 1974; Nauta, 1979). He noted fibers decussating in the interhabenular commissure to innervate the contralateral HL, as well as other fibers crossing in the supramammillary decussation and in the massa intermedia to reach the CM contralaterally (Nauta, 1974; Nauta, 1979). Various rétrograde cell labeling and anterograde tract-tracing investigations hâve confirmed the existence of contralateral pallidofugal projections in the cat (McBride, 1981; Ilinsky et al., 1982; Nakano et al., 1983) and monkeys (DeVito and Anderson, 1982; Hazrati and Parent, 1991; Parent et al., 1999). However, such contralateral pallidofugal fibers were not alluded to in certain studies in monkeys (Nauta and Mehler, 1966; Kuo and Carpenter, 1973; Kim et al., 1976; Sidibé et al., 1997), and their existence has been categorically denied in another primate study (Fénelon et al., 1990).

Our previous double rétrograde fluorescent labeling studies hâve revealed retrogradely labeled neurons in the contralateral GPi following injections in the ventral tier thalamic nuclei and PPN (Parent et al, 1999). They accounted for about 10-20% of those found ipsilaterally and about one-third of thèse retrogradely labeled neurons in the contralateral GPi were double labeled (Parent et al., 1999). Thèse findings support the existence of pallidal neurons that project to the ventrolateral thalamus or the PPN contralaterally, but

132 also provide évidence for neurons that project contralaterally to both of thèse sites via axon collaterals (Parent et al., 1999).

In the présent study, some collaterals of single motor axons that arborized within the ventrolateral thalamus, the CM/Pf and the PPN ipsilaterally were found to cross the midline in the supramammillary decussation, the rostral pôle of the CM and the midbrain tegmentum. The decussating collaterals could easily be followed in the contralateral CM, but were much more difficult to trace in the contralateral ventrolateral thalamus and PPN because of their faint labeling. Collaterals of single limbic axons were easily traceable in the interhabenular commissure up to the contralateral HL, where they arborized less densely but similarly than ipsilaterally.

Thèse results reveal that the pallidofugal projections System is composed of single axons that are not only highly collateralized, but also partly bilateralized in primates. The pallidofugal fibers that arborize in the contralateral thalamus and/or brainstem may play a major rôle in the functional organization of the basai ganglia. Such decussating axons may explain, for example the bilatéral effects often seen after unilatéral stereotaxic surgical interventions at the globus pallidus level to alleviate the various motor symptoms that characterized Parkinson's disease (Lang, 2000).

3.6.7

Pallidal inputs to Forel's H field and zona incerta

In support of a previous suggestion made following autoradio graphie studies of the pallidofugal fibers in the cat (Nauta, 1979) and monkey (DeVito and Anderson, 1982; Sidibé et al., 1997), the présent study has confïrmed the présence of axonal varicosities in the FH or prerubral field (nucleus campi Foreli) and Zi. In fact, our data reveal that the FH should be considered as one of the major récipient structure of the pallidal outflow in primates. Virtually every fiber that passes through or bifurcates within that région displays some varicosities en passant or emits short and varicose collaterals. The total number of varicosities provide by the pallidofugal fibers as they traverse the FH is about 2 or 3 times

133

as large as that found in the PPN. Although not formally quantified, the pallidal terminais in the Zi appear to be much less numerous than in the FH.

Obviously more information on the overall connections of the FH and the zona incerta is needed to better appreciate the significance of the pallidal input to thèse two important components of the subthalamic région. We hope that the single-axon tracing studies of thèse two structures that are currently undergoing in our laboratory will help to fill this gap.

3.6.8

Comparison with other basai ganglia components

Our previous single-axon tracing studies in monkey hâve shown that the striatum (Parent et al, 1995), the GPe (Sato et al., 2000a) and the STN (Sato et al., 2000b) are composed of neurons that can be divided into numerous différent types on the basis of their targets sites and patterns of axonal collateralization. Furthermore, thèse various types of projection neurons did not to occupy any spécifie seetors of the striatum, GPe or STN, but were rather uniformly distributed throughout their parent nucleus. By comparison, the GPi, with its numerous motor neurons lying within the core of the nucleus and its less abundant limbic neurons scattered at the periphery, differs strikingly from the other basai ganglia components investigated thus far. Furthermore, the GPi harbors the most widely arborized neurons ever encountered within the various basai ganglia components. Through their broadly distributed axonal network, each GPi motor neurons is able to exert a profound effect upon vast collections of neurons that belong to functionally différent neuronal Systems.

Despite major différences in the overall spreading of their axonal arborization, the neurons of the GPi and those of the GPe appear to share a remarkable morphological feature. In both cases the axonal arborization is maximally oriented along the sagittal plane, that is, along the same plane as their dendrites. Previous Golgi-staining studies in primates hâve revealed that the dendritic arborization of both GPe and GPi neurons form a typical disklike structure, the largest surface of which is oriented along the sagittal plane, that is,

134 perpendicular to the incoming striatopallidal fibers (Percheron et al., 1984b). The présent single-axon study of the GPi and our previous investigation of the GPe (Sato et al, 2000a) reveal that a similar morphological trait characterizes the axonal arborization of the neurons of the two pallidal segments. The axonal arborization of the most widely distributed GPi motor neurons disclosed in the présent study (type Ib neuron) was covering an area of about 140 mm2 in the sagittal plane, compared to approximately 30 mm2 in the frontal and horizontal planes. Hence, the axonal arborization of such GPi neurons is about 5 times more extended along the sagittal plane than along the frontal and horizontal planes. The same ratio was found for the axonal arborization of the most widely distributed GPe neuron (type I, which projects to GPi, STN and SNr), despite the fact that the total terminal arbor of this GPe neuron was about 3 times less widespread than that of the GPi neuron. The axonal arborization of this type of GPe neurons covered an area of about 50 mm2 in the sagittal plane, compared to approximately 10 mm2 in the frontal and horizontal planes (Sato et al., 2000a).

Thèse findings indicate that the highly collateralized GPi projections to the ventrolateral thalamus, the CM/Pf and the PPN form in fact elongated units that extend rostrocaudally from the thalamus to the brainstem (Fig. 3.9). Similarly, the highly collateralized GPe projections to the GPi, STN and SNr form identical elongated units located laterally to those formed by the GPi (Fig. 3.9). The exact functional significance of this striking columnar arrangement of the GPi and GPe terminal fields that parallels that of their dendritic fields is at présent unknown. However, as it appears to be a morphological trait common to ail pallidal neurons, this organizational feature should be taken into account when interpreting the functional organization of the primate basai ganglia.

Acknowledgments: The authors express their sincère gratitude to Carole Émond, René Boucher and Cynthia Moore for skilful technical assistance. Martin Parent was holding a Studentship from the FCAR/FRSQ.

Figure 3.1 A, Patterns of neural activity that characterizes the external (upper row) and internai (lower row) segment of the pallidum, as recorded during a single brain pénétration with a glass injection micropipette. B, An injection site in the medial part of the GPi with a dense core of BDA precipitate (arrowhead). Two distinctly labeled neurons (arrows) can be seen dorsomedial to this injection site. C, Higher magnifïcation of the Golgi-like labeled neurons pointed by arrows in B.

135

A

€

'v.

lOOyrp

136

Figure 3.2 Examples of axon trajectories of type I neurons within the GPi as viewed in frontal plane. The stereotaxic coordinates (according to the atlas of Szabo and Cowan, 1984) of both the cell bodies and the levels at which the axon of thèse neurons exits the GPi are indicated. Note the présence of neuron # 1 in red, the axon of which gives rise to two branches within the GPi, one that exits through the lenticular fasciculus (la) and the other via the ansa lenticularis (lb) at a considérable distance from its cell body.

137

138

Figure 3.3 Axonal arborization of a type la GPi neuron, as viewed on sagittal (A), frontal (B) and horizontal (C) planes. The number of axonal varicosities observed in each target site is indicated in parenthèses in A and the arrows in A, B and C indicate the location of the cell body, which is shown photographically (arrow) in D.

139

A /

VApc

/ (468)

VAdc VM total axonal length: 5.86 cm Ar

FH \ ,(142)

GPi

I)

PPN \ lmm

c

»

/GPi

140

•

Figure 3.4 Axonal arborization of a type Ib GPi neuron, as viewed on sagittal (A), frontal (B) and horizontal (C) planes. The number of axonal varicosities observed in each target site is indicated in parenthèses in A, whereas the arrow in A, B and C indicates the location of the cell body. The three main axonal branches of this neuron are illustrated in blue, red and green, respectively. D, Photomicrograph showing terminal varicosities confîned to the calbindin-rich territory of the VAdc. The dashed Une traces the limit between a CB rich (CB+) sector containing labeled varicose axons and a CB poor (CB-) sector of the VAdc devoid of such fibers. Arrows point to CB+ cell bodies in the CB rich sector of the VAdc.

141

total axonal length: 27.22 cm

Jy

PPN

\(57) lmm x

142

/

Figure 3.5 Photomicrographs of terminal arborization of GPi axons in the VAdc (A), the Pf (B), the PPN(C), and FH (D).

143

A

B

>

.S.

c

100(xm

144

Figure 3.6 A, Axonal arborization of a type Ha GPi neuron, as seen on sagittal plane. The arrow indicates the location of the cell body. B, Drawmg showing a higher power view of the terminal field in the HL; the number of axonal varicosities is indicated in parenthèses. C, The dense axonal arborization in the HL. The inset shows an axon that cross the midline via the habenular commissure.

145

A

146

Figure 3.7 A-F, Transverse half-sections through the GPi showing the localization of GPi neurons whose axons were connected to their parent cell bodies. They represent equally spaced (850 /xm apart) frontal sections displayed in a rostrocaudal order. The red and black squares represent respectively type la and Ib neurons, whereas the blue and green triangles indicate respectively type Ha and Ilb neurons. The numbers refer to figures where those neurons are illustrated.

147

A

148

Figure 3.8 A, Frontal section through the middle portion of the GPi to illustrate the distribution of the rétro gradely labeled neurons (blue circles) after FB injection into the HL. B, A few FB labeled pallidohabenular neurons. The inset shows the injection site (blue area).

149

150

Figure 3.9 Diagram illustrating the typical columnar arrangement of the axonal arborization of GPi and GPe neurons, as seen in the frontal plane. The two types of terminal field form elongated bands (gray areas) that lie parallel to one another, as well as parallel to the dendritic arborization of the neurons of origin.

151

PPN

152

CHAPITRE 4 THE PALLIDOFUGAL MOTOR FIBER SYSTEM IN PRIMATES

CHAPITRE 4

THE PALLIDOFUGAL MOTOR FIBER SYSTEM IN PRIMATES

Martin Parent and André Parent

Laboratoire de Neurobiologie Centre de recherche Université Laval Robert-Giffard 2601, de la Canardière, Beauport, Québec Canada G1J 2G3

Parkinsonism and Related Disorders (2004) 10:203-211

4.1

RESUME

L'organisation du système pallidofuge provenant du segment interne du globus pallidus (GPi) chez le singe cynomolgus (Macaca fascicularis) a été investiguée par l'utilisation d'une technique de traçage axonal unitaire. Chez le primate, le GPi est composé majoritairement de neurones dotés d'un axone hautement collatéralisé innervant les neurones prémoteurs situés dans les noyaux du tiers ventral du thalamus, dans le complexe thalamique centre médian/parafasciculaire ainsi que dans le noyau pédonculopontin du tegmentum mésencéphalique. Ces axones suivent un long et tortueux trajet à l'intérieur même du GPi pour ensuite émerger soit par l'anse lenticulaire (AL) ou par le faisceau lenticulaire (LF), et ce, indépendamment de la localisation de leur corps cellulaire d'origine. D'autres axones pallidofuges émergent par le pôle médian du GPi, à une distance variable de l'AL, située ventralement, et du LF, situé dorsalement. Pratiquement tous les axones pallidofuges cheminent à travers le champ H de Forel pour atteindre le thalamus et le tronc cérébral. Ils émettent plusieurs courtes collatérales et varicosités axonales dans cette région subthalamique qui constitue une cible majeure des axones du GPi. Contrairement à ce que l'on croyait, l'AL et le LF ne forment pas des entités anatomiques indépendantes, chacune composée d'axones provenant de territoires fonctionnels distincts du pallidum. Nos résultats indiquent plutôt que ces deux faisceaux de fibres constituent les limites ventrales et dorsales d'un continuum morphologique contenant une multitude d'axones pallidofuges provenant de toutes les régions du GPi. Ces données doivent être prises en considération afin d'améliorer la compréhension des données cliniques obtenues suite aux microstimulations intracérébrales à haute fréquence effectuées dans le pallidum et les régions subthalamiques afin d'atténuer les symptômes moteurs de la maladie de Parkinson.

4.2

ABSTRACT

The organization of the pallidofugal fiber system originating from the internai segment of the globus pallidus (GPi) in cynomolgus monkeys {Macaca fascicularis) was studied by means of a single-axon tracing method. The primate GPi is composed of a majority of neurons endowed with a highly collateralized axon that projects to the premotor neurons located in the ventral tier thalamic nuclei, the centre-médian/parafascicular thalamic complex and the brainstem pedunculopontine nucleus. Thèse axons often follow a long and tortuous course within the GPi and then émerge either through the ansa lenticularis (AL) or the lenticular fasciculus (LF), irrespective of the location of their parent cell body in the GPi. Other pallidofugal axons exit through the medial pôle of the GPi, at various distances between the AL ventrally and the LF dorsally. Virtually ail pallidofugal axons course through Forel's field H, on their way to the thalamus and brainstem. They émit numerous short collaterals and boutons en passant in this sector of the subthalamic région, which stands out as a major target of GPi axons. Our results indicate that AL and LF do not form separate anatomical entities, each carrying axons originating from distinct functional pallidal territories, as commonly believed. Instead, thèse two fascicles form the ventral and dorsal borders of a morphological continuum that harbors a multitude of pallidofugal axons arising from ail sectors of the GPi. This type of information should be taken into account when interpreting data from deep brain stimulation applied to pallidal and subthalamic régions in Parkinson's disease.

157

4.3

INTRODUCTION

Constantin von Monakow, who first gave a detailed account of the ansa lenticularis (Linsenkernschlinge), referred to this structure as being "the sum ofthefiber masses which corne from the région of the lentiform nucleus, penetrate the cérébral peduncle, and gain the subthalamic région and the medial division of the thalamus" (von Monakow, 1895). Von Monakow subdivided this massive fiber System into three différent components: (1) a dorsal division, commonly termed lenticular fasciculus (LF) or fïeld H2 of Forel (Forel, 1877); (2) a middle division, often described separately as fasciculus subthalamicus; and (3) a ventral division, called the ansa lenticularis (AL) sensus striction by later authors. Originally, von Monakow believed that the ansa lenticularis had a mixed striatal and pallidal origin. This concept was invalidated by Ranson and his colleagues, who demonstrated that the AL (von Monakow's ventral division) and LF (von Monakow's dorsal division) in monkeys arise only from the internai segments of the globus pallidus (GPi), whereas the fasciculus subthalamicus (von Monakow's middle division) originates mainly from the external segment of the globus pallidus (GPe) (Ranson et al., 1941; Nauta and Mehler, 1966; Carpenter and Strominger, 1967). In fact, Ranson and coworkers used the more restricted définition of the AL originally put forward by Theodor Meynert (Meynert, 1872) instead of von Monakow's broader view, and the same will be done in the présent study.

The AL courses ventromedially around the posterior limb of the internai capsule and then bifurcates posteriorly to enter Forel's fïeld H. Fibers of the LF émerge dorsomedially at a more caudal level then those of the AL. The LF fibers cross through the internai capsule and then swept caudally to reach the AL in Forel's fïeld H. Ultimately, the AL and LF fibers merge to form the thalamic fasciculus (or Forel's fïeld Hl) located dorsal to the zona incerta. Cécile Vogt (Vogt, 1909) was the first to note that the pallidofugal fibers merge within Hl, but was unable to détermine in which direction thèse fibers were coursing. In 1940, Papez correctly assumed that the fibers run from the pallidum to the thalamus by

158 passing successively through fields H2, H and Hl. He also rightly foresaw that Forel's field H might be an important synaptic relay station for the pallidofugal fibers (Papez and Stotler, 1940). In human and nonhuman primates, the GPi can be subdivided into a latéral (GPi-1) and a medial (GPi-m) portion by the fibers of the accessory medullary lamina. Carpenter and collaborators hâve used this subdivision of the GPi to summarize the results of their tract-tracing experiments in the form of an attractive scheme that is still in use today (Parent, 1996). This view assumes that the fibers of the AL arise predominantly from GPi-1, whereas those form the LF émerge from the GPi-m (Kuo and Carpenter, 1973; Kim et al., 1976; Carpenter, 1981).

The GPi and the subthalamic nucleus (STN), which become hyperactive in Parkinson's disease,

are

currently

major

neurosurgical

targets

for

the

treatment

of this

neurodegenerative disease (Lozano et al., 2002). The most frequently used neurosurgical approach to silence the GPi or the STN involves high frequency stimulation (also termed deep brain stimulation or DBS) of thèse two forebrain nuclei, as well as the fibers that interconnect them (Lozano et al., 2002). Récent studies hâve determined that the most effective DBS location to alleviate motor symptoms in Parkinson's disease is the anterodorsal portion of the STN, with current spreading over fibers coursing nearby this nucleus (Hamel et al., 2003; Patel et al, 2003; Yelnik et al., 2003). It has been suggested that the fibers involved in such a case might be the pallidothalamic bundle (including Forel's fields H and Hl) (Saint-Cyr et al, 2002; Voges et al, 2002), but a detailed knowledge of the organization of the pallidofugal projections is needed to confirm or infirm this suggestion.

We therefore thought it useful to reinvestigate the organization of the pallidofugal fiber System in monkeys with the help of a single-axon tracing method that allows a detailed reconstruction of entire axonal projection of neurons located in différent parts of the GPi. A spécial attention was paid to the initial course of the pallidofugal axons within the GPi itself, as well as to the more distal trajectory of the axons as they emerged from the GPi en route to the thalamus. This study has revealed that the organization of the pallidofugal fiber

159

System in primates does not follow the simple GPi-l/AL and GPi-m/FL dichotomy, as. portrayed in the current literature.

4.4

MATERIALS AND METHODS

4.4.1

Préparation of the animais

A total of eleven adult cynomolgus monkeys (Macaca fasciciilaris) of both sexes, with a body weight that ranged from 3-4 kg, were used in the présent study. Ail surgi cal and animal care procédures adhered to the guidelines for the use and care of expérimental animais of the Canadian Council of Animal Care and Laval University's Animal Care Committee approved our expérimental protocol. The animais were first anesthetized with ketamin (75 mg/kg) plus xylazine (5 mg/kg) and their head placed in a specifically designed stereotaxic apparatus. After trépanation, a radiopaque solution (Omnipac or Iohexol, 0.8 ml of a 65% solution, Nicomed Imaging, Brandford, Ontario, Canada) was injected through a microsyringe into the right latéral ventricle. A few minutes after the injection, latéral and frontal X-ray pictures of the ventricular System were taken to precisely localize the baseline formed by the anterior and posterior commissures in each animal (Percheron, 1975).

4.4.2

Injection procédures

Two to three weeks after ventriculography, the animais were anesthetized as above and placed in the same stereotaxic apparatus. They were then maintained under propofol (10 mg/ml, i.v.) anesthesia while bilatéral microiontophoretic injections of anterograde tracer, biotin dextran aminé (BDA, Molecular probes, Eugène, Or), were being made in différent portions of the GPi. In ail cases, only two injections were made in each GPi. The target was aimed at by using the stereotaxic coordinates of the atlas of Szabo and Cowan (Szabo and Cowan,

1984),

as

modifîed

by

the

data

collected

from

ventriculography.

160 Microiontophoretic labeling was carried out with glass micropipettes (tip diameter 2-3 jxm). filled with a solution of potassium acétate (0,5M) plus 2% BDA. Thèse électrodes had impédance ranging between 10-15 MQ and were used to monitor the extracellular activity of the neuronal populations encountered during the pénétration of the micropipette. Once in the chosen target, the micropipette was connected to a high compliance iontophoresis device (NeuroData) and the tracer was injected by passing positive current puises of 300400 nA (ls on/ ls off) for 25 min.

4.4.3

Tracer révélation and cytochrome oxidase staining

After a survival period of 8-10 days, the animais were deeply anesthetized with sodium pentobarbital and perfused transcardially with 1 liter of saline solution (0.9% NaCl) followed by 2 liters of a fixative solution containing 4% paraformaldehyde in phosphate buffer (PB, 0.1M, pH 7.4) and 1 liter of 10% sucrose solution in PB. The brains were dissected out and placed in a cryoprotective solution composed of 1/3 paraformaldehyde (4% solution in PB) and 2/3 sucrose (30% solution in PB) for 24h at 4°C. They were eut along the sagittal (7 monkeys) or frontal plane (4 monkeys) at 70 /xm using a freezing microtome. The sections were collected serially in phosphate buffer saline (PBS, 0.1M, pH 7.4) and processed for the visualization of BDA according to the avidine-biotineperoxydase method (ABC, Vector Labs, Burlingame, CA). In brief, The sections were incubated overnight at 4°C in a solution containing ABC diluted 1:100 in PBS (0.1M, pH 7.4), plus 1% normal rabbit sérum and 1% Triton X-100. They were then rinsed twice in PBS and once in Tris buffer. The bound peroxidase was revealed by incubating the sections in a solution containing 0.025% 3,3'diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO), 0.3% nickel-ammonium sulfate, 0.008% cobalt chloride and 0.008% hydrogen peroxide in 0.05M Tris buffer, pH 7.6, for 10-15 minutes at room température. The reaction was terminated by a rinse in Tris buffer followed by two rinses in PBS.

To help identifying nuclei and structures that harbored labeled neurons and axons, most sections were counterstained for cytochrome oxidase, according to the histochemical protocol of Wong-Riley (Wong-Riley, 1979). The counterstaining was performed before

161 BDA révélation, and nickel-cobalt-intensified DAB (dark blue reaction) and unintensified, DAB (diffuse brown precipitate) were used to reveal BDA and cytochrome oxidase, respectively.

4.4.4

Material analysis

Ail sections were mounted on gelatin-coated slides, dehydrated in graded alcohols, cleared in toluène, and coverslipped with Permount. They were examined under a Nikon light microscope equipped with a caméra lucida. Détails of single BDA-labeled neurons were drawn at 200X and 400X magnifications. The terminal fields and cell body of the labeled neurons were mapped at lower magnifications to détermine their topographie location according to the atlas of Szabo and Cowan (Szabo and Cowan, 1984). Most axons were entirely reconstructed along either the sagittal or frontal planes with the help of a caméra lucida.

Furthermore,

a

computerized

image-analysis

System

(Neurolucida,

MicroBrightField, Inc., Colchester, VT) was used to reconstruct in three dimensions the GPi and the initial axonal trajectory of GPi neurons. The photomicrographs were digitally captured (AGFA Studiocam, Woburn, NA) and handled with the Adobe Photoshop software (version 7.0, Adobe, San Jpse, CA).

4.5

RESULTS

The injection procédure used in the présent study produced very small injection sites involving no more than 5 to 10 GPi neurons per site. Most of the injection loci had a dense core of BDA precipitate surrounded by several neurons labeled in a Golgi-like manner. Thèse singly labeled GPi neurons had a spindle-shaped, ovoid or triangular cell body, with 2-5 long and poorly ramifïed primary dendrites (Fig. 4.1 B, C). Intensely labeled axons emerged from either the core of the injection sites or from individually labeled neurons located at the periphery of the injection loci. The axons exited from either the cell body or a

162 primary dendrite and, since they could easily be followed individually throughout each section, they were entirely reconstructed along the sagittal or frontal planes with a caméra lucida. Based on their target sites and axonal branching patterns, the primate GPi is composed of: (1) a large number (90%) of "motor neurons" whose axon projects profusely to the ventral tier thalamic nuclei, the centre médian/parafascicular thalamic complex (CM/Pf) and pedunculopontine tegmental nucleus (PPN) (Fig. 4.1 A); and (2) a smaller number (10%) of "limbic neurons" whose axon arborizes principally within the latéral habenula. Previous rétrograde cell-labeling experiments hâve revealed that the limbic neurons abound in the anterior pôle of the GPi, but are confmed to the periphery of the nucleus at more caudal levels (Parent et al., 1999; Parent et al., 2001). The axonal trajectory of thèse limbic GPi neurons hâve been described in détail elsewhere, so they will not be considered further hère.

Twenty-six axons of motor neurons were traced entirely from the cell body in the GPi to their terminal fields. Thèse axons émit a few collaterals that crossed the midline at the level of the CM/Pf or the supramammillary decussation. Thèse decussating fîbers innervated the same structures contralaterally and the pattern of innervation was similar on both sides, except that the labeled fîbers were much less abundant contralaterally than ipsilaterally. The main axon of GPi motor neurons emitted short and varicose collaterals in the rostral part of Forel's fîeld H. It then swept caudally and descended toward the brainstem tegmentum (PPN) providing, en passant, a collatéral to the retrorubral area (RRA) (Fig. 4.1 A). The terminal field in the PPN consisted of a small number of varicose terminal branches that emitted a few collaterals oriented at right angles from the main axonal branch (Fig. 4.2 D).

Before reaching the brainstem, the main axon of motor GPi neurons gave at least one axonal branch that ascended via the thalamic fasciculus (Hl) and branched into 10-15 long and thin collaterals (Fig. 4.2 A) that reached the ventral anterior and ventral latéral thalamic nuclei (VA/VL), where they formed typical glomerule-like terminal clusters (Fig. 4.2 B).

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Another major collatéral attained the CM/Pf, where it branched in the form of loops oriented along the longest extent of CM/Pf (Fig. 4.2 C). Most of thèse fibers arborized within the medial part of the CM and the latéral part of the Pf. Some axons of motor GPi neurons also emitted 1 or 2 thin collaterals that arborized poorly within, and sometimes beyond, the somatodendritic domain of their parent cell body. Thèse local collaterals exhibited vancosities of various sizes reminiscent of boutons en passant or boutons terminaux. Besides local collaterals, the main axon of motor neurons often follow a complex and tortuous course in the GPi and branched either within or just after leaving the pallidum. Pallidofugal axons emerged either through the AL or the LF, irrespective of the position of their parent cell body in the GPi (Fig. 4.3). Some axons arising from cell bodies located in the GPi-1 or the GPi-m emerged at approximately the same rostrocaudal level as their cell body (e.g. neurons # 3 in yellow and # 4 in red in figure 4.3 C). Other axons did not run through the AL or LF but exited directly through the medial pôle of the GPi (neuron # 2 in figure 4.3). Some axons coursed along a considérable distance rostrally or caudally within the GPi before exiting the nucleus via either the LF or the AL. Neuron # 3, whose cell body was located in the GPi-1, had an axon (yellow) that exited through the AL, whereas neuron # 4, whose cell body was located in the GPi-m, had an axon (red) that also exit through the AL at a rostro-caudal plane corresponding to that of its cell body (Fig. 4.3). Neuron # 1 gave rise to an axon that bifurcated within the GPi. One of the two major collaterals of this axon exited the GPi through the LF caudally, at about the same level as its parent cell body (la, Fig. 4.3), whereas the other coursed for about 3 mm rostrally and ventrally and emerged via the AL (lb, Fig. 4.3). Cell bodies located in the anterior half of the GPi (e.g. neurons # 1 and 3 in figure 4.4 C) gave rise to axons that exited either through the LF and the AL. Likewise, neurons located in the posterior half of the GPi (e.g. neurons # 6 and 2 in figure 4.4 C) gave rise to axons that coursed in the LF or the AL. The GPi motor neurons that had the most profusely arborized axon prevailed ventrally in the caudalmost portion of the GPi and their axons exited through either the AL or the LF.

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4.6

DISCUSSION

The présent study has provided the first detailed picture of the initial axonal trajectory of motor GPi neurons in primates. Our single-axon tracing study reveals that that the pallidofugal fiber System is much more widely distributed than what can be inferred from the current AL-LF scheme. The functional significance of thèse findings vvill now be discussed in the light of previous data gathered principally in nonhuman primates.

4.6.1

Définition of motor and limbic pallidal neurons

In the présent study, we used a notion of motor and limbic pallidal neurons that has nothing to do with the functional subdivision the globus pallidus into the so-called "associative", "sensorimotor" and "limbic" territories, as proposed by Smith and collaborators (Shink et al, 1997; Sidibé et al., 1997; Baron et al., 2001). This tripartite subdivision of the GPi assumes that the striatal organization is directly transposed at pallidal level. Hence, by virtue of the topography of the striatopallidal projection, the dorsorriedial third, the ventrolateral two-thirds and the rostroventromedial pôle of the GPi would correspond, respectively, to the associative, sensorimotor and limbic territories of the GPi (Shink et al., 1997; Sidibé et al, 1997; Baron et al., 2001). Hère, the GPi neurons were defined following a detailed study of their axonal projections, which showed that they target the premotor neurons located in the VA/VL and PPN nuclei. They were not characterized on the basis of an a priori location of their cell body of origin in any putative functional pallidal territories. Thus, the motor neurons, as defined in the présent study, may réside anywhere within the large, central portion of the GPi, including within the so-called associative or sensorimotor territories. However, they are unlikely to occur in significant number in the rostral pôle of the GPi and at the periphery of this nucleus more caudally, because thèse pallidal sectors are principally populated by neurons that deserve the epithet limbic inasmuch as their axon targets essentially the habenula (Parent et al., 1999; Parent et al., 2001).

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4.6.2

Organization of the pallidofugal fiber System

The current concept of the organization of the pallidofugal projection is largely based on the results of tract-tracing studies undertaken in macaque monkeys by Carpenter and colleagues, who used anterograde degeneration methods and autoradiographic axonal labeling procédures (Kuo and Carpenter, 1973; Kim et al., 1976; Carpenter, 1981). Thèse investigators concluded that fibers in the AL arise predominantly from the GPi-1 and pass ventromedially through portions of the GPi-m, whereas fibers in the LF were said to originate mainly from GPi-m and to project dorsomedially through the internai capsule (Fig. 4.4 A, B). This attractive scheme was challenged recently by the results of an anterograde labeling studies in squirrel monkeys that involved large injections of BDA, mainly in the posterior aspects of the GPi (Baron et al., 2001). This investigation led to the suggestions: (1) that pallidofugal fibers originating from neurons in the caudal half of the GPi exit from the medial border of the nucleus, without traveling to any significant extent along the rostrocaudal plane, and (2) that the AL is largely composed of axons that originate from neurons located in the rostral half of the GPi (Baron et al., 2001).

The data cited above were obtained with methods that hâve obvious technical limitations. For instance, the lack of sensibility and précision of the anterograde degeneration method is notorious, whereas the difficulty to follow axons labeled with tritiated amino acids, particularly their terminal portion, has led to the abandon of this procédure. Likewise, studies that involve large injections of BDA are hindered by the fiber-of-passage problem and also by the impossibility of visualizing axons that course within the injection site. Thèse limitations were circumvented hère by labeling only small (5-10 cells) pools of GPi neurons and by reconstructing only axons that could be directly connected to their parent cell bodies and whose trajectory, including its initial portion in the GPi, was clearly visible and did not intermingle with other labeled axons. This approach has allowed us to provide a detailed three-dimensional reconstruction of the entire axonal arborization, including the initial intrapallidal segment, of single neurons located in various sectors of the GPi.

Our data reveal that motor axons émerge through either the AL or the LF, irrespective of the location of their cell body in the GPi. Some motor neurons, whose cell body was

166 located in the anterior portion of the GPi, had an axon that followed a long caudal course, before exiting via the LF. This finding is incongruent with the notion that the AL is largely composed of axons that originate from neurons located in the rostral half of the GPi, as advocated earlier on the basis of data obtained following large BDA injections within the GPi of squirrel monkeys (Baron et al., 2001). Our study also revealed motor neurons with an axon that followed a rather straight, intrapallidal course and exited through the medial pôle of the GPi, at various distances between the LF dorsally and the AL ventrally. Furthermore, some neurons located on each side of the accessory medullary lamina had their respective axon coursing in the AL. We also detected a neuron with an axon that bifurcated within the GPi; one of its branch emerged through the LF at about the same anteroposterior level than that of its parent cell body, but the other branch coursed for more than 3 mm within the GPi before emerging via the AL rostrally.

Thèse findings reveal that pallidal axons and collaterals follow rather complex and tortuous courses within the GPi so that the location of the cell body within the nucleus cannot be considered as a faithful predictor of where the pallidal axon (or its multiple branches) will émerge. Our results indicate that, instead of being considered as two separate anatomical entities, each carrying axons originating from distinct functional pallidal territories, the AL and the FL should be viewed as the ventral and dorsal borders of a morphological continuum that harbors a multitude of pallidofugal axons arising from ail sectors of the GPi (Fig. 4.4 C, D).

4.6.3

Functional considérations

The tracing of the more distal portion of pallidofugal fibers has revealed that ail GPi motor axons are widely distributed and highly collateralized. For example, ail pallidothalamic fibers that we traced were collaterals of single axons that also innervated the PPN and, in about 50% of the cases, the CM/Pf as well. This form of organization implies that motor GPi neurons send efferent copies of the same neural information to the small cell clusters in the ventral tier thalamic nuclei, and to more diffuse cell populations in the PPN and CM/Pf. In that sensé, the GPi is not différent from ail the other major components of the primate

167 basai ganglia, which were shown to be composed of neurons endowed with a highly collateralized axon (Parent et al., 2000; Parent et al., 2001; Parent and Parent, 2002). Thèse fmdings indicate that the primate basai ganglia must be viewed as a highly distributed System in which each component can interact with the others in a multifarious fashion.

The fact that each GPi labeled axon expanded over a large portion of the ventral tier thalamic nuclei, irrespective of the location of their parent cell body in the GPi, argues against the idea of a direct transposition at thalamic level of the putative pallidal functional territories (Sidibé et al., 1997). Instead, the major organizational feature of the pallidothalamic System appears to be the fact that each of its axon gives rise to 5-10 glomerule-like terminal plexuses that are widely distributed in the ventral tier nuclei. This organizational pattern allows neural information integrated within GPi motor neurons to be processed in parallel by each of thèse small thalamic cell clusters before being conveyed to the cérébral cortex. This mode of organization is opposite to the sériai type of processing used by most classical somatosensory Systems. In such a case, each axon carried a spécifie type of information that is transmitted to a single thalamic unit, which in turn projects to a spécifie cortical domain.

Studies on localization of DBS électrodes in human subthalamic région hâve revealed that clinically effective stimulation in cases of Parkinson's disease was commonly directed at the anterodorsal sector of the STN, with current spreading into surrounding région, such as Forel's fields (Saint-Cyr et al., 2002; Hamel et al., 2003; Yelnik et al., 2003). Such a spreading of electric current is likely to affect the hyperactive pallidofugal axons that course fields Hl and H2, but it might also influence field H, which is one of the major récipient structure of the pallidal outflow in primates. Virtually every single motor fiber passes through or bifurcates within this complex région, where they émit numerous short and varicose collaterals or display boutons en passant. The total number of varicosities provide by the pallidofugal axons as they traverse the field H is about two or three times as large as that found in the PPN. Obviously, more information on the overall connections of field H is needed to better appreciate the significance of the pallidal input to this important component of the subthalamic région. Altogether, our fmdings suggest that, in addition to a

168

direct action on the STN, the bénéficiai effects of DBS in Parkinson's disease might also, imply stimulation of the pallidofugal axons that form a fiber continuum bordered dorsocaudally by the LF and ventro-rostrally by the AL, as well as an effect on the pallidal terminal fïeld H.

Acknowledgements : This study was supported by grant MOP-5781 of the Canadian Institûtes for Heath Research (CIHR). Martin Parent was the récipient of a Studentship from the National Science and Engineering Research Council (NSERC).

Figure 4.1 A, Schematic illustration of the axonal arborization of a typical GPi motor neuron that projects to the VA/VL, CM/Pf, PPN, field H (FH), and RRA, as viewed on a sagittal plane. This axon did not run through the AL or the LF, but exited directly through the medial pôle of the GPi. The axon was entirely reconstructed from sériai sections using caméra lucida. B, C, Photomicrographs showing two examples of Golgi-like labeled neurons in the GPi.

169

B

w

(

-c^

^%

^

v

+J

170

Figure 4.2 A, Caméra lucida drawing of the terminal axonal arborization of an individually labeled GPi motor neuron in the VA/VL. B, Typical glomerule-like clusters in the VA/VL. C, axonal arborization of the same GPi neuron in the CM/Pf. D, terminal field in the PPN that consisted of a small number of varicose terminal branches that emitted a few collaterals oriented at right angles from the main axonal branch. The number of axonal varicosities observed in each target site is indicated in parenthèses in A, C and D.

171

172

Figure 4.3 A-C, Computerized three-dimensional reconstruction of the initial axonal trajectory of four GPi motor neurons, as seen in frontal (A), medial (B) and dorsal (C) plane. The axon #1, in black, gives rise to two branches within the GPi, one (la) exiting through the LF at the same rostro-caudal plane as its parent cell body in the GPi-1, and another (lb) emerging via the AL at a considérable distance from its cell body. The axon #2, in blue, originating from a neuron in GPi-1, does not run through the AL or LF, but exits directly through the medial pôle of the GPi. The axons #3 (in yellow) and #4 (in red), whose parent cell bodies are located respectively in GPi-1 and GPi-m, exit both through the AL.

173

A

lmm

B

lmm

174

Figure 4.4 Schematic drawings comparing the current model of the organization of the pallidofugal fiber (A, B) with a revised view based on the data obtained in the présent study (C, D). The trajectory of the fibers is drawn in relation to the location of the AL and FL, as seen in horizontal (A, C) and frontal plane (B, D). According to the current scheme (A, B), fibers of the AL (red) arise from the outer portion of the GPi (latéral to the accessory medullary lamina indicated by a dashed Une), whereas those of the LF (blue) originate in the inner portion of the GPi. In contrast, our data (C, D) indicate that pallidal motor axons emerged through either the AL or the LF, irrespective of the location of their cell bodies in the GPi. Some motor axons also emerged from the medial pôle of the GPi, at various distance between the AL ventrally and the LF dorsally, hence forming a sort of fiber continuum.

175

A

D

176

CHAPITRE 5 SINGLE-AXON TRACING STUDY OF CORTICOSTRIATAL PROJECTIONS ARISING FROM PRIMARY MOTOR CORTEX IN PRIMATES

CHAPITRE 5

SINGLE-AXON TRACING STUDY OF CORTICOSTRIATAL PROJECTIONS ARISING FROM PRIMARY MOTOR CORTEX IN PRIMATES

Martin Parent and André Parent

Laboratoire de Neurobiologie Centre de recherche Université Laval Robert-Giffard 2601, de la Canardière, Beauport, Québec Canada G1J2G3

The Journal of Comparative Neurology (2006) 496:202-213.

5.1

RESUME

Les projections efférentes provenant de la région du cortex moteur primaire (Ml) associée au membre antérieur chez le singe cynomolgus (Macaca fascicularis) ont été investiguées par injections microiontophorétiques de biotine dextran aminé. Ces microinjections, centrées sur la couche V, ont permis de marquer antérogradement 42 axones corticofuges. La reconstruction unitaire de l'ensemble de ces neurones démontre que, chez le primate, le striatum reçoit à la fois une innervation directe et indirecte en provenance de Ml. La projection corticostriée directe provient d'axones de faible diamètre qui n'émettent aucune collatérale lors de leur course sinueuse vers le striatum ipsilatéral. Ces axones se divisent en entrant dans la partie dorsolatérale du putamen post-commissural, une région qui correspond au territoire sensorimoteur du striatum. La projection corticostriée indirecte, quant à elle, provient d'une fine collatérale axonale émise dans la corona radiata par un axone de plus fort diamètre qui descend vers le tronc cérébral. Cette collatérale entre par la partie dorsomédiane du putamen et commence à se diviser seulement lorsqu'elle atteint la région dorsolatérale du putamen. Dans ce territoire sensorimoteur du striatum, elle se divise en 2-4 branches axonales dotées de petites varicosités axonales également réparties sur l'axone. Au sein du striatum, les axones corticostriés directs et indirects se divisent de façon modérée mais occupent tout de même un vaste territoire striatal rostrocaudal dans lequel ils contactent en passant plusieurs neurones striataux. Ces données démontrent que, contrairement à ce que l'on croyait, le système corticostrié moteur n'est pas exclusivement formé d'axones dédiés uniquement au striatum. Ce système est aussi composé de fines collatérales provenant d'axones de plus fort diamètre en route vers le tronc cérébral. Ces projections corticostriées indirectes permettent au striatum de recevoir une copie de l'information neuronale envoyée vers le tronc cérébral et la moelle épinière par les neurones pyramidaux de la couche V de Ml.

5.2

ABSTRACT

The axonal projections arising from the forelimb area of the primary motor cortex (Ml) in cynomolgus monkeys (Macaca fascicularis) were studied following microiontophoretic injections of biotinylated dextran aminé under electrophysiological guidance. The microinjections were centered upon layer V and a total of 42 anterogradely labeled corticofugal axons were reconstructed from sériai frontal or sagittal sections with a caméra lucida. Our investigation shows that the primate striatum receives both direct and indirect projections from Ml. The direct corticostriatal projection is formed by axons that remain uniformly thin and unbranched throughout their sinuous trajectory to the ipsilateral striatum. They divide as they enter the dorsolateral sector of the post-commissural putamen, the so-called sensorimotor striatal territory. The indirect corticostriatal projection dérives from a thin collatéral emitted within the corona radiata by thick, long-range fibers that descend toward the brainstem. The collatéral enters the putamen dorsomedially and remained unbranched until it reaches the dorsolateral sector of the putamen, where it brakes out into 2-4 axonal branches displaying small and equally spaced varicosities. Both direct and indirect corticostriatal axons branch moderately, but occupy vast rostrocaudal striatal territories, where they appear to contact en passant several widely distributed striatal neurons. Thèse findings reveal that, in contrast to current beliefs, the primate motor corticostriatal System is not exclusively formed by axons dedicated solely to the striatum. It also comprises collaterals from long-range corticofugal axons, which can thus provide to the striatum a copy of the neural information that is being conveyed to the brainstem and/or spinal cord.

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5.3

INTRODUCTION

Among the various striatal afférents, those arising from the cérébral cortex are by far the most prominent. The corticostriatal pathway is a massive projection linking virtually the entire neocortex with the striatum, the latter being considered as the major input stage of the basai ganglia. The question of knowing if corticostriatal projection system is independent from the long-range fibers system that targets the brainstem or the spinal cord has been a matter of controversy since the beginning of the 20th century. At that time, observations made on Golgi-stained material in mice led Ramôn y Cajal to conclude that the corticostriatal pathway was made of collaterals of fibers descending in the internai capsule (Ramôn y Cajal, 1909, 1911). This view was supported by more récent observations on Golgi-stained material (Webster, 1961) and by electrophysiological recording and intracellular labeling experiments (Donoghue and Kitai, 1981; Cowan and Wilson, 1994) in rats. The corticostriatal projection system in rodents is currently viewed as being composed of collaterals of long-range corticofugal axons and of corticostriatal neurons dedicated mainly to the striatum, but which also project elsewhere in the telencephalon (Donoghue and Kitai, 1981; Landry et al., 1984; Wilson, 1987; Cowan and Wilson, 1994; Lévesque et al., 1996a; Lévesque et al., 1996b; Lévesque and Parent, 1998; Zheng and Wilson, 2002; Reiner et al., 2003).

In primates, the first accurate depiction of the corticostriatal projection stems from the pioneering autoradiographic tracing studies undertaken in cynomolgus monkeys by Heinz Kùnzle some 30 years ago (Kunzle, 1975). Thèse studies provided fïrst-hand information on the topographical organization of the terminal fïelds of corticostriatal fibers in primates, but were much less informative regarding the trajectory and branching pattern of corticofugal fibers, which are difficult to delineate in autoradiographic material. Nevertheless, following a detailed examination of his expérimental material, Kunzle concluded that, rather than being formed by collaterals of long-range corticofugal fibers, corticostriatal fibers are most likely part of a distinct system solely dedicated to the striatum

182

(Kùnzle, 1975). This view is supported by the results of a rétrograde cell labeling study in squirrel monkeys indicating that the corticostriatal axons are unlikely to be collaterals of axons projecting to other sites (Jones et al., 1977). A similar conclusion was reached by more récent electrophysiological investigations in monkeys (Bauswein et al., 1989; Turner and DeLong, 2000). Thèse studies reported that collatéral branching of long-range corticofugal axons to the monkey putamen is infrequent and suggested that the putamen receives a cortical message that is significantly différent from that sent down to the brainstem and spinal cord in primates.

In view of the rôle that the primary motor cortex (Ml) plays in the input of the major récipient structure of the basai ganglia, we thought it worthwhile to investigate, at the single cell level, the trajectory and arborization of corticostriatal axons arising from Ml in primates. This issue was addressed by using a straightforward procédure that allows the injection of an anterograde tracer in very small subsets of electrophysiologically identified neurons located in lamina V of Ml and the subséquent tracing of single anterogradely labeled axons. This research has yielded novel findings that are important to reach a more complète understanding of the anatomical and functional organization of primate basai ganglia.

5.4 5.4.1

MATERIALS AND METHODS Injection procédures

A total of nine adult cynomolgus monkeys (Macaca fascicularis) of both sexes, with a body weight that ranged from 3-4 kg, were used in the présent study. The work was performed in accordance with the Canadian Guide for the Care and Use of Laboratory Animais and ail surgical and animal care procédures were approved by the Institutional Animal Care Committee of Laval University. The animais were first anesthetized with ketamine (75 mg/kg) plus xylazine (5 mg/kg) and their head placed in a specifically designed stereotaxic apparatus. They were then maintained under propofol (10 mg/ml, i.v.) anesthesia, while microiontophoretic injections of biotin dextran aminé (BDA, Molecular

183 Probes, Eugène, OR) were being made bilaterally mainly in the forelimb area of the primary motor cortex, as identified on physiological maps established by Woolsey (1958), Kwan et al. (1978) and Stepniewska et al. (1993) (Fig. 5.1 A). Because corticostriatal projections were found to originate mainly frora layer V in primates (Jones et al., 1977; Saint-Cyr et al., 1990), BDA injections were centered upon layer V of Ml. In some cases, neurons located in supragranular layers were also labeled. However, thèse neurons were very few in number, their staining was weak and their axon could not be traced outside the cérébral cortex. They most likely represent neurons whose neuronal processes (axons or dendrites) hâve transported retrogradely a small amount of tracer from the periphery of the main injection loci.

In most cases four injections were made, two on each side of the brain. In one animal, however, the two injections were placed on the same side of the brain. We used the stereotaxic coordinates of the atlas of Szabo and Cowan (1984) and microiontophoretic labeling was carried out with glass micropipettes (tip diameter 2-3 fini) filled with a solution of potassium acétate (0.5M) plus 2% BDA. Thèse électrodes had impédance ranging between 10-15 MCI and were used to monitor the extracellular activity of the neuronal populations encountered during the pénétration of the micropipette. Layer V of Ml was easily recognizable by the characteristic bursting firing pattern of its neurons under ketamine-xylazine anesthesia. Once in the chosen target, the micropipette was connected to a high compliance iontophoresis device (NeuroData) and the tracer was injected by passing positive current puises of 300-400 nA (ls on/ ls off) for 25 min.

5.4.2

Tracer visualization and cytochrome oxidase staining

After a survival period of 8-10 days, the animais were deeply anesthetized with sodium pentobarbital and perfused transcardially with 1 liter of saline solution (0.9%) followed by 2 liters of a fixative solution containing 4% paraformaldehyde in phosphate buffer (PB, 0.1M, pH 7.4) and 1 liter of 10% sucrose solution in PB. The brains were dissected out and placed in a cryoprotective solution composed of 1/3 paraformaldehyde (4% solution in PB)

184

and 2/3 sucrose (30% solution in PB) for 24h at 4°C. The brains were then eut along the sagittal (2 monkeys) or frontal (7 monkeys) plane at 70 [ira using a freezing microtome. The sections were collected serially in phosphate buffer saline (PBS, 0.1M, pH 7.4) and processed for the visualization of BDA according to the avidine-biotin-peroxydase method (ABC Elite kit, Vector Labs, Burlingame, CA) with 3,3'diaminobenzidine (DAB; Sigma, St. Louis, MO) as the chromogen. In brief, the sections were incubated overnight at 4°C in a solution containing ABC diluted 1:100 in 0.1M PBS, pH 7.4, plus 1% normal rabbit sérum and 1% triton X-100. They were then rinsed twice in PBS and once in Tris buffer. The bound peroxidase was revealed by incubating the sections in a solution containing 0.025% DAB, 0.3% nickel-ammonium sulfate, and 0.008% hydrogen peroxide in 0.05M Tris buffer, pH 7.6, for 10-15 minutes at room température. The reaction was terminated by a rinse in Tris buffer followed by two rinses in PBS.

To help identifying cortical layers, nuclei and structures that harbored labeled neurons and axons, sections were counterstained for cytochrome oxidase, according to the histochemical protocol of Wong-Riley (Wong-Riley, 1979). The counterstaining was performed before BDA révélation, and nickel-cobalt-intensified DAB (dark blue reaction) and unintensified DAB (diffuse brown precipitate) were used to reveal BDA and cytochrome oxidase, respectively.

5.4.3

Material analysis

Ail sections were mounted on gelatin-coated slides, dehydrated in graded alcohols, cleared in toluène, and coverslipped with Permount. They were examined under a Nikon light microscope equipped with a caméra lucida. Détails of single BDA-labeled neurons were hand-drawn at 200X and 400X magnifications. The terminal fields and cell body of the labeled neurons were mapped at lower magnifications to détermine their topographie location according to the atlas of Szabo and Cowan (Szabo and Cowan, 1984). The photomicrographs were digitally captured (Leica DC 300 caméra, Wetzlar, Germany) and

185 processed with the Adobe Photoshop software (version 7.0, Adobe, San José, CA). Axons were reconstructed along either the sagittal or frontal plane with the help of a caméra lucida.

In a typical experiment, not more than 5 corticostnatal axons could be clearly visualized from each injection locus and among thèse 5 axons only those that were not closely intermingled with one another as they descend through the subcortical white matter were retained for a more detailed analysis. The terminal arborization of each of the latter axons at the striatum level was carefully examined and only axons that had a terminal field that did not overlap with those of other axons were retained in our sample. Following detailed examination of axons emerging from a total of 34 injection loci, including some axons that could be directly linked to their cell body of origin, we found only 31 corticostriatal axons that meet thèse highly stringent inclusion criteria. Thèse corticostriatal axons were charted individually by tracing ail of their branches and collaterals through sériai brain sections allowing a complète reconstruction of their entire axonal arborization. A neuron was considered to project toward the brainstem when its labeled axon could be traced into the cérébral peduncle. Each axonal varicosity encountered along the various branches of individual axons was precisely charted and the total number of such varicosities was determined to estimate the strength of the synaptic input provided by a single corticostriatal axon. Ail brain sections in which the axon and its multiple branches traveled had to be preserved and carefully examined to establish the direct continuity between the various segments of a single axon. The fact that each charted axonal segment belongs to the same axon was ensured by moving microscopically up and down through the différent brain sections in which thèse axonal segments coursed. The rare cases in which the various axonal segments could not be linked with one another with certainty were excluded from our sample. Occasionally, however, the most distal portions of certain axon became too faintly labeled to be accurately traced, although they were clearly in continuity with the rest of the axonal branching. Thèse short axonal segments and the varicosities they displayed were not taken into account in our overall reconstructions. The alignment of axonal segments in adjacent sections was made by properly adjusting the contour of each section at low magnification. Furthermore, we used various landmarks visible at high

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magnification, such as blood vessels and brain nuclei, the latter being delineated with cytochrome oxidase, as further means to ensure the proper alignment of the various axonal profiles. Our previous single-axon tracing investigations of the various basai ganglia components in primates (Sato et al., 2000a; Sato et al, 2000b; Parent et al., 2001; Parent and Parent, 2005) should be consulted for further technical détails.

5.5 5.5.1

RESULTS General labeling features

The Ml sector investigated hère corresponds mainly to the forelimb area, as defmed anatomically using physiological maps of monkey cérébral cortex (Woolsey, 1958; Kwan et al., 1978; Stepniewska et al., 1993) (Fig. 5.1 A). Because rétrograde cell labeling studies indicated that most of corticostriatal projections anse from layer V (Jones et al., 1977; Saint-Cyr et al., 1990), the injection sites were centered upon this cortical layer (Fig. 5.1 B). Most injection loci had a dense core composed of BDA precipitate surrounded by several neurons labeled in a Golgi-like manner (Fig. 5.1 C). Thèse labeled Ml neurons had a triangular cell body ranging between 15 and 30 jum in diameter from which emerged an apical dendrite and 2-5 horizontal basai dendrites arborizing as far as 400 to 700 /rai away from the cell body. The latter feature could explain why labeled neurons were occasionally found at some distance from the core of the injection sites. Intensely labeled axons could be seen to émerge from either the core of the injection sites or from individually labeled neurons located peripherally. In the latter case, the axons emerged from the basis of the cell body and reach the subcortical white matter where they initiate their sinuous downward course toward subcortical grey matter.

Forty-two corticofugal axons were traced in the présent study. Many of thèse axons could not be connected to their parent cell body (see below), but a detailed analysis of the material was made to ensure that thèse axons were emerging from the core of the cortical injection loci. Because ail injection sites were confined to the cortical gray matter, it is

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highly unlikely that some fibers of passage might hâve taken up the tracer. Ail the main structures that project to Ml, principally the thalamus, were nevertheless carefully scanned for the présence of retrogradely labeled neurons. However, no such retrogradely labeled neurons were encountered probably because of the very small amount of BDA delivered in Ml injection sites.

5.5.2

Axonal trajectory

Based on their target sites and axonal branching patterns, we identified two différent types of corticostriatal projection neurons in the primate Ml. Neurons of the first type had an axon that projected directly to the striatum, whereas neurons of the second type innervated indirectly the striatum through collaterals emitted by long-range axons projecting toward the brainstem.

The axonal arborization of 21 neurons that belong to the first neuronal type were reconstructed from sériai sections. In thèse cases, the axons did not intermingle with other labeled axons and could thus be easily followed individually through each section. Two of thèse axons were connected to their cell bodies of origin. They lie in the upper part of layer V of Ml, within the dense core of an injection site. Direct corticostriatal projections were formed by axons that remained uniformly thin and unbranched throughout their trajectory to the ipsilateral striatum. Thèse axons exited the cérébral cortex and followed a tortuous course within the subcortical white matter. They then entered the putamen through its dorsal or latéral aspects. Once into the putamen, the major axonal branch broke out into 2-5 smaller collaterals that arborized scarcely but widely in the dorsolateral sector of the portion of the putamen that lies posterior to the anterior commissure, which will be referred to hère as the post-commissural putamen (Fig. 5.2 A). Thèse small intraputamenal axonal branches were endowed with rather equally spaced varicosities of very small size, and their distal ends often displayed pedunculated varicosities (Fig. 5.4 C). In some cases the axon divided just before it entered the putamen (Fig. 5.2 B). Furthermore, although the bulk of the axonal arborization of the first type Ml neurons was restricted to the post-commissural

188 putamen, terminal arborization extending within the portion of the putamen located in front of the anterior commissure (pre-commissural putamen) was also observed (Fig. 5.2 C). The three Ml neurons illustrated in figure 2 yielded respectively 98, 60 and 122 varicosities in the putamen. Thèse numbers are likely to be an underestimate of the real values because, occasionally, some of the most distal axonal segments displayed a rather faint labeling, which hindered the visualization of axonal varicosities at thèse levels.

Some neurons of the first type (3 out of 21) had an axon that projected also to the claustrum. In thèse cases, the main axonal branch coursed through the latéral aspect of the putamen and emitted a thin collatéral that pierced the putamen laterally and arborized sparsely in the dorsolateral sector of the claustrum.

The second type of Ml corticostnatal projection neurons targeted indirectly the striatum. Ten axons of this neuronal subtype were reconstructed and one of them could be directly connected to its parent cell body, as illustrated in figure 5.3. The cell body of this second type neuron was larger and more deeply located in layer V than the two cell bodies to which axons of the first type could be connected. Its axon descended through the subcortical white matter and remained unbranched until it reached the corona radiata. There, it emitted a thin collatéral (Fig. 5.3 C) that entered the medial aspect of the putamen and remained unbranched until it reached the dorsolateral sector of the structure. There, it formed several very thin and long collaterals displaying elongated varicosities of the boutons-en-passant type (Figs. 5.3 B, 5.4 C). The axon illustrated in figure 5.3 yielded only 51 varicosities in the putamen (Fig. 5.3 B), many of which were in close contact with cell bodies of striatal neurons. The major axonal branch remained in the internai capsule, became faintly labeled and was lost as it reached the cérébral peduncle, below the substantia nigra. Fibers passing trough the substantia nigra as they descended toward the brainstem were also noted, but no collatéral or varicosity was observed in the substantia nigra itself. None of the direct and indirect corticostriatal axons traced in the présent study emitted collatéral to the thalamus or the subthalamic nucleus.

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5.5.3

Contralateral corticostriatal projections

Some corticofugal axons crossing the midline and innervating the contralateral putamen were also observed in the présent study. Six contralateral corticostriatal axons were reconstructed in material frora the monkey that was injected on one side of the brain only. Two examples of the trajectory of thèse axons are provided in figure 5.4. In the first example, the axon followed a very long but rather straightforward trajectory through the corpus callosum before reaching the contralateral putamen. Along its way to the putamen, the axon traversed the caudate nucleus, where it emitted 15 axonal varicosities of the en passant type (arrows in figure 5.4 A), but the bulk of varicosities (256) was located within the dorsolateral sector of the contralateral post-commissural putamen. The number of axonal varicosities yielded at the putamen level by this contralaterally projecting axon was significantly higher than that of ipsilaterally projecting corticostriatal axons. In the second example, the axon also crossed the midline through the corpus callosum and emitted a collatéral as it exited this commissural fiber System (Fig. 5.4 B). The collatéral ascended toward the contralateral Ml and was lost just before it reached the cérébral cortex because of its faint labeling. The main axonal branch coursed around the caudate nucleus and arborized within the dorsolateral sector of the contralateral post-commissural putamen. The other contralaterally projecting axons also decussated via the corpus callosum and arborized in the dorsolateral sector of the contralateral putamen, but thèse axons did not émit collaterals to the contralateral cérébral cortex.

The 6 contralateral axons reconstructed in the présent study could not be linked to their ipsilateral counterparts because, for unknown reasons, thèse axons became progressively faintly labeled as they penetrated more and more deeply within the corpus callosum. This decrease in staining intensity reached its peak at the midline level, where a direct continuity between the ipsilateral and contralateral portions of the decussating axons could no longer be established with certainty. Hence, although both ipsilateral and contralateral portions of decussating axons were intensely stained, the callosal segment of the axons was very weakly labeled. It is for this reason, and not because decussating axons were too numerous, that we could not connect the ipsilateral and contralateral portions of the axons with a

190 suffïcient degree of confidence. Since the decussating corticostriatal axons could not be linked to their ipsilateral counterparts, it was not possible to détermine if thèse axons project only to the contralateral putamen or to the putamen on both sides of the brain. It was also impossible to know with certainty whether the contralateral axons that we charted belonged to the direct or the indirect type, because their cortical neurons of origin, which could hâve had a branch that descend to the brainstem, could not be identified.

5.5.4

Corticosubthalamic and corticorubral projections

Among the corticofugal axons that hâve been reconstructed in the présent study, 5 were found to be in direct relationship with the subthalamic nucleus and/or the red nucleus. One of thèse axons gave rise to a collatéral that arborized sparsely within the subthalamic nucleus before invading the cérébral peduncle. Another axon coursed through the subthalamic nucleus and ran dorsally within the lenticular fasciculus (H2) to finally reach the parvicellular part of the red nucleus where they arborized discretely. This axon yielded some varicosities of the en passant type within the subthalamic nucleus before arborizing in the red nucleus. Two other axons passed through the subthalamic nucleus without yielding any terminais there, and arborized sparsely within the parvicellular division of the red nucleus. Finally, one axon reached the red nucleus without coursing through the subthalamic nucleus. After having provided some terminais in the red nucleus, the major axonal branch of this axon continued its downward course and invaded the cérébral peduncle. Because of their faint labeling, thèse axons could not be followed further down than the midbrain level. They did not appear to project to the subthalamic nucleus nor to the red nucleus on the other side of the brain, and none of them were found to project to the striatum.

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5.6

DISCUSSION

The présent study has provided the first detailed description of the trajectory of individually labeled corticostriatal neurons in monkeys. Our fmdings call for a revision of the current view of the morphological organization of the corticostriatal projection System in primates. The functional significance of our fmdings will now be discussed in the light of relevant data gathered principally in primates.

5.6.1

The corticostriatal projection: direct, indirect, or both ?

The corticostriatal projection System in primates is currently viewed as a distinct set of neurons whose axons are dedicated solely to the striatum. This concept emerged from autoradiographic tracing studies (Kiinzle, 1975) and was further supported by rétrograde cell labeling experiments, which led to the conclusion that the corticostriatal projections arises directly from relatively small pyramidal cells restricted to upper half of layer V and not from collaterals of larger and more deeply located pyramidal cells, the axon of which descends within the cérébral peduncle (Jones et al., 1977). The fïndings of the présent single-axon tracing study challenge this current view of the organization of the corticostriatal projection System in primates. Our investigation reveals that neurons in the primary motor cortex of cynomolgus monkeys project to the striatum both directly and indirectly. The direct projection is made up of very thin axons solely devoted to the striatum, whereas the indirect projection consists of fine collaterals emerging from thick, long-range axons that project downward to the brainstem. Thèse results indicate that the primary motor cortex in primates has a dual access to the striatum; on one hand, it can influence directly the striatum through a specifically dedicated channel (the direct projection) and, on the other hand, it can send to the striatum a copy of the motor signal that is being conveyed to the brainstem and/or spinal cord (the indirect projection).

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The possibility that some of the corticostriatal axons described hère as belonging to the direct type might in fact originate from collaterals that émerge very close from the cell body of origin can not entirely be ruled out. However, the existence of a direct corticostriatal projection system in primates is ascertained by the fact that at least two axons that project directly and solely to the striatum were connected to their parent cell body. Thèse two axons remained uniformly thin and unbranched along their entire sinuous course to the striatum, as it was the case for the other direct axons that were not linked to their parent cell body. Moreover, there is ample support in the current literature for the existence of a direct corticostriatal projection in primates (see références in the Introduction), but this is not the case for the indirect corticostriatal projection, whose description represents one of the original contributions of the présent study. Axons that project indirectly to the striatum can easily be distinguished from the directly projecting ones. They consist of thick axons that descend through the subcortical white matter and remain unbranched until they reach the corona radiata. It is only at this low level that they émit a collatéral that runs toward the striatum and this pattern was the same for ail axons of the indirect type that were reconstructed in the présent study.

Our morphological data are at odds with the results of electrophysiological experiments that failed to detect Ml neurons responding to antidromic stimulation of both the cérébral peduncle and the striatum in primates (Bauswein et al., 1989; Turner and DeLong, 2000). Such a discrepancy could reflect limitations inhérent to the electrophysiological approach. For example, the failure to identify double antidromically invaded Ml neurons could simply be due to a misplacement of the two électrodes that hâve to be in perfect register to stimulate the striatal and peduncular segments of the same axon. The différence in conduction velocity that is likely to exist between the thick, long-range, peduncular axons and the thin, striatal collaterals also might hâve complicated the interprétations of antidromic invasion studies. The long-range axons that branch to the striatum could also hâve been missed simply because they are much less numerous than the direct corticostriatal projection neurons. Indeed, the indirect neurons (N = 10) were half less numerous than the direct ones (N = 21) in the sample analyzed in the présent study. However, this sample is too small to yield a valid conclusion as to the exact proportion of

193

the two types of corticostriatal projection neurons in primates. Further studies are needed to résolve this issue. The antidromic invasion studies cited above hâve revealed that, by comparison to longrange corticofugal neurons, the direct corticostriatal projection neurons display a minimal spontaneous activity and possess a slow conducting axon, indicating that the putamen receives a cortical message that is significantly différent from that sent down to the pyramidal tract (Bauswein et al., 1989; Turner and DeLong, 2000). The direct corticostriatal projection neurons were also found to be involved in aspects of motor behavior that are différent from those devoted to long-range corticofugal neurons. Thèse electrophysiological findings coupled to the présent morphological results suggest that the primary motor cortex in primates uses two distinct projection Systems (direct and indirect) and two différent neural coding Systems to convey to the striatum complementary information regarding the initiation and exécution of motor acts. Studies in rodents hâve raised the possibility that the two main types of corticostriatal projections preferentially target différent types of striatal projection neurons (Lei et al., 2004). Whether the direct and indirect cortical inputs to striatum described hère for primates differ in their targeting of striatal projection neurons, as distinguished by neurochemistry or connectivity, is unknown.

5.6.2

The corticostriatal projection: cellular origin and termination pattern

The laminar distribution of the cell bodies of origin of the two types of corticostriatal projection neurons encountered in this study is diffïcult to ascertain because many reconstructed axons could not be directly linked to their parent cell body. However, a close examination of neurons whose axon could be connected to their parent cell body suggests that pyramidal cells of the direct type are slightly smaller and more dorsally located in layer V than those of the indirect type, in agreement with previous findings obtained in primates with rétrograde cell labeling techniques (Jones et al., 1977; Arikuni and Kubota, 1986). Thèse morphological findings coupled to data revealing the unique physiological properties

194 of corticostriatal neurons further support the notion that the direct and indirect corticostriatal projection Systems described hère are morphologically and physiologically distinct from one another. However, given the small sample of axons analysed hère, more experiments are obviously needed to more firmly establish the laminar distribution and the size of the cell bodies that give rise to the direct and indirect corticostriatal projections in monkeys. Because of the very weak staining of their callosal segment, we could not link with certainty the ipsilateral and the contralateral portions of the decussating corticostriatal axons. It is therefore impossible to know if thèse axons arborize bilaterally in the striatum or émit collaterals to other subcortical structures. The same technical limitation has hampered the tracing of labeled axons below the midbrain level. Further experiments with longer survival periods should help providing a more detailed view of the distal segments of such long axons. However, because the axonal tracer is présent in a limited quantity and is rapidly transported to the terminal portion of the axonal arbor, long survival periods might lead to a faint labeling of the proximal segments of axons, so that the thin collaterals that innervate the striatum could be missed in such a condition (Lacroix et al., 2004).

Direct and indirect corticostriatal projections terminate chiefly in the dorsolateral sector of the post-commissural putamen, which is the main portion of the so-called sensorimotor striatal territory, the name of which dérives from the fact that this striatal région is selectively innervated by somatosensory and primary motor cortices (Parent, 1990). Corticostriatal axons that target the contralateral striatum also arborize in the sensorimotor striatal territory. Thèse findings concur with the results of earlier neuroanatomical studies, which hâve shown that the corticostriatal projection from primary motor and sensorimotor cortices in monkeys projects exclusively to the putamen where a somatotopic représentation of the leg, arm and face occurs in the form of obliquely arranged strips (Flaherty and Graybiel, 1993). The corticostriatal terminais are heterogeneously distributed within thèse putamenal bands, being particularly abundant in small areas of the striatal matrix (matrisomes) but absent from striosomes (Flaherty and Graybiel, 1993). A précise topographie distribution of the cortical motor input in the sensorimotor striatal territory is

195 important because this territory harbors neurons directly involved in the exécution of movements (Parent, 1990; Flaherty and Graybiel, 1993). The direct and indirect corticostriatal axons displayed terminal varicosities that had a similarly small diameter, but terminais of the indirect axons were often more elongated than those of the direct axons. Overall, the terminal pattern of both the direct and indirect corticostriatal projection was similar. After entering the striatum, the major axonal branch usually broke out into 2-5 thin collaterals that arborized scarcely but widely in the sensonmotor striatal territory. Thèse long and straight axonal branches were endowed with rather equally spaced varicosities of very small size, and their distal ends often displayed pedunculated varicosities. This mode of termination resemble very much the so-called "cruciform axodendritic" pattern described by Ramôn y Cajal at the beginning of the 19th century following Golgi studies of the corticostriatal projection in rodents (Ramôn y Cajal, 1909, 1911). The cruciform pattern refers to cortical fibers taking a relatively straight course through striatal tissue crossing over dendrites and making synapses with them en passant. This pattern of arborization suggests that projection arising from very small cortical areas may extend through large région of the striatum, implying that any given striatal cell receive convergent inputs from multiple corticostriatal axons (Parent and Hazrati, 1995a).

5.6.3

A comparison between corticostriatal and thalamostriatal projections

Both thalamostriatal and corticostriatal axons target médium spiny projection neurons located in the extrastriosomal matrix compartment of the striatum (Flaherty and Graybiel, 1993). However, most synaptic contacts of thalamic origin occur on dendritic shafts while those from the cortex are found mainly on the head of dendritic spines of médium spiny neurons (Frotscher et al, 1981; Somogyi et al., 1981; Sadikot et al, 1992b; Smith et al., 1994). This ultrastructural arrangement suggests that thalamostriatal synapses can modulate, in a highly précise mariner, the activity of médium spiny striatal neurons that receive cortical input. The number of varicosities observed in a target site can give an idea

196 of the relative strength of an axonal projection. The ipsilaterally organized corticostriatal axons reconstructed in the présent study yielded relatively few varicosities at the putamen level. In accordance with previous data obtained in the rat (Zheng and Wilson, 2002), the contralaterally projecting axons in monkeys displayed a larger number of axonal varicosities than the ipsilaterally projecting axons. Neurons of the centre-median-parafascicular thalamic complex also project to the dorsolateral part of the post-commissural putamen, but the number of terminais yielded by single thalamostriatal axons (1,621 - 3,139) is markedly greater that that of single corticostriatal axons arborizing within the same sensorimotor striatal territory, as revealed by single-axon tracing study in monkeys (Parent and Parent, 2005). Thèse values underline the strong and prominent aspects of the thalamostriatal projection, which is often neglected in the current scheme of basai ganglia organization. The thalamostriatal projection appears also more focused than the corticostriatal projection in primates. Thalamostriatal axons form dense terminal clusters concentrated in a relatively small portion of the striatum, indicating that a relatively small number of médium spiny striatal neurons are contacted by numerous terminais of thalamic origin. In contrast, the scarce and widely distributed corticostriatal innervation suggests that each Ml neurons provide only a few axonal varicosities to médium spiny striatal neurons scattered over a vast région of the putamen.

5.6.4

Corticosubthalamic and corticoclaustral projections

The subthalamic nucleus occupies a crucial anatomical and functional position in basai ganglia circuitry (Parent and Hazrati, 1995b). Our previous single-axon tracing studies in primates hâve revealed that neurons that composed this subcortical structure are endowed with a highly collateralized and widely distributed axon, which allows them to exert a direct excitatory influence upon the neurons of the two major output structures of the basai ganglia, namely the internai segment of the globus pallidus and the pars reticulata of the substantia nigra (Sato et al., 2000b). Besides the central place it occupies in basai circuitry, the subthalamic nucleus is also regarded as a further input station of the basai ganglia that acts in parallel with the striatum (Mink and Thach, 1993; Kita, 1994; Mink, 1996; Levy et

197

al, 1997; Nambu et al., 2000). In monkeys, the primary motor cortex is known to project to the dorsolateral sector of the subthalamic nucleus, where it displays a somatotopic organization, with a clear représentation of the leg, arm and face (Kiïnzle, 1978; Hartmannvon Monakow et al., 1981). In the présent study, labeled axons that target the subthalamic nucleus did not émit collaterals to the striatum. This finding suggests that the striatum and the subthalamic nucleus in primates are targeted by two distinct sets of Ml cortical neurons. Hence, the subthalamic nucleus can bypass the striatum and convey directly the powerful excitatory effect of the motor cortex to the output structures of the basai ganglia (see Nambu et al., 2000).

Corticofugal axons coursing within the subthalamic area often gave terminal varicosities of the en passant type within the subthalamic nucleus while heading to the red nucleus, which appears to be their main terminal site. However, in one case, the subthalamic innervation was found to dérive from a collatéral emitted by long-range corticofugal axon that invaded the cérébral peduncle. Evidence that cortical input to the subthalamic nucleus dérives from long-range axons has also been obtained in rats (Donoghue and Kitai, 1981) and cats (Iwahori, 1978; Giuffrida et al., 1985). Single-axon tracing studies specifically devoted to the corticosubthalamic projections are obviously needed to better understand the organisation of this neuronal System, which plays a crucial rôle in the organization of the basai ganglia in both health and disease.

Three of the 21 axons of the direct corticostriatal type were found to émit a thin collatéral that arborized sparsely in the claustrum. The latter structure is reciprocally linked with virtually ail areas of the cérébral cortex (Riche and Lanoir, 1978; Pearson et al., 1982; Sherk, 1986; Minciacchi et al., 1991) and comprises discrète somesthetic, visual, auditory and limbic zones (Oison and Graybiel, 1980; LeVay and Sherk, 1981). The claustrum is thus a crucial relay in a complex polymodal corticosubcortical circuitry conveying and integrating neuronal information of limbic and somatosensory types. Projections from various areas of the precentral cortex to both striatum and claustrum hâve been documented in various species, including primates (Kemp and Powell, 1970; Kûnzle and Akert, 1977; Jurgens, 1984; Selemon and Goldman-Rakic, 1988; Stanton et al, 1988; Lévesque and

198

Parent, 1998). However, the existence in primates of single Ml neurons with an axon that branches to both striatum and claustrum is hère demonstrated for the first time. This finding suggests that a copy of the same neural information is sent to spécifie subsets of claustral and striatal neurons through the direct corticostriatal projection system. The functional signifïcance of such a morphological orgamzation is unknown, but it suggests that the striatum and the claustrum might hâve more in common than previously thought.

5.6.5

Concluding remarks

The présent study has provided the first detailed description of the organization of the corticostriatal system originating in the primary motor cortex of primates at the single axon level. One of the major findings of this investigation is the démonstration of an indirect corticostriatal projection consisting of collaterals emitted by long-range corticofugal axons. In addition, single Ml neurons with a thin axon that projects directly to primate striatum hâve also been traced in détail for the first time. The terminal arborizations of thèse indirect and direct corticostriatal axons occupy the same dorsolateral sector of the putamen, which corresponds to the sensorimotor striatal territory. Thèse two types of corticostriatal neurons appear to possess distinct physiological properties and we hypothesize that they are involved in the control of différent aspects of motor behavior.

Acknowledgments : The authors express their sincère gratitude to Doris Côté, Cyntia Tremblay, Martin Lévesque, and Catherine Couture for skilful technical assistance. Martin Parent was supported by a Studentship from the Natural Sciences and Engineering Research Council (NSERC) of Canada.

Figure 5.1 A, Location of injection sites (white circles) in the primary motor cortex (Ml) of the cynomolgus monkey. The injection loci are plotted onto this single dorsal view of the macaque brain to give an idea of the portion of Ml that has been injected in the présent study. B, High magnification view of a BDA injection site in Ml centered upon the ventral two thirds of layer V. The injection site comprises a dense core of BDA precipitate surrounded by distinctly labeled neurons. C, Photomicrograph showing typical pyramidal, Golgi-like, BDA-labeled neurons in primate Ml. D, Photomicrograph depicting a typical injection site in Ml, as seen on a frontal section.

199

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Figure 5.2 A, B, Composite two-dimension reconstructions from sériai frontal sections of two single BDA-labeled Ml neurons projecting directly to the dorsolateral sector of the putamen. The cell bodies lie in the upper part of layer V of Ml (arrows). Thèse composite reconstructions were obtained by superposing ail sériai sections that contained labeled profiles onto a single two-dimension frame. This procédure inevitably leads to some image distortion due to the tortuous three-dimension course of the axon and also because structures in which the axon courses and arborizes are not necessarily at the same plane than the one chosen for the illustration. Hence, the limits of the various brain nuclei, which were purposefully delineated by dashed lines, should be taken as mère indications of the location of thèse structures. C, Sagittal reconstruction of another axon that projects directly to the dorsolateral sector of the post-commissural putamen. Its axonal arborization occupies a vast striatal territory. The number of axonal varicosities observed at putamen level is indicated in parenthèses in A, B and C.

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A

B

202

Figure 5.3 A, Frontal plane reconstruction of the axonal arbonzation of a Ml neuron that projects to the striatum, via a thin collatéral emitted in the corona radiata by a thick long-range axon that courses toward the brainstem. The parent cell body lies in the lower part of layer V (arrow). B, Higher power view of the axon terminal arborization, which is restricted to the dorsolateral sector of the post-commissural putamen. The number of varicosities emitted in the putamen by this single neuron is indicated in parenthesis. C, Photomicrograph showing the thin striatal projecting collatéral emitted by the thick long-range axon that courses toward the brainstem. The arrow points to the branching point.

203

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Figure 5.4 A, Composite two-dimension reconstruction of an axonal branch that crosses the midline by passing through the corpus callosum. This branch emits axonal varicosities as it passed through the caudate nucleus (arrows). It splits into two main collaterals as it enters the putamen. The axonal arbonzation is mainly restricted to the dorsolateral part of the postcommissural putamen and the number of varicosities is indicated in parenthesis. B, Caméra lucida drawing of another axonal branch that crosses the midline. This particular axon, which was traced in the unilaterally injected monkey, crosses within the corpus callosum and splits into two collaterals, one that ascends toward the contralateral Ml and the other that invades the contralateral putamen. C, Photomicrograph showing the terminal arbonzation of a labeled axon in the dorsolateral part of the putamen. The distal segment of this axonal branch displays rather equally spaced varicosities of small size, many of which are pedunculated (arrows). The inset offers a higher power view of some pedunculated varicosities (arrowheads). The varicosities pointed by the arrowheads lie on a particularly long peduncle.

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206

CHAPITRE 6

SINGLE-AXON TRACING AND THREE-DIMENSIONAL RECONSTRUCTION OF CENTRE MÉDIAN-PARAFASCICULAR THALAMIC NEURONS IN PRIMATES

CHAPITRE 6

SINGLE-AXON TRACING AND THREE-DIMENSIONAL RECONSTRUCTION OF CENTRE MÉDIAN-PARAFASCICULAR THALAMIC NEURONS IN PRIMATES

Martin Parent and André Parent

Laboratoire de Neurobiologie Centre de recherche Université Laval Robert-Giffard 2601, de la Canardière, Beauport, Québec Canada G1J2G3

The Journal of Comparative Neurology (2005) 481:127-144.

6.1

RESUME

Les projections efférentes du complexe thalamique centre médian (CM)/parafasciculaire (Pf) ont été investiguées chez le singe écureuil par injection microiontophorétique de biotine dextran aminé. Un total de 29 axones reliés à leur corps cellulaire d'origine ont été entièrement reconstruits à l'aide de sections sériées et d'une caméra lucida. Nos données démontrent que, chez le primate, trois types de neurones de projection composent les noyaux CM et Pf: (1) des neurones qui projettent densément mais à des régions circonscrites du striatum; (2) des neurones qui s'arborisent de manière diffuse dans le cortex cérébral; et (3) des neurones qui innervent à la fois le striatum et le cortex cérébral. L'innervation striatale provenant du CM se présente sous la forme de bouquets denses de varicosités axonales. Ces dernières sont souvent pédonculées et forment ensemble des bandes obliques dans la partie dorsolatérale du putamen (territoire sensorimoteur du striatum). Le même type d'arborisation striatale est présentée par les neurones du Pf mais cette fois, dans la tête du noyau caudé (territoire associatif du striatum). Les neurones du CM qui ciblent le cortex cérébral s'arborisent principalement dans les aires corticales motrices et prémotrices alors que les neurones du Pf projettent essentiellement aux aires corticales préfrontales. Dans les deux cas, l'innervation corticale est plus dense dans les couches V et VI que dans la couche I. L'analyse tridimensionnelle des neurones reconstruits révèle que leur arborisation dendritique et axonale s'étend principalement dans le plan sagittal. Dans l'ensemble, nos résultats démontrent que, contrairement au rongeur où pratiquement tous les neurones du Pf projettent à la fois au cortex cérébral et au striatum, le complexe thalamique CM/Pf du primate est composé de trois types différents de neurones de projection, ce qui indique un haut niveau d'organisation de ce complexe intralaminaire.

6.2

ABSTRACT

The axonal projections from the centre médian (CM)/parafascicular (Pf) thalamic complex in squirrel monkeys were studied following microiontophoretic injections of biotinylated dextran aminé under electrophysiological guidance. A total of 29 axons connected to their parent cell body were entirely reconstructed from sériai sections with a caméra lucida. Our investigation shows that the CM and Pf nuclei in primates comprise three types of projection neurons: (1) neurons that innervate densely and focally the striatum; (2) neurons that arborize diffusely in the cérébral cortex; and (3) neurons that innervate both striatum and cérébral cortex. Striatal innervation of CM origin consists of dense clusters of axon terminais exhibiting pedunculated varicosities and forming oblique bands in the dorsolateral sector of putamen (sensorimotor striatal temtory). The sarne type of striatal innervation occurs in the head of caudate nucleus (associative striatal temtory) in cases of Pf labeled neurons. The CM neurons that target cérébral cortex arborize principally in motor and premotor areas, whereas Pf neurons innervate chiefly prefrontal areas. Cortical innervation from both nuclei is much more profuse in layers V and VI than in layer I. Our 3D reconstruction studies show that dendritic and axonal arborizations of CM neurons extend essentially along the sagittal plane. Thèse results revealed that, in contrast to rodents where virtually ail Pf neurons project to both striatum and cortex, the primate CM/Pf complex comprises several types of highly patterned projection neurons. As such, this complex might be considered as an intégral part of the widely distributed basai ganglia neuronal System.

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6.3

INTRODUCTION

The centre médian (CM) and parafascicular (Pf) nuclei hâve traditionally been regarded as a single complex that represents the essential part of the so-called caudal intralaminar nuclei of the thalamus (Jones, 1985). Although a Pf nucleus has been recognized in ail mammals, a well-differentiated CM nucleus is believed to hâve appeared later in évolution and to hâve enlarged in parallel with the development of the putamen (Mehler, 1966; Mehler, 1981). Hodologically, the CM/Pf complex is closely related to the basai ganglia, as exemplified by its massive projection to the striatum (Cowan and Powell, 1956; Parent and De Bellefeuille, 1983; Smith and Parent, 1986; Nakano et al., 1990). Indeed, our previous experiments involving bulk injections of anterograde tracers in the CM/Pf complex of primates hâve shown that this structure projects to virtually ail sectors of the striatum, including the caudate nucleus, putamen and ventral striatum (Sadikot et al., 1992a; Sadikot et al., 1992b). The close relationship between the CM/Pf complex and the basai ganglia is further supported by the démonstration that CM neurons degenerate in Parkinson's disease, a pathology characterized by a massive loss of the dopaminergic innervation of the striatum (Henderson et al., 2000).

As part of the intralaminar nuclei, the CM/Pf complex is also known to project diffusely to wide areas of the cérébral cortex (Jones and Leavitt, 1974) and this thalamocortical projection is believed to play a crucial rôle in various state-dependent functions, such as arousal and attention mechanisms (Jones, 1998; Steriade, 2000; Jones, 2001). The CM/Pf complex is thus strategically positioned to modulate gênerai brain activities as well as motor behavior by acting at the cortical and the basai ganglia level. Despite its critical rôle in the modulation of cortical and basai ganglia activities, relatively little is known about the microcircuitry underlying such a dual function of the CM/Pf complex. For instance, it is not yet clear if the action of CM/Pf neurons at cortical and basai ganglia levels is mediated by single neurons that project to both targets or by two distinct

212 sets of neurons, one projecting to the striatum and the other to the cérébral cortex. In the rat, single-axon tracing studies hâve indicated that virtually ail Pf neurons project to both striatum and cérébral cortex (Deschênes et al., 1996). In the cat, double rétrograde celllabeling experiments hâve led to the conclusion that the Pf nucleus comprises neurons that project to both sites, whereas the CM nucleus harbors neurons that innervate either the striatum or the cérébral cortex (Royce, 1983; Macchi et al., 1984). In the squirrel monkey, similar double rétrograde cell-labeling studies hâve indicated that neurons in the CM nucleus that project to the cérébral cortex are distinct from those that target the striatum (Sadikot et al., 1992a).

This discrepancy might reflect genuine species différences in the organization of the CM/Pf efferent projection System, but it might also be due to inhérent limitations in the methodological approaches. For example, one of the major drawbacks of the double rétrograde cell-labeling technique is the false single-labeled neurons that might be encountered if the two injections sites are not in perfect register, that is, if the two tracers do not reach in equal amount the two terminal fïelds of the same axon. This limitation is particularly acute when the two terminal fïelds, whose exact topographie location can hardly be known in advance, are of unequal size, which is likely to be the case for striatal and cortical terminal fïelds of CM/Pf axons.

In the présent study, the important issue of knowing how the caudal intralaminar nuclei affect the cérébral cortex and basai ganglia was addressed by using a much more straightforward procédure that allows microiontophoretic injections of anterograde tracers in very small subsets of electrophysiologically identifîed neurons in the CM/Pf complex of squirrel monkeys. Singly labeled thalamofugal axons, as well as the somatodendritic domains of thèse Golgi-like labeled neurons, were entirely reconstructed from sériai sections and a three-dimensional rendering of the entire network was obtained with the help of a computerized image-analysis System. This approach has allowed us to provide a detailed picture of the organization of the thalamofugal projection System that anses from the CM/Pf complex in primates.

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6.4 6.4.1

MATERIALS AND METHODS Préparation ofthe animais

A total of 12 adult squirrel monkeys (Saimiri sciureus) of both sexes, with a body weight that ranged from 0.8-1.5 kg, were used in the présent study. The animais were raised in a markedly enriched environment for approximately 3 years before being used for expérimentation. They lived in a colony of 25-30 individuals housed in a single large animal facility room in which they could play with various toys and used a complex system of ropes and shelves to move around and rest. Animal work was performed in accordance with the Canadian Guide for the Care and Use of Laboratory animais and ail surgical and animal care procédures were approved by the Institutional Animal Care Committee of Laval University. The animais were first anesthetized with ketamine (75 mg/kg, i.m.) plus xylazine (5 mg/kg, i.m.) and their head placed in a specifically designed stereotaxic apparatus. After trépanation, a radiopaque solution (Omnipac or Iohexol, 0.8 ml of a 65% solution, Nicomed Imaging, Brandford, Ontario, Canada) was injected through a microsyringe into the right latéral ventricle. A few minutes after the injection, latéral and frontal X-ray pictures ofthe ventricular System were taken to precisely localize the baseline formed by the anterior and posterior commissures in each animal (Percheron, 1975). The animais were kept in isolation under close surveillance by the animal keepers until they fully recover from anesthesia.

6.4.2

Injection procédures

One to three weeks after ventriculography, the animais were anesthetized as above and placed in the same stereotaxic apparatus. They were then maintained under propofol (10 mg/ml, i.v.) anesthesia while microiontophoretic injections of biotin dextran aminé (BDA, Molecular Probes, Eugène, OR) were being made bilaterally in différent parts ofthe CM/Pf complex. In ail cases only two injections were made in each CM/Pf complex. We used the stereotaxic coordinates ofthe atlas of Emmers and Akert (1963) corrected according to the

214 data collected from ventriculography. Microiontophoretic labeling was carried out with glass micropipettes (tip diameter 2-3 jxm) filled with a solution of potassium acétate (0.5M) plus 2% BDA. Thèse électrodes had impédance ranging between 10-15 MQ and were used to monitor the extracellular activity of the neuronal populations encountered during the pénétration of the micropipette. The central latéral (CL) thalamic nucleus was easily recognizable by the characteristic spontaneous firing pattern of its neurons under propofol anesthesia, which includes regular discharges at 3 Hz (Fig. 6.1 A, B). The CM neurons discharged irregularly (Fig. 6.1 A, C) and were thus easily recognizable. The Pf neurons also discharged irregularly but their firing pattern was not formally recorded and analyzed in the présent study. Once in the chosen target, the micropipette was connected to a high compliance iontophoresis device (NeuroData) and the tracer was injected by passing positive current puises of 300-400 nA (ls on/ ls off) for 25 min. After surgery, the monkeys were isolated and kept under close surveillance by the animal keepers until they fully recover from anesthesia. They remained isolated and fed ad libitum until sacrifice.

6.4.3

Tracer visualization and cytochrome oxidase staining

After a survival period of 8-10 days, the animais were deeply anesthetized with a mixture of ketamine (150 mg/kg, i.m.) and xylazine (10 mg/kg, i.m.) and perfused transcardially with 1 liter of saline solution (0.9%) followed by 2 liters of a fixative solution containing 4% paraformaldehyde in phosphate buffer (PB, 0.1M, pH 7.4) and 1 liter of 10% sucrose solution in PB. The brains were dissected out and placed in a cryoprotective solution composed of 1/3 paraformaldehyde (4% solution in PB) and 2/3 sucrose (30% solution in PB) for 24h at 4°C. The brains were then eut along the sagittal (10 monkeys) or frontal (2 monkeys) plane at 70 /xm using a freezing microtome. The sections were collected serially in phosphate buffer saline (PBS, 0.1M, pH 7.4) and processed for the visualization of BDA according to the avidin-biotin-peroxidase method (ABC, Vector Labs, Burlingame, CA) with 3,3'diaminobenzidine (DAB) as the chromogen (Sato et al., 2000a; Sato et al., 2000b).

215 To help identifying nuclei and structures that harbored labeled neurons and axons, most sections were counterstained for cytochrome oxidase, according to the histochemical protocol of Wong-Riley (1979). The counterstaining was performed before BDA révélation, and nickel-cobalt-intensified DAB (dark blue reaction) and unintensified DAB (diffuse brown precipitate) were used to reveal BDA and cytochrome oxidase, respectively.

6.4.4

Material analysis

Ail sections were mounted on gelatin-coated slides, dehydrated in graded alcohols, cleared in toluène, and coverslipped with Permount. They were examined under a Nikon light microscope equipped with a caméra lucida. Détails of single BDA-labeled neurons were drawn at 200X and 400X magnifications. The terminal fields and cell body of the labeled neurons were mapped at lower magnifications to détermine their topographie location according to the atlas of Emmers and Akert (1963). The photomicrographs were digitally captured (Leica DC 300 caméra, Wetzlar, Germany) and processed with the Adobe Photoshop software (version 7.0, Adobe, San José, CA). Axons were entirely reconstructed along either the sagittal or frontal planes with the help of a caméra lucida. Composite twodimension figures showing the axonal trajectones were made by superposition of a séries of sections, each containing at least one axonal segment. Although very useful to illustrate the entire course of the axons, this procédure inevitably leads to a slight distortion of the image because of the tortuous course of the axons. Furthermore, the structures in which the axon course and/or arborizes, which were delineated hère by dashed lines, do not always lie within the plane that has been chosen for the illustration. For this reason, a computerized image-analysis System (Neurolucida, MicroBrightField, Inc., Colchester, VT) was used to reconstruct in three dimensions the entire axonal trajectory and terminal arborization of 3 CM neurons that correspond to the 3 différent neuronal types encountered within this thalamic nucleus. The same System was employed to gather quantitative estimâtes of the number of terminal boutons and total axonal length, as well as to reconstruct the somatodendritic domain of thèse CM neurons. It was also used to measure the perikaryal area of CM and Pf neurons. The neurons selected for measurement had to hâve a minimum

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of two primary dendrites visible in the same plane section. We used an unpaired Mest to verify if the différence in the mean penkaryal area of CM and Pf neurons was statistically significant.

6.5 6.5.1

RESULTS Nuclei délimitation and gênerai labeling features

There has always been some controversy regarding the précise délimitation between the CM nucleus laterally and the Pf nucleus medially. In addition to obvious topographie considérations, one of the major criteria to trace the limit between thèse two nuclei is that Pf neurons are slightly larger and more compactly arranged than CM neurons (Olszewski, 1952; Jones, 1985). In the présent study, CM neurons were found to hâve a mean perikayal area of 186.5 ± 64.9fim2 (n=20), while the value for Pf neurons was 232.3 ± 91.1/xm2 (n=9). However, the différence between CM and Pf neurons in regard to their mean perikaryal area was not statistically significant; the two-tailed P value was 0.1751 when using a 95% confidence interval. Some investigators hâve proposed a tripartite division of the caudal intralaminar nuclei in monkeys, based on cytological and hodological criteria (Percheron et al., 1991; Percheron, 2004). However, since counterstaining with cytochrome oxidase did not allow us to delineate 3 components in the CM/Pf complex of the squirrel monkey, we adhered to the commonly accepted dual subdivision of this complex, as depicted in the atlas of Emmers and Akert (1963). We also purposefully avoid injecting into the so-called subparafascicular nucleus. First described by Aronson and Papez (Aronson and Papez, 1934) in macaques, this nucleus lies between the CM/Pf complex and the midbrain tegmentum and has occasionally been considered as an extended part of the CM/Pf complex by virtue of its projections to the ventral part of the striatum, as well as to various extrastriatal structures (Sadikot et al., 1992a).

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In the présent study, the BDA deliveries produced small injection sites involving no more than 5 to 10 CM/Pf neurons per site. Most injection loci had a dense core composed of BDA precipitate surrounded by several neurons labeled in a Golgi-like manner (Fig. 6.2 A). Thèse labeled CM/Pf neurons had a triangular or ovoid cell body from which emerged several long, poorly branched and sparsely spined dendrites (Fig. 6.2 B, C). Threedimensional reconstruction revealed that the somatodendritic domain of CM neurons had its greatest extent along the sagittal plane. Dendrites of CM/Pf neurons were rather long and could extend as much as 1 mm in the anterior-posterior or dorsal-ventral axis. This feature could explain why labeled neurons were occasionally found at some distance from the core of the injection sites. The dendrites were endowed with characteristic axon like processes or appendages that were more abundant on CM then on Pf neurons (Fig. 6.2 B). Also, Pf neurons had fewer and shorter primary dendrites than CM neurons. Intensely labeled axons could be seen to émerge from either the core of the injection sites or from individually labeled neurons located peripherally (Fig. 6.2 A). In the latter case, the axons exited from either the cell body or a primary dendrite. Thèse axons could easily be followed individually throughout each section and were thus entirely reconstructed along the sagittal or frontal planes with a caméra lucida. Because labeled axons traveled in parallel and did not intermingle, there was very little possibility of confusing one axon with another.

Only neurons whose axon could be connected to their cell body of origin were retained in the présent study. A total of 29 such axons were traced entirely from their parent cell body to their terminal fields. Twenty of thèse labeled neurons had their cell body in the CM nucleus and 9 in the Pf nucleus. Three CM neurons that correspond to the 3 différent neuronal types encountered within this thalamic center (see below) were further analyzed with the Neurolucida image-analysis system to generate a three-dimension rendering of thèse neurons, to analyze their axonal and dendritic branching patterns and to count the number of axonal varicosities observed in each target site. Those numbers are important to assess the relative strength of each collatéral projection.

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6.5.2 6.5.2.1

Axonal trajectory Centre médian thalamic nucleus

Based on their target sites and axonal branching patterns, we identifiée 3 différent neuronal types in the primate CM nucleus. The first type had an axon that projected profusely to the dorsolateral part of the post-commissural putamen (n=6). The second type of neurons innervated layers I, V and VI of motor and premotor cortex (n=7). Finally, the third type of neurons projected to the putamen and the motor or premotor cortex (n=7). The axonal arborization of 6 neurons that belong to the first neuronal type were entirely reconstructed from sériai sections. Thèse axons exited the CM nucleus through its anterior part and coursed through the ventral posteromedial nucleus and/or the ventral posteroinferior nucleus and then crossed the reticular thalamic nucleus and bifurcated within the internai capsule. Half of the axons (3/6) gave rise to a collatéral that arborized poorly in the reticular thalamic nucleus. The major axonal segment ran through the globus pallidus to enter ventrally the dorsolateral sector of the post-commissural putamen or coursed within the internai capsule and along the dorsal surface of the putamen to penetrate this structure dorsally and aborize in the same putamenal sector. The neuron illustrated in figure 6.3 had a main axonal branch that divided within the internai capsule, with one collatéral entering the putamen through its dorsal aspect and another penetrating the same structure more ventrally. Once into the putamen, each major axonal branch broke out into 3-5 smaller collaterals. Thèse thin branches divided into numerous shorter collaterals that bear typical pedunculated or club-like varicosities organized as dense clusters of axon terminais. Thèse clusters formed oblique bands restricted to spécifie domains of the dorsolateral région of the post-commissural putamen (Fig. 6.3 B, C). The axon of the neuron illustrated in figure 6.4 provided a total of 3,139 varicosities in the putamen. Interestingly, this single axon emitted one terminal cluster in the medial portion of the putamen but several others in the dorsolateral sector of the structure (Fig. 6.4 B, C). The striatal arborization was 4 times more extended along the sagittal plane than the frontal plane. The parent cell body was located in the central portion of CM nucleus (Fig. 6.4 D, Fig. 6.10 B). It displayed long dendrites that were 3 times more extended along the sagittal

219 plane than the frontal plane (Fig. 6.4 E-G) and were endowed with characteristic appendages (Fig. 6.4 E). This neuron had a total axonal length of 7.59 cm. Two CM neurons that projected to the putamen also provided a collatéral that swept laterally to reach the claustrum where it emitted a few varicosities.

The second type of CM neurons projected sparsely to the motor and premotor cortical areas, but did not arborize in the striatum. In this case, the axon ascended via the white matter of the corona radiata to the motor cortex, where it arborized moderately in layers V and VI, and much less profusely in layer I. The axons of 7 neurons of this type were entirely reconstructed and 2 examples are illustrated hère (Figs. 6.5, 6.6). In figure 6.5, the cell body indicated by an arrow was located in the latéral part of the CM nucleus (Figs. 6.5 B, 6.10 C). It had long dendrites that chiefly extended along the anteroposterior axis (Fig. 6.5 D) and were endowed with characteristic appendages (Fig. 6.5 A). This cell body emitted an axon that exited the CM nucleus dorsally en route to the internai capsule. This axon remained unbranched until it reached the corona radiata. There, it divided itself into 2 collaterals that branched in the white matter before arborizing in layer VI and V of the motor cortex. At this level, we observed multiple isolated axonal varicosities reminiscent of terminal boutons, as well as axonal varicosities aligned in the form of boutons en passant. This particular neuron provided 130 varicosities in layer V, 168 varicosities in layer VI, and 58 varicosities of the en passant type in the cérébral white matter. This particular CM neuron had a total axonal length of 6.14 cm.

The neuron illustrated on a sagittal plane in figure 6.6 had a cell body located in the latéral and anterior sector of the CM nucleus (Fig. 6.10 A). The axon emerged from a primary dendrite (Fig. 6.6 B) and exited through the anterior part of the CM nucleus. It emitted one collatéral that arborized poorly in the anterior portion of the thalamic reticular nucleus (22 boutons). The major axonal segment remained undivided until it reached the cortical white matter. There, 3 major branches entered the motor cortex to innervate rather massively layers V (445 boutons) and layer VI (361 boutons), but much less prominently layer I (10 boutons). Many of thèse axonal varicosities were often in close contact with cortical cell bodies (Fig. 6.6 D).

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The third type of CM neurons projected to both putamen and cérébral cortex. Seven neurons displaying this pattern of axonal coUateralization were entirely reconstructed and only 3 of them arborized rather poorly in the reticular thalamic nucleus. Two neurons of the third type are illustrated on sagittal plane in figure 6.7 and another that has been entirely reconstructed in three dimensions with a computerized image analysis system is depicted in figure 6.8. The cell body of the neuron shown in figure 6.7A was located in the central core of the CM nucleus (Fig. 6.10 C); it gave rise to an axon that supplied the reticular thalamic nucleus with 2 small collaterals. The main axonal branch reached the internai capsule, passed through the external segment of the globus pallidus (GPe) and divided into 2 collaterals within the external medullary lamina. One of thèse collaterals entered the putamen where it branched and formed 4 major clusters of closely packed terminal varicosities. Thèse clusters were restricted to spécifie domains in the dorsolateral sector of the post-commissural putamen. The other collatéral ascended up to the cortical white matter, swept caudally and, after a rather long and linear course beneath the cortical gray matter, invaded the motor cortex. At this level, the terminal axonal arborization was diffuse but still restricted to layers I, V and VI. The axon of the neuron shown in figure 6.7B emitted 2 collaterals that departed at right angle in the internai capsule. Both of thèse collaterals arborized within dorsolateral putamen, where they formed a total of 1,621 axonal varicosities. The remaining axonal segment reached the corona radiata, but became too faintly labeled and was lost as it entered the cortical white matter. The neuron illustrated in three dimensions in figure 6.8 had a major axonal segment that penetrated the putamen through its dorsal aspect. Its axonal arborization field occupied the dorsolateral sector of the post-commissural putamen, was more extended along the sagittal than frontal plane (Fig.6.8 A, B), and comprised a total of 2,276 axonal varicosities. By comparison, the same axonal branch emitted only 19 axonal varicosities in the reticular thalamic nucleus. The axon of this neuron could be followed up to the cérébral white matter but became too faintly labeled to be followed in détail in the cérébral cortex itself. In contrast to the fainting that occurred in the most terminal portions of some of the axons that reached the cérébral cortex, the proximal segments of the thalamocortical axons were always intensely labeled and easy to visualize. Furthermore, the main axon of neurons that projected to both

221 striatum and cortex always branched within white matter areas, principally the internai capsule, so that it is highly unlikely that axonal branches heading toward the striatum or the cérébral cortex might hâve been missed or that the number of axons projecting to both striatum and cortex might hâve been underestimated. Although often thinner than the thalamostriatal projection, the thalamocortical branch was always intensely labeled, at least until it reaches the white mater of the cérébral cortex and, in most cases, the labeling remained sufficiently intense to delineate their entire cortical arborization.

6.5.2.2

Parafascicular thalamic nucleus

The Pf nucleus was found to harbored 3 types of projection neurons similar to those disclosed in the CM nucleus. A total of 9 Pf neurons were entirely reconstructed from sériai sections. Five of them projected only to the striatum, one neuron projected only to cérébral cortex and 3 neurons innervated both the cérébral cortex and the striatum. In contrast to CM neurons that targeted the dorsolateral sector of the post-commissural putamen, Pf neurons projected principally to the head of the caudate nucleus.

Figure 6.9A shows a Pf neuron that projected to striatum but not to cortex, whereas figure 6.9B depicts a Pf neuron that targeted both striatum and cortex. The neuron illustrated in figure 6.9A had a cell body located in the ventral part of the Pf nucleus (Fig. 6.10 B). Its dendritic arborization was less extended than that of CM neurons (Fig. 6.9 A inset) and it remained confined within the boundaries of the Pf nucleus (Fig. 6.9 A). The cell body emitted an axon that exited the Pf dorsally, invaded the internai capsule, and coursed along the dorsal surface of the GPe. It then penetrated the putamen, giving rise to a short collatéral that swept ventrally and arborized poorly in that structure and also in the GPe. The main axonal segment continued its course dorsally and finally reached the caudate nucleus. There it broke out into several short terminal collaterals that formed several dense clusters of pedunculated axonal varicosities scattered throughout the head of the caudate nucleus.

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The main axonal branch of the Pf neuron that projected to both cérébral cortex and striatum reached the frontal cortex after having followed a very long but rather straightforward course through the internai capsule, the head of the caudate nucleus, and the white matter underlying the frontal cortex (Fig. 6.9 B). Along its way, the axon sent a short collatéral that arborized poorly in the GPe. Collaterals were also emitted as the axon passed through the caudate nucleus. However, thèse striatal collaterals where poorly arborized by comparison to those that emerged from Pf neurons that target solely the striatum (Fig. 6.9 A). Only 2 neurons whose parent cell body was located in the Pf nucleus were found to innervate the GPe. In one of thèse 2 cases (a neuron that projected only to the frontal cortex), the axon emitted one thin collatéral that arborized poorly within the somatodendritic domain of its parent neuron. This local collatéral exhibited some varicosities of various sizes reminiscent of boutons-en-passant or boutons terminaux.

6.5.3

Topographical distribution

The plotting of each type of CM and Pf neurons (Fig. 6.10) has revealed that the 3 types of CM/Pf projection neurons identified in the présent study did not occupy spécifie terri tories in the CM/Pf complex, which thus appears as a heterogeneous entity in terms of the distribution of its projection neurons. Although there was a tendency for neurons projecting only to the cérébral cortex to be located more laterally in the CM nucleus, the location of the cell bodies of neurons in the CM/Pf complex could not be considered a good predictor of the type of axonal arborization displayed by thèse neurons nor of their target sites.

6.6

DISCUSSION

The présent study has provided the first detailed account of the axonal branching patterns of CM/Pf neurons in primates. Our investigation has demonstrated that, in contrast to rodents where virtually ail Pf neurons project to both striatum and cortex, the CM and Pf

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nuclei in primates comprise 3 types of projection neurons. Neurons of the first type émit an axon that arbonzes densely and focally in the striatum, which is the major input structure of the basai ganglia. Neurons of the second type innervate only the motor and premotor cérébral cortex in a diffuse manner. Finally, neurons of the third type display an axon that branches to both striatum and cérébral cortex. The functional significance of thèse findings will now be discussed in the light of the data from the current literature.

6.6.1

Somatodendritic morphology

The CM and Pf neurons were found to differ slightly in respect to the morphological features of their somatodendritic domain. While CM neurons had a medium-sized cell body that displayed long and widely branched dendrites endowed with characteristic appendages, Pf neurons had a slightly larger cell body with fewer primary dendrites, many of which devoid of appendages. The exact rôle of thèse peculiar dendritic appendages is unknown, but they could markedly enhance the integrative capacity of intralaminar thalamic neurons. The fact that the morphological traits of injected CM/Pf neurons correspond to those reported in Golgi studies of primate CM/Pf complex (Hazlett et al., 1976; Fénelon et al., 1994) validâtes the présent injection approach as a means to investigate the somatodendritic domain of single projection neurons.

6.6.2

Thalamostriatal projections

The pioneering rétrograde cell degeneration studies of Cowan and Powell (1956) hâve clearly established that the entire intralaminar nuclei project densely to the striatum in monkeys. We now know that the CM/Pf complex constitutes the major source of thalamostriatal inputs, although rostral intralaminar, as well as non-intralaminar nuclei, also contribute significantly to striatal innervation (Parent et al., 1983; Smith and Parent, 1986; Nakano et al, 1990; Sadikot et al., 1990; Sadikot et al, 1992a; McFarland and Haber, 2001). Furthermore, both light and électron microscopic studies hâve revealed that the

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thalamostriatal projections arising from CM/Pf neurons target chiefly the matrix compartment of the striatum in squirrel monkey (Sadikot et al., 1992b). In the présent study, single CM neurons were found to arborize exclusively in the dorsolateral sector of the post-commissural putamen, which receives its major cortical input from the primary sensory and motor cortical areas and thus corresponds to the so-called sensorimotor striatal territory (Parent and Hazrati, 1995a). In contrast, single Pf neurons target principally the head of the caudate nucleus, which receives its main cortical input from associative cortical areas and is thus considered as part of what has been termed the associative striatal territory (Parent and Hazrati, 1995a). Hence, the CM/Pf complex can influence the entire dorsal striatum through a séries of topographically organized complementary projections. Ail axons that targeted the striatum were found to arborize either in the sensorimotor territory (CM neurons) or associative territory (Pf neurons), a finding that confions the overall topographical organization of the thalamostriatal projections in primates determined earlier by bulk injections of anterograde tracers (Nakano et al., 1990; Sadikot et al., 1992a). However, it must be noted that a least one Pf neuron projecting massively to the caudate nucleus was found to provide collaterals to the dorsolateral sector of the post-commissural putamen.

The présent démonstration of CM and Pf neurons that branch to the striatum and the cérébral cortex is at odds with the results of a previous rétrograde cell-labeling study where no double-labeled neurons were detected in the CM/Pf complex of squirrel monkeys following injections of one fluorescent tracer in the cérébral cortex and of another in the striatum (Sadikot et al., 1992a). This discrepancy is likely due to limitations inhérent to the double rétrograde cell-labeling technique, such as the présence of false single-labeled neurons when the 2 injections sites are not in perfect register (see Introduction). A more récent single-axon tracing study in rats has revealed that virtually ail neurons in the Pf nucleus project to both striatum and cérébral cortex (Deschênes et al., 1996). This is obviously not the case in squirrel monkeys where more than half of the CM/Pf neurons (19/29) were found to project solely to the striatum (11/29) or the cérébral cortex (8/29). Such a différence between the two studies is unlikely to be due to technical variations since the same methodological approach was used in both cases. It most likely reflects an important species différence between

225 rodents and primates in regard to the organization of the thalamostriatal projection. The single-axon tracing data currently available suggest that the rat Pf nucleus is composed of a unique population of multipotential neurons that branch to several sites, whereas the primate CM/Pf complex harbors specialized neuronal subpopulations with distinct, highly spécifie axonal branching patterns. The large number of varicosities displayed by single axons at striatal level reveals that the thalamofugal projection is largely oriented toward the striatum. The fact that thèse axonal varicosities form dense clusters concentrated in a relatively small portion of the striatum suggests that axon terminais of thalamic origin contact a relatively small number of médium spiny neurons. This pattern of organization implies that single CM/Pf neurons might act as a strong driving force upon small subsets of striatal projection neurons.

Our three-dimensional analyses hâve demonstrated, for the first time, that the dendritic and axonal arborizations of CM/Pf neurons are more extended along the sagittal plane than along the frontal or horizontal plane. A similar pattern of organization had been detected earlier following single-cell labeling studies of external (GPe) and internai (GPi) segments of the globus pallidus in primates (Sato et al., 2000a; Parent et al., 2001). The axonal arborization of GPe and GPi neurons was 5 times more extended along the sagittal plane than along the frontal and horizontal planes. The functional significance of such a band-like arrangement is presently unknown, but the wide sagittal extent of the dendritic and axonal arborizations of thèse basai ganglia components offers a strikingly extended réceptive field capable of integrating neural information from various levels of the neuraxis.

Both thalamostriatal and corticostriatal axons are known to target médium spiny projection neurons located in the matrix compartment of the striatum. However, most synaptic contacts of thalamic origin occur on the dendritic shaft while those from the cortex are largely found on the head of dendritic spines of médium spiny neurons (Frotscher et al., 1981; Somogyi et al., 1981; Sadikot et al, 1992b; Smith et al., 1994). This ultrastructural arrangement suggests that thalamostriatal synapses can modulate, in a highly précise manner, the activity of médium spiny neurons that receive cortical input.

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6.6.3

Thalamocortical projections

The intralaminar and midline thalamic nuclei, often referred to as "non-specific thalamic nuclei," hâve long been though to play an important rôle in awareness by exerting a global influence on cortical functioning (Jones, 1998; Steriade, 2000; Jones, 2001). Hère, we described, for the first time, the pattern of terminal axonal arborization of single thalamocortical neurons in primates. This pattern of arborization was remarkably différent from that of thalamostriatal projections. Instead of forming dense clusters, as in the striatum, CM/Pf axons that reached the cérébral cortex arborize diffusely and in a widespread manner. This mode of termination allows single CM/Pf neurons to provide a few axon terminais to a large number of cortical neurons and thus to modulate the activity of large cortical neuronal assemblies. However, the fact that the axonal varicosities are often in close contact with the cell body of cortical neurons and the possibility that several CM/Pf neurons target cortical neurons belonging to the same neuronal assembly point to the functional importance of the thalamocortical projection.

Autoradiographic tract-tracing studies in cat hâve reported that the cortical projection arising from the CM/Pf complex arborizes primarily in cortical layers I and III, with projection to layer I being more extensive than that to layer III (Royce and Mourey, 1985). In the présent study, axons from CM/Pf neurons were found to arborize much more profusely within layers V and VI than in layer I of the cérébral cortex. However, it must be realized that collaterals in layer I are the most distal segments of the thalamocortical axons so that a significant number of such collaterals might hâve been undetected because thèse extrême portions of labeled axons often stain faintly. Our results are nevertheless in agreement with an earlier study that involved bulk Phaseolus vw/garâ-leucoagglutinin (PHA-L) injections in the rat Pf nucleus, which showed that Pf axons arborized principally in layers V and VI and much less so in layer I of the rodent frontal cortex (Berendse and Groenewegen, 1991). Thèse findings indicate that CM/Pf neurons exert an important excitatory influence principally on long-range cortical projection neurons, including those

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that give rise to the massive corticothalamic projection System, which plays a crucial rôle in various state-dependent events (Jones, 1998; Steriade, 2000; Jones, 2001).

6.6.4

Other CM/Pf projections

Beside striatal and cortical projections, some CM/Pf axons were found to émit a few poorly branched collaterals to the GPe, the claustrum and principally the thalamic reticular nucleus. Several anatomical studies (Jones, 1975a; Cornwall and Phillipson, 1988; Kolmac and Mitrofanis, 1997; Tsumori et al., 2000) hâve revealed that the reticular thalamic nucleus, whose neurons use the inhibitory transmitter GABA (Oertel and Mugnaini, 1984), is reciprocally linked to the CM/Pf complex, the neurons of which probably utilize the excitatory transmitter glutamate (Streit, 1980; Christie et al., 1987; Fuller et al., 1987). Hence, the collaterals that CM/Pf ascending axons émit in the reticular nucleus may be viewed as part of a intrathalamic feedback loop that modulâtes the excitatory influence exerted upon the striatum and/or the cérébral cortex by CM/Pf neurons.

A previous investigation has revealed that, besides the striatum and cérébral cortex, some anterogradely labeled fibers can be detected in the subthalamic nucleus, GPi, substantia nigra, ventral tegmental area, amygdala, hypothalamus and substantia inominata following bulk injections of PHA-L in the CM/Pf complex of squirrel monkeys (Sadikot et al., 1992a). The présence of anterogradely labeled fibers in such a wide variety of subcortical structures might reflect the involvement of fibers of passages or the inclusion in the injection sites of the subparafascicular nucleus, which is known to project to a large number of subcortical structures (Ottersen and Ben-Ari, 1979; Mehler, 1980; Saint-Cyr and Courville, 1981; Bentivoglio and Molinari, 1984; Royce and Mourey, 1985; Grove, 1988; Sadikot et al., 1992a). This nucleus has been carefully avoid in the présent single-cell labeling study and no axon collaterals hâve been seen to aborize to any significant degree in the above-mentioned subcortical structures. However, we cannot entirely rule out the existence of such widely projecting CM/Pf neurons because of neuron sampling limitation inhérent to the approach used in the présent study. In rodents, a double rétrograde

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fluorescent cell-labeling study has indicated that projections to the subthalamic nucleus and striatum arise from separate Pf neuronal populations (Féger et al., 1994). In contrast, Pf neurons providing collaterals to the subthalamic nucleus, globus pallidus and entopeduncular nucleus on their way to the striatum hâve been visualized in single-axon tracing studies (Deschênes et al, 1996). This discrepancy may reflect the limitations of the double rétrograde cell-labeling method (see above) compared to the single-axon tracing procédure.

6.6.5

Functional considérations

The thalamostriatal projection originating from CM/Pf neurons is part of a three-synaptic loop that involves: (1) the striatopallidal projection, which anses from neurons located in the sensorimotor striatal temtory and project to the core of the GPi; (2) the pallidothalamic projection, which originates from neurons in the core of the GPi and project to the CM/Pf thalamic complex; and (3) the thalamostriatal projection, which stems from CM neurons that project back to the sensorimotor striatal territory (Nauta and Mehler, 1966; Hazrati and Parent, 1992; Sadikot et al., 1992a; Parent et al., 2001). The fact that a significant number of CM neurons that innervate the sensorimotor striatal territory also project to the cérébral cortex implies that this loop is part of an open circuitry rather than a close one. Hence, CM neurons endowed with an axon that projects to both striatum and cortex can no longer be considered as simple feedback éléments; their branched axon allows them to influence neurons in the sensorimotor striatal territory directly, as well as indirectly via a relay in the motor cortex, which projects back to the sensorimotor striatal territory.

In face of neurons endowed with an axon that provides collaterals to différent targets, like those présent in primate CM/Pf complex, it becomes important to evaluate the relative "strength" of each projection of thèse single neurons. In the présent study, we noted that CM/Pf neurons that target only the striatum exhibited a much larger number of axonal varicosities in this structure than neurons that project to both striatum and cortex. Likewise, CM/Pf neurons that innervate only the cérébral cortex arborized much more profusely at

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this level than neurons that project to both cortex and striatum. It is as if each CM/Pf neuron could exhibit only a fixed number of axon terminais so that in cases where the amount of axonal varicosities is high in the striatum it will be low in the cortex and vice versa. Such a rule appears to apply also to the nigrostriatal neuronal System, where the degree of axonal branching in the striatum was found to be inversely proportional to that in extrastriatal structures (Gauthier et al., 1999; Prensa and Parent, 2001). In conclusion, the présent study has shown that the CM/Pf complex in primates is not a monolithic and homogeneous entity. This thalamic complex is composed of at least 3 types of neurons with différent axonal branching patterns that allow this brain center to influence its différent target sites in a highly spécifie fashion. Our previous single-axon tracing studies hâve shown that virtually ail components of primate basai ganglia are composed of différent types of neurons endowed with a highly collateralized axon (Parent et al., 2000; Parent and Parent, 2002). By virtue of its highly patterned efferent projection System, the CM/Pf complex should be considered as an intégral part of this widely distributed neuronal System.

Acknowledgment : The authors express their sincère gratitude to Doris Côté, Cyntia Tremblay, Martin Lévesque, Catherine Couture and Cynthia Moore for skilful technical assistance. Martin Parent was holding a Studentship from the Natural Sciences and Engineering Research Council of Canada (NSERC).

Figure 6.1 A, Patterns of neural activity that characterizes the central latéral nucleus (CL, upper row) and the centre médian nucleus (CM, lower row) of the squirrel monkey (Saimiri sciureus), as recorded under propofol anesthesia during a single brain pénétration with a glass injection micropipette. B, C, Interspike interval histograms of the neural activity recorded in the CL nucleus (B) and in the CM nucleus (C). The firing pattern in CL nucleus is much more rhythmic than the one in CM nucleus.

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Figure 6.2 A, Example of a BDA injection site in the caudal portion of the CM nucleus, as seen on a sagittal section. B, C, Photomicrographs showing Golgi-like BDA-labeled neurons in the primate CM nucleus. The CM neurons are elongated with smooth and poorly branched dendrites that display characteristic appendages.

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Figure 6.3 A, Composite two-dimension reconstruction from sériai sagittal sections of a single BDAlabeled CM neuron that projects densely to the latéral sector of the putamen (PUT), but not to the cortex. This particular neuron also provides a weak innervation of the reticular thalamic nucleus (R). The arrow indicates the location of the cell body in the CM nucleus. The somatodendritic domain of the neuron is illustrated at a higher magnification in the inset. The composite reconstruction shown hère was obtained by superposing ail sériai sections that contained labeled profiles onto a single two-dimension frame. This way of doing inevitably leads to some image distortion because of the tortuous three-dimension course of the axon and also because the structures in which the axon courses and arborizes are not necessarily at the same plane than the one chosen for the illustration. Hence, the limits of the various structures, which hâve been purposefully delineated by dashed lines, should be taken as mère indications of the location of thèse structures. Thèse outlines hâve been added simply to facilitate the reading of the illustration. This word of caution also applies to ail composite reconstructions shown in figures 6.4 to 6.9. B, Photomicrograph of the distal segment of the thalamostriatal axon that forms dense terminal fields arranged as oblique bands confined to the dorsolateral portion of the putamen, which represents the sensorimotor striatal territory. C, Higher magnification view of a cluster-like terminal field within the sensorimotor striatum. Some of the typical pedunculated axon terminais are visible at a higher magnification in the inset.

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Figure 6.4 A-C, Axonal arborization of a CM neuron that projects only to the striatum, as viewed on sagittal (A), frontal (B) and horizontal (C) planes. The number of axonal varicosities observed in the putamen (PUT) is indicated in parenthèses in A, arrows in A, B and C point to the cell body, and the total axonal length is indicated in C. D, The exact location of this labeled neuron is shown in a fontal plane drawing of the CM/Pf complex. E-G, The somatodendntic domain of the labeled neuron is depicted on sagittal (E), frontal (F) and horizontal (G) planes. The arrows point to the initial segment of the axon and the inset in E shows a photomicrograph of the cell body.

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Figure 6.5 A-C, Axonal and dendritic arborizations of a CM neuron that projects to layers I, V and VI of the motor cortex, but not to the striatum, as viewed on sagittal (A), frontal (B) and horizontal (C) planes. The number of axonal varicosities observed at the cortical level is indicated in parenthèses in A and the arrows in A, B and C point to the cell body. The location of this labeled neuron is shown in a frontal plane drawing of the CM/Pf complex that has been inserted in the central portion of figure B. D, Polar diagram of the dendritic arborization of the labeled neuron. It shows that dendrites of this neuron are maximally extended along the anterior-posterior plane and minimally arborized along the mediallateral plane.

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total axonal length: 6.14 cm lOOum

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Figure 6.6 A, Caméra lucida drawing of a neuron located in the anterior-lateral sector of the CM nucleus and whose axon projects to layers I, V and VI of the motor cortex and to the reticular thalamic nucleus (R), but not to the striatum. The axon is drawn on sagittal plane, the number of axonal varicosities observed at each target site is indicated in parenthesis and the arrow points to the cell body. B, Somatodendritic domain of the neuron illustrated in A. Arrows point to the initial segment of the axon. C, This photomicrograph that shows an axonal varicosity in close apposition to a cortical cell body located in layer V of the primary motor cortex.

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Figure 6.7 A, B, Caméra lucida reconstructions of two labeled CM neurons projecting to striatum and cortex, as seen on a sagittal plane. The axon of the neuron illustrated in A courses within the white matter (wm) and innervâtes rather diffusely layers I, V and VI of the motor cortex and more densely the dorsolateral sector of the putamen. The block of cérébral cortex illustrated hère has been drawn at a higher magnification than the subcortical structures to better visualize the terminal arborization. The neuron depicted in B has an axon that heads toward the motor cortex, but could not be traced entirely because the labeling of the axon within the cortical white matter became too faint. The number of axonal varicosities observed in the putamen is indicated in parenthèses. The arrows in A and B point to the cell bodies.

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Figure 6.9 A, B, Caméra lucida reconstructions on sagittal plane of two BDA-labeled Pf neurons, one projecting to the striatum only (A) and the other to both the striatum and the cérébral cortex (B). The axon of the neuron illustrated in A innervâtes profusely the caudate nucleus, but it also aborizes weakly in the external pallidum and putamen. The inset provides a higher magnification view of the somatodendritic domain of the neuron (arrows point to the initial segment of the axon). Neuron depicted in B has an axon that arborizes rather weakly in the caudate nucleus, but gives rise to a long branch that reaches the frontal cortex. The single arrows in A and B indicate the location of cell bodies.

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Figure 6.10 Transverse half-sections through the CM/Pf complex showing the localization of neurons, the axon of which was connected to parent cell body. The drawings represent equally spaced (500 /im apart) frontal sections displayed in a rostrocaudal order form A to D. The anterior-posterior (AP) stereotaxic coordinates of sections, according to the atlas of Emmers and Akert (1963), are indicated in the upper right portion of each figure. The Pf appears in gray, whereas the CM is in white. Blue circles represent neurons that innervate only the striatum, red triangles indicate neurons that project only to the cérébral cortex, whereas green squares depict neurons that project to both cortex and striatum. The numbers refer to figures where those neurons are illustrated.

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CHAPITRE 7 CONCLUSIONS GÉNÉRALES

CHAPITRE 7

L'ensemble des résultats expérimentaux présentés dans cette thèse démontre que la coUatéralisation axonale est une propriété intrinsèque des ganglions de la base ainsi que des noyaux qui y sont étroitement associés. La technique de marquage et de reconstruction neuronale unitaire utilisée depuis maintenant plus d'une dizaine d'années dans notre laboratoire nous amène à changer la vision que nous avions de l'organisation hodologique de ces structures sous-corticales impliquées dans le comportement psychomoteur. Cette nouvelle façon de concevoir ce système neuronal s'oppose à celle couramment retrouvée dans la littérature qui, de façon simpliste, expose les différentes composantes des ganglions de la base comme étant des éléments reliés en série sous la forme d'une boucle dans laquelle l'information neuronale est canalisée et ségréguée fonctionnellement.

7.1

Les axones pallidofuges et la microstimulation intracérébrale à haute fréquence

Nos résultats indiquent clairement que la vaste majorité des neurones du pallidum interne (GPi) ont un axone qui est hautement collatéralisé, un même neurone moteur du GPi étant en mesure d'envoyer une copie de l'information neuronale qu'il vient de traiter aux neurones prémoteurs situés dans thalamus et dans le tronc cérébral. Les neurones pallidaux limbiques, quant à eux projettent d'une manière beaucoup plus focalisée sur l'habénula latérale. Ces neurones, pouvant agir telle une interface entre les systèmes moteur et limbique, ainsi que l'existence de projections pallidofuges controlatérales pourraient expliquer l'apparition de déficits cognitifs suite aux chirurgies bilatérales impliquant le GPi.

Bien que la stimulation intracérébrale à haute fréquence du GPi soit efficace afin d'atténuer les symptômes moteurs qui caractérisent la maladie de Parkinson (Burchiel et al., 1999;

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Obeso et al., 2001; Rodriguez-Oroz et al, 2005), le STN est présentement considéré comme étant la meilleure cible (Limousin et al., 1998). Notre compréhension du mécanisme d'action de ce traitement demeure limitée. On suppose généralement que la stimulation à haute fréquence engendre une inactivation fonctionnelle des neurones localisés près de la microélectrode. Cette inactivation pourrait résulter soit d'une inhibition directe des neurones du STN, c'est-à-dire de la très forte dépolarisation de leurs membranes cellulaires, ou encore d'une stimulation plus ou moins locale des terminaisons synaptiques inhibitrices. On sait que l'atténuation maximale des symptômes moteurs de la maladie de Parkinson est atteinte lorsque la microélectrode est implantée dans la région antérodorsale du STN (Saint-Cyr et al., 2002; Patel et al., 2003; Yelnik et al., 2003; Zonenshayn et al, 2004; Nowinski et al., 2005). Tel que dévoilé par traçage unitaire, les axones pallidofuges émergent du GPi par un faisceau de fibres formant un continuum dont la limite ventroantérieure est l'anse lenticulaire et la limite dorsocaudale est le faisceau lenticulaire (champ H2 de Forel). Tous les axones moteurs du GPi cheminent ensuite à travers le champ H de Forel et émettent dans cette région subthalamique plusieurs courtes collatérales possédant de nombreuses varicosités axonales. Le nombre total de varicosités axonales compté dans le champ H de Forel est de 2 à 3 fois supérieur à celui retrouvé dans le noyau pédonculopontin (PPN) ce qui en fait une cible importante des axones pallidofuges. Les collatérales qui se dirigent vers le tiers ventral du thalamus empruntent le faisceau thalamique (champ Hl de Forel) pour se rediviser et s'arboriser sous la forme de bouquets denses de varicosités axonales au sein des noyaux ventral antérieur (VA) et ventral latéral (VL) du thalamus. La description précise du trajet emprunté par les axones moteurs du GPi suggère que la localisation de la microélectrode dans la partie antérodorsale du STN lors de microstimulations intracérébrales à haute fréquence permet au courant électrique de diffuser et d'influencer non seulement les neurones du STN mais aussi (1) les champs Hl et H2 de Forel composés en partie des axones pallidofuges GABAergiques hyperactifs dans la maladie de Parkinson et (2) le champ H de Forel lequel constitue une cible importante des axones pallidofuges chez le primate. En collaboration avec le Dr Cameron Mclntyre de la Cleveland Clinic Foundation en Ohio, nous avons réalisé un modèle informatique permettant d'étudier la propagation du signal électrique à la suite de stimulations intracérébrales à haute fréquence du STN chez le singe macaque rendu parkinsonien suite à

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l'injection del-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). Ce modèle est basé sur (1) un atlas anatomique tridimensionnel des ganglions de la base du macaque, (2) la modélisation de l'électrode de stimulation ainsi que de la propagation du courant à travers le médium tissulaire et (3) les propriétés biophysiques neuronales modelisées appliquées à des reconstructions anatomiques tridimensionnelles individuelles de neurones du GPi et du STN. Ce modèle a permis de générer des données qui indiquent que les paramètres de stimulation cliniquement efficaces permettent au courant d'activer non seulement les neurones du STN mais aussi les fibres pallidofuges du faisceau lenticulaire (Miocinovic et al., 2004) (Fig. 7.1). Ce modèle pourra servir de cadre permettant de combiner les analyses théoriques et expérimentales concernant l'effet de la stimulation intracérébrale à haute fréquence du STN à l'intérieur duquel la position de l'électrode ainsi que les paramètres de stimulation pourront être manipulés systématiquement afin de prédire (1) l'effet exercé sur les neurones du STN, (2) l'effet exercé sur les neurones du GPi et (3) les bienfaits cliniques de l'intervention.

Figure 7.1 A, B, Modèle informatisé de l'effet d'une stimulation intracérébrale de la partie antérodorsale du noyau subthalamique sur les axones pallidofuges et les neurones du noyau subthalamique. Ce modèle est basé sur des reconstructions tridimensionnelles de neurones du noyau subthalamique et du pallidum interne réalisées chez le macaque. Les données générées suggèrent que les paramètres cliniquement efficaces (fréquence de 136Hz, durée de 0.09ms, amplitude de 3V) permettent d'activer à la fois les axones pallidofuges du faisceau lenticulaire et les neurones du STN. Une stimulation bipolaire de 2V (C) et de 3V (D) active respectivement 3% et 28% des neurones du noyau subthalamique (illustrés en rouge). Une stimulation de 2V (E) et de 3V (F) active respectivement 28% et 52 % des axones pallidofuges (illustrés en rouge). Des études effectuées chez le singe macaque rendu parkinsonien suite à l'intoxication au MPTP démontrent que l'amélioration de la rigidité et la bradykinésie est obtenue suite à des stimulations bipolaires d'une amplitude de 3V, une amplitude de 2V n'ayant aucun effet bénéfique. (Miocinovic et al., 2004)

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7.2

L'innervation glutamatergique striatale

Les projections corticostriées et thalamostriées sont les deux principales afférences glutamatergiques striatales (Parent, 1996). L'étude par traçage unitaire des efférences du cortex moteur primaire a changé la vision que nous avions de la projection corticostriée chez le primate. En effet, contrairement à ce que l'on croyait, nous avons démontré que le cortex moteur primaire peut influencer de façon directe et indirecte le striatum. Cette affirmation est basée sur la découverte de deux populations distinctes de neurones corticostriés au sein du cortex moteur primaire soit : (1) des neurones possédant un axone fin qui innerve uniquement le striatum et (2) des neurones qui projettent indirectement au striatum, par le biais d'une collatérale émise par un axone de fort diamètre se dirigeant vers le tronc cérébral. Par ailleurs, nos recherches concernant les efférences du complexe thalamique centre médian/parafasciculaire (CM/Pf) ont permis de décrire trois types neuronaux principaux composant ce complexe, soit : (1) des neurones qui innervent uniquement le striatum, (2) des axones qui s'arborisent de façon diffuse dans le cortex cérébral, et finalement (3) des neurones qui projettent à la fois au cortex cérébral et au striatum par le biais d'un jeu complexe de collatérales axonales.

Les neurones épineux de taille moyenne situés dans la matrice striatale du putamen postcommissural (territoire sensorimoteur du striatum) sont ciblés à la fois par les axones provenant du noyau thalamique CM et par ceux du cortex moteur primaire (Sadikot et al., 1992a; Sadikot et al., 1992b; Flaherty et Graybiel, 1993). Il existe cependant une différence importante quant à l'organisation ultrastructurale de ces innervations glutamatergiques. Des études en microscopie électronique ont démontré que les contacts synaptiques originant du CM/Pf se retrouvent majoritairement sur les dendrites des neurones épineux de taille moyenne du striatum alors que les contacts synaptiques établis par les axones corticostriés abondent principalement sur la tête des épines dendritiques de ces mêmes neurones (Frotscher et al, 1981; Somogyi et al., 1981; Sadikot et al, 1992b; Smith et al., 1994). Les cinquième et sixième chapitres de cette thèse montrent clairement que l'organisation des champs terminaux de ces deux afférences glutamatergiques diffère au niveau neuronal

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unitaire. Tout d'abord, chacun des axones corticostriés reconstruits en provenance du cortex moteur primaire émet une quantité relativement faible de varicosités axonales (51256) au sein du striatum sensorimoteur alors que le nombre de terminaisons axonales émis dans ce même territoire striatal par un seul axone thalamostrié est de loin supérieur (1,621 3,139). Cette observation souligne l'importance fonctionnelle de l'entrée synaptique glutamatergique en provenance des neurones du CM/Pf. Malgré le fait que les neurones du CM/Pf exercent un effet de rétroaction positive important sur le striatum, la projection thalamostriee est souvent négligée dans le schème organisationnel actuel des ganglions de la base. Cette projection forme de denses bouquets de terminaisons axonales restreints à certaines portions du striatum sensorimoteur, ce qui signifie que de petits groupes composés d'un nombre relativement faible de neurones épineux de taille moyenne du striatum sont contactés par plusieurs terminaisons d'origine thalamique. À l'opposé, l'innervation en provenance du cortex moteur primaire est relativement diffuse et distribuée sur une vaste portion du territoire sensorimoteur du striatum. Ceci suggère que chaque neurone du cortex moteur primaire émet seulement quelques varicosités axonales qui influencent cependant plusieurs neurones striataux répartis à travers une vaste région du putamen dorsolatéral. Ce faible nombre de varicosités axonales n'enlève rien à l'importance fonctionnelle globale de la projection corticostriée qui bénéficie de la contribution d'un très grand nombre de neurones corticofuges.

7.3

Orientation spatiale des arborisations axonales et dendritiques

L'analyse tridimensionnelle des reconstructions axonales des neurones du GPi, du segment externe du pallidum et du CM/Pf a permis de déceler une organisation des arborisations dendritiques et des arborisations axonales étendues dans l'axe antéropostérieur (Sato et al., 2000a; Parent et al., 2001; Parent et Parent, 2005). On ne connaît pas la signification fonctionnelle exacte de cet arrangement en bandes allongées. Cependant, on peut émettre l'hypothèse que l'organisation étendue dans l'axe antéropostérieur de l'arborisation dendritique peut contribuer à accroître le champ récepteur d'un neurone, lui permettant

258

ainsi d'intégrer une multitude d'informations neuronales provenant de systèmes neuronaux complexes qui recouvrent plusieurs étages du névraxe.

7.4

Différences interspécifiques

L'utilisation de la technique de reconstruction neuronale unitaire a permis de visualiser directement l'organisation neuromorphologique entière de neurones composant certaines structures intimement liées anatomiquement aux ganglions de la base chez le primate. Plusieurs données accumulées à l'aide de cette technique sont présentées dans la thèse. Elles ne laissent aucun doute quant au fort degré de collatéralisation axonale qui caractérise les neurones appartenant à la plupart de ces structures. En comparant les résultats obtenus par traçage unitaire chez le primate à ceux obtenus par la même technique chez le rongeur, on constate qu'il existe des différences interspécifiques importantes en ce qui a trait à l'organisation des connexions neuroanatomiques des ganglions de la base. Par exemple, l'étude par traçage unitaire des projections efférentes du cortex moteur primaire chez le rat démontre que toutes les projections corticostriées ipsilatérales sont formées d'une collatérale émise par un axone projetant vers le tronc cérébral (Lévesque et al., 1996b; Lévesque et Parent, 1998). Chez le primate, nous avons clairement identifié deux types de projections corticostriées homolatérales distinctes au sein du cortex moteur, soit (1) une projection composée d'axones dédiés uniquement au striatum et (2) une autre composée d'axones projetant de façon indirecte au striatum via une collatérale émise par un axone de plus fort diamètre se dirigeant vers le tronc cérébral. La projection thalamostriée provenant du noyau Pf chez le rat semble être composée uniquement d'axones innervant à la fois le striatum et le cortex cérébral (Deschênes et al., 1996). Chez le primate, nous avons décrit trois types de neurones de projection au sein du CM/Pf : (1) le premier type est composé de neurones qui ciblent uniquement le striatum, (2) le deuxième type comprend des neurones qui projettent seulement au cortex cérébral et (3) le troisième type est formé de neurones qui innervent à la fois le striatum et le cortex cérébral. De la même façon, le striatum, les segments interne et externe du pallidum et le STN chez le primate sont des structures

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composées de plusieurs types de neurones de projection (Sato et al., 2000a; Sato et al., 2000b; Parent et al., 2001; Lévesque et Parent, 2005). Contrairement aux rongeurs où l'on retrouve souvent qu'un seul type de neurone de projection, les données obtenues chez le primate démontrent que chacune des structures étudiées est composée de plusieurs types neuronaux dont certains possèdent un axone hautement collatéralisé. On ne connaît pas la signification fonctionnelle de cette différence interspécifique. Cependant, elle pourrait refléter l'acquisition chez le primate de systèmes neuronaux davantage spécialisés afin de permettre l'acquisition d'un répertoire moteur hautement sophistiqué et caractéristique du primate.

7.5

La ségrégation de l'information neuronale

Le schème actuel de l'organisation anatomo-fonctionnelle des ganglions de la base suppose que l'information neuronale demeure, jusqu'à un certain point, ségréguée dans la boucle cortico-ganglions de la base-thalamo-corticale. En effet, il existe vraisemblablement un certain niveau de ségrégation de l'information neuronale au sein de la circuiterie des ganglions de la base. Par exemple, les territoires fonctionnels de structures recevant une innervation corticale directe comme le striatum (Kiinzle, 1975; Selemon et Goldman-Rakic, 1985) et le STN (Monakow et al., 1978; Nambu et al., 1996) sont très bien établis. Cependant, il est incertain que cette ségrégation fonctionnelle de l'information neuronale soit maintenue à des niveaux situés plus en aval dans la boucle cortico-ganglions de la base-thalamo-corticale. Dans le troisième chapitre de la présente thèse, nous avons rapporté l'existence de neurones limbiques et moteurs au sein du GPi. Mentionnons que l'utilisation de ces termes n'a rien à voir avec les divisions fonctionnelles du pallidum proposées par Smith et collaborateurs (Shink et al., 1997; Sidibé et al., 1997; Baron et al., 2001). Cette division tripartite du GPi suppose que les territoires fonctionnels du striatum soient directement transposés au pallidum. Selon ces auteurs, le tiers dorsomédian, les deux-tiers ventrolatéraux et le pôle rostroventromédian représenteraient respectivement les territoires associatif, sensorimoteur et limbique du GPi (Shink et al., 1997; Sidibé et al., 1997; Baron

260

et al., 2001). Dans l'article présenté au troisième chapitre, le qualificatif moteur ou limbique a été attribué aux neurones du GPi suite à la reconstruction unitaire entière de leur axone ainsi qu'à l'identification de la nature de leurs cibles. Les appellations moteur et limbique ne sont donc pas attribuées selon la localisation de leur corps cellulaire d'origine dans le GPi. Les neurones pallidofuges moteurs, tel que définis dans notre étude, ont leur corps cellulaire localisé dans la portion centrale du GPi qui comprend, en partie, les territoires sensorimoteur et associatif du pallidum, tel que déterminés par les projections striatopallidales. Quant à eux, les corps cellulaires des neurones limbiques sont davantage concentrés dans le pôle rostral du GPi ainsi qu'en périphérie du noyau pour ce qui est de la partie caudale du GPi.

Les axones moteurs du GPi que nous avons reconstruits innervent un vaste territoire pallidal du tiers ventral du thalamus et ce, sans égard à la localisation de leur corps cellulaire d'origine dans le GPi. Ces données vont à rencontre d'une transposition directe des territoires fonctionnels du pallidum sur le thalamus (Hoover et Strick, 1993; Sidibé et al., 1997). De plus, les neurones moteurs du GPi innervent à la fois les noyaux CM et Pf. Telles que révélées par traçage unitaire, les efférences du complexe thalamique CM/Pf sont en retour ségréguées au niveau du striatum, le CM innervant le territoire sensorimoteur du striatum et le Pf, le territoire associatif.

Les études présentées dans cette thèse suggèrent qu'il existe un certain niveau de ségrégation de l'information neuronale et ce, principalement au niveau des voies d'entrée et de ré-entrée de l'information neuronale soit respectivement la projection corticostriée et thalamostriée. La projection pallidofuge, quant à elle, semble permettre une intégration importante entre les différentes modalités fonctionnelles. Cette affirmation est supportée par l'arborisation axonale hautement coUatéralisée des neurones du GPi mais aussi par la morphologie des arborisations dendritiques qui se présentent sous la forme de disques plats de très grande dimension orientés parallèlement les uns aux autres ainsi qu'au bord latéral de chaque segment pallidal (Percheron et al., 1984a; Percheron et al., 1984b; Yelnik et al., 1984; Parent et Hazrati, 1995a). L'orientation du domaine somatodendritique de ces neurones, perpendiculaire aux axones striatofuges, indique que les neurones du GPi sont en

261

mesure d'intégrer

de l'information

neuronale provenant de différents

territoires

fonctionnels du striatum.

7.6

Rôles fonctionnels de la coUateralisation axonale

Le niveau de coUateralisation axonale présent dans les ganglions de la base du primate tel que décrit dans cette thèse signifie qu'un même neurone est en mesure d'acheminer l'information neuronale qu'il a intégrée à plusieurs structures cibles. On ne connaît pas précisément le rôle fonctionnel de cette organisation neuroanatomique. Cependant, on peut présager qu'elle permet aux composantes des ganglions de la base d'interagir de manière très précise les unes avec les autres. De cette façon, les différentes structures associées aux ganglions de la base sont en mesure de réguler adéquatement le flot de l'information corticale à travers les structures sous corticales.

7.6.1

Le concept de divergence

Un important degré de coUateralisation axonale constitue un substratum morphologique permettant la divergence de l'information neuronale et la diversification de cette information au sein du système des ganglions de la base. Par exemple, chaque axone pallidofuge qui entre dans le tiers ventral du thalamus émet des collatérales caractérisées par plusieurs bouquets de varicosités axonales répartis sur un vaste territoire du tiers ventral du thalamus. Cette organisation permet à un seul axone pallidothalamique de transmettre une copie de l'information neuronale à plusieurs groupes composés d'une vingtaine de neurones thalamocorticaux (Ilinsky et Kultas-Ilinsky, 1987; Arecchi-Bouchhioua et al., 1996; Ilinsky et al., 1997) répartis sur l'ensemble du territoire thalamique pallidal. Cette information peut ensuite être traitée de façon différente par chacun de ces groupes de neurones thalamocorticaux avant d'être acheminée vers le cortex cérébral. En considérant ces observations, il semble que la coUateralisation axonale permette un traitement de l'information neuronale en «parallèle » comparativement à un traitement en « série » qui caractérise la plupart des systèmes somatosensoriels classiques (Deschênes et al., 2005). Ce

262 mode de traitement de l'information contribue non seulement à augmenter la diversité de l'information neuronale au sein des ganglions de la base, mais aussi à accroître le niveau d'intégration de cette information. De plus, un traitement de l'information neuronale en parallèle permet une certaine redondance de l'information transmise par un neurone. De cette façon, un neurone dont l'axone est hautement collatéralisé augmente ses propres chances de voir la commande efférente qu'il envoie exercer une réponse significative sur certaines de ses structures cibles.

7.6.2

La plasticité

On peut émettre l'hypothèse que la collatéralisation axonale contribue à la plasticité neuronale, une des caractéristiques fondamentales de l'organisation du système nerveux central. En effet, on peut facilement imaginer que l'effet exercé par un neurone projetant à plusieurs sites peut être modulé par le renforcement de synapses au sein de ces structures cibles. De cette façon, le système neuronal que constituent les ganglions de la base serait facilement en mesure de moduler la copie de l'information neuronale acheminée par un même neurone en modifiant l'efficacité de certaines connexions synaptiques déjà présentes au sein des structures cibles. Un système hautement collatéralisé comme celui des ganglions de la base constitue certainement un avantage lorsque survient une lésion cérébrale. On peut présager que la collatéralisation axonale ainsi que les projections controlatérales peuvent permettre de contrebalancer beaucoup plus facilement certains changements pathologiques conséquents à une lésion cérébrale.

7.7

La collatéralisation axonale et la vulnérabilité face aux processus neurotoxiques et neurodégénératifs

Les études effectuées par traçage unitaire chez le primate démontrent que pratiquement toutes les composantes des ganglions de la base ainsi que les structures associées (cortex cérébral, striatum, segments externe et interne du pallidum, STN, CM/Pf) sont composées

263 de plusieurs types de neurones de projection qui possèdent un axone long et hautement collatéralisé (Sato et al., 2000a; Sato et al., 2000b; Parent et al., 2001; Lévesque et Parent, 2005; Parent et Parent, 2005). Le meilleur exemple d'une telle organisation est celui des neurones moteurs du GPi qui s'arborisent profusément dans le thalamus et le tronc cérébral. Le corps cellulaire de ces neurones d'environ 30 /xm de diamètre est en mesure de supporter un axone d'une longueur totale pouvant atteindre près de 30 cm. Dans ce cas, le ratio entre le diamètre du corps cellulaire et la longueur axonale totale est d'environ 1 sur 10 000. À la lumière de ces données, nous émettons l'hypothèse que la production énergétique et métabolique nécessaire pour assurer l'intégrité d'axones aussi longs représente un désavantage puisqu'elle pourrait rendre plus vulnérables ces neurones dont l'axone est hautement collatéralisé face à certains processus neurodégénératifs ou neurotoxiques. D'ailleurs, les neurones du GPi sont les premiers à être affectés dans les cas d'encéphalopathie toxique (Laplane et al., 1984). Des études d'imagerie cérébrale et des analyses faites sur du tissu post-mortem humain ont révélé d'importantes lésions restreintes au pallidum chez des individus ayant tenté de se suicider par intoxication au disulfurame (Mesiwala et Loeser, 2001), au cyanure (Feldman et Feldman, 1990), au monoxyde de carbone (Tom et al., 1996; Ruszkiewicz et al., 1997) ainsi qu'aux barbituriques (Strassmann et al., 1969). Notre hypothèse est confortée par le lien étroit qui existe entre le degré de collatéralisation axonale d'un neurone et sa vulnérabilité face à certains processus neurodégénératifs. Chez des singes rendus parkinsoniens, on a rapporté que les neurones nigrostriés qui s'arborisent profusément dans le striatum sont davantage affectés par l'intoxication au MPTP que les neurones nigrostriés qui branchent plutôt dans les structures extrastriatales (Lavoie et al., 1992). D'autre part, les neurones striataux de projections sont spécifiquement vulnérables dans la maladie de Huntington alors que les neurones non épineux dont l'axone demeure au sein du striatum sont épargnés (Cicchetti et al., 2000).

7.8

Hypothèse d'un nombre maximal de varicosités axonales

L'article présenté dans le sixième chapitre de cette thèse rapporte que les axones des neurones du CM/Pf qui ciblent uniquement le striatum émettent au sein de cette structure

264 un plus grand nombre de varicosités axonales que ceux qui projettent à la fois au striatum et au cortex cérébral. De même, les neurones du CM/Pf qui innervent seulement le cortex cérébral s'arborisent davantage dans cette structure comparativement aux neurones qui projettent à la fois au cortex cérébral et au striatum. Rappelons que les neurones limbiques du GPi branchent peu avant d'atteindre l'habénula latérale. Ils émettent cependant une quantité impressionnante de varicosités axonales dans ce noyau. À l'inverse, les neurones moteurs du GPi dont l'axone branche davantage, émettent dans chacune des structures cibles, une quantité moindre de varicosités axonales. Ces observations nous laissent croire qu'un neurone donné ne peut émettre qu'une certaine quantité de varicosités axonales. Donc si la quantité de varicosités axonales observée dans une structure cible donnée est très élevée, elle sera faible dans les autres noyaux ciblés par le même neurone. Cette règle semble aussi s'appliquer à la projection nigrostriée composée de neurones dont le degré de branchement axonal dans le striatum est inversement proportionnel à celui retrouvé dans les structures extrastriatales (Gauthier et al., 1999; Prensa et Parent, 2001); il pourrait donc s'agir d'un principe organisationnel valable pour l'ensemble des composantes des ganglions de la base.

Le fait qu'un neurone donné émet un nombre maximal de varicosités axonales pourrait s'expliquer par certains mécanismes mis en place au cours du développement ayant pour effet de limiter le branchement axonal d'un neurone ainsi que le nombre de varicosités axonales. À titre d'exemple, pensons à une régulation très précise de l'expression de récepteurs pour certains signaux moléculaires responsables du branchement axonal. Ces récepteurs seraient donc distribués parmi les différentes collatérales axonales en croissance. Le branchement axonal ainsi que le nombre de varicosités observées dans chacune des structures cibles résulteraient donc d'une combinaison entre (1) des facteurs intrinsèques, telle que la régulation précise de l'expression de récepteurs, et (2) des facteurs extrinsèques, comme la présence dans les structures cibles de signaux moléculaires de différentes natures responsables du branchement axonal. En empêchant une croissance axonale excessive et l'apparition d'un trop grand nombre de varicosités axonales, le système limiterait ainsi la quantité d'énergie nécessaire au maintient de l'intégrité axonal, ce qui favoriserait la

265

résistance des neurones possédant un axone hautement collatéralisé face à certains, processus neurotoxiques et neurodégénératifs.

7.9

Conclusions

Les ganglions de la base sont un ensemble de structures sous-corticales qui jouent un rôle crucial dans le comportement psychomoteur, comme en font foi les troubles moteurs et cognitifs caractéristiques des maladies neurodégénératives associées aux ganglions de la base, telles que la maladie de Parkinson et la chorée de Huntington. Dans la présente thèse, nous avons décrit de la façon la plus détaillée possible la microcircuiterie liant certaines composantes des ganglions de la base. L'utilisation d'une technique de pointe permettant le marquage et la reconstruction unitaire complète de neurones a permis de déceler un niveau de collatéralisation axonale jusqu'alors insoupçonné au sein des ganglions de la base du primate. Cette organisation neuroanatomique permet une interaction extrêmement précise entre les différentes composantes des ganglions de la base et assure un contrôle approprié du flot de l'information neuronale qui circule le long de la boucle cortico-ganglions de la base-thalamo-corticale. À la lumière des données présentées dans cette thèse, les ganglions de la base nous apparaissent maintenant comme un vaste réseau neuronal s'étendant sur plusieurs étages du névraxe et au sein duquel chaque composante est en mesure d'interagir avec les autres de manière précise et appropriée. Ces données jettent un éclairage nouveau sur l'organisation anatomique et fonctionnelle des ganglions de la base des primates et nous permettent d'envisager avec plus de confiance et de certitudes le traitement des pathologies rattachées à cet ensemble de structures sous-corticales.

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ANNEXE 1 LISTE DES CONTRIBUTIONS Articles publiés dans des revues avec comité de pairs Parent M, Parent A. Single-axon tracing study of the corticostriatal projections arising from primary motor cortex in primates. J Comp Neurol 496:202-213. Parent M, Parent A. 2005. Single-axon tracing and three-dimensional reconstruction of centre median-parafascicular thalamic neurons in primates. J Comp Neurol 481:127144. Parent M, Parent A. 2004. The pallidofugal motor fïber system in primates. Parkinsonism & Related Disorders 10: 203-211. Parent M, Parent, A. 2002. Axonal collateralization in primate basai ganglia and related thalamic nuclei. Thalamus & Related Systems 2: 71-86. Parent A, Parent M, Leroux-Hugon V. 2002 Jules Bernard Luys: a singular figure of 19th century neurology. Can J Neurol Sci 29: 282-288. Parent M, Lévesque M, Parent A. 2001. Two types of projection neurons in the internai pallidum of primates: Single-axon tracing and three-dimensional reconstruction. J Comp Neurol 439: 162-175. Parent A, Sato F, Wu Y, Gauthier J, Lévesque M, Parent M. 2000. Organization of the basai ganglia: the importance of axonal collateralization. Trends Neurosci 23 (Suppl.) Basai Ganglia, Parkinson's disease and levadopa therapy : S20-S27. Parent A, Lévesque M, Parent M. 2000. A re-evaluation of the current model of the basai ganglia. Parkinsonism & Related Disorders 7: 193-198. Sato F, Parent M, Lévesque M, Parent A. 2000. Axonal branching pattern of neurons of the subthalamic nucleus in primates. J Comp Neurol 424 : 142-152. Parent M, Lévesque M, Parent A. 1999. The pallidofugal projection system in primates : Evidence for neurons that branch ipsilaterally and contralaterally to the thalamus and brainstem. J Chem Neuroanat 16: 153-165. Gauthier J, Parent M, Lévesque M, Parent A. 1999. The axonal arborization of single nigrostriatal axons in rats. Brain Res 834: 228-232.

292 Parent A, Parent M, Charara A. 1999. Glutamatergic inputs to midbrain dopaminergic neurons in primates. Parkisonism & Related Disorders 5: 193-201. Parent A, Parent M, Lévesque M. 1999. Basai ganglia and Parkinson's disease : An anatomical perspective. Neurscience News 2 : 19-28.

Articles sous-presse Parent M, Parent A. Relationship between axonal collateralization and neuronal degeneration in basai ganglia. J Neural Transmission, sous presse. Miocinovic S, Parent M, Butson CR, Hahn PJ, Russo GS, Vitek JL, Mclntyre CC. Computational analysis of the activation of the subthalamic nucleus and lenticular fasciculus during therapeutic deep brain stimulation. J Neurophysiology, sous presse.

Contributions à un ouvrage collectif Parent M, Parent A. La maladie de Parkinson. In : Guidoin R, editor. L'homme réparé : Succès et limites de l'implantologie. Paris : John Libbey Eurotext. sous presse Parent A, Sato F, Parent M, Lévesque M. 1999. Basai ganglia anatomy and functional organization. In: Krauss JK, Jankovic J, Grossman RG, editors. Priciples of Surgery for Parkinson's Disease and Movement Disorders. Philaddelphia: Lippincott, Williams & Wilkins. p 48-55.

Communications à titre de conférencier invité Parent M. 2006. La réponse neuronale à la stimulation intracérébrale à haute fréquence du noyau subthalamique. Hôpital de l'Enfant-Jésus, Québec. Parent M. 2006. Three dimensional reconstruction of basai ganglia neurons in primates. Cleveland Clinic Foundation, Ohio. Parent, M. 2005. Anatomical and functional organization of primate basai ganglia. Centre de recherche Hôpital Douglas, Université McGill, Montréal.

293

Résumés publiés Miocinovic S, Parent M, Parent A, Russo GS, Vitek JL, Mclntyre C. 2005. Designed parameters for sélective deep brain stimulation of the subthalamic nucleus. Program No. 331.5. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2005. Online (Washington). Miocinovic S, Parent M, Parent A, Butson CR, Russo GS, Vitek JL, Mclntyre CC. 2005. Theoretical Design of Sélective Deep Brain Stimulation Parameters for the Subthalamic Nucleus. NIH and DBS Consortium, 36. (Bethesda). Parent M, Parent A. 2005. Axonal collateralization and neuronal degeneration in basai ganglia. Poster session. Proc. of the 16th International Congress on Parkinson's Disease and Related Disorders, 16: 81. Online (Berlin). Parent M, Parent A. 2004. Thalamic and cortical inputs to the primate striatum: singleaxon tracing studies. Program No. 754.5. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online (San Diego). Miocinovic S, Parent M, Parent A, Mclntyre CC. 2004. Electrical stimulation of the subthalamic nucleus: Model-based analysis of a 3D reconstructed neuron to intracellular and extracellular stimulation. Program No. 70.27. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online (San Diego). Parent M, Parent A. 2004. Traçage unitaire des afférences striatales en provenance du thalamus et du cortex moteur primaire chez le primate. Journée de la recherche de la faculté de médecine de l'Université Laval, p 134, (Québec). Parent M, Parent A. 2003. Thalamic and cortical inputs to the striatum : a single-axon tracing study in primates. IBRO 8, 1215, (Prague). Parent M, Lévesque M, Parent A. 2003. Étude par traçage unitaire des projections efférentes du centre médian chez le primate. Journée de la recherche de la faculté de médecine de l'Université Laval, p 80, (Québec). Parent M, Lévesque M, Parent A. 2002. Single-axon tracing of the centre médian efferent projections in primates. Program No. 460.1. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002. Online (Orlando). Parent M, Lévesque M, Parent A. 2002. The organization of the pallidofugal fiber System in primates. CCNS 37, H-03, (Vancouver). Parent M, Parent A. 2002. Étude par traçage unitaire des projections corticostriées en provenance du cortex moteur primaire chez le primate. Ann de l'ACFAS 70 : 108, (Québec).

294 Parent M, Lévesque M, Parent A. 2001. Single-axon tracing study of the projections from the primary motor cortex to the striatum in primates. . Program No. 69.6. 2001 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2001. Online (San Diego). Parent M, Lévesque M, Parent A. 2001 Traçage unitaire et reconstruction tridimensionnelle des neurones moteurs et limbiques du pallidum interne. Journée de la recherche de la faculté de médecine de l'Université Laval, p 65, (Québec). Parent M, Lévesque M, Parent A. 2000. Single-axon tracing study of the internai pallidum. Program No. 361.9. 2000 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2000. Online (La Nouvelle-Orléans). Parent M, Lévesque M, Parent A. 2000. La projection pallido-habénulaire chez le primate: une interface entre les systèmes moteur et limbique. Ann de l'ACFAS 68: 158, (Montréal) Parent M, Lévesque M, Parent A. 1999. Étude par marquage unitaire des projections efférentes du pallidum interne chez le primate. Ann de l'ACFAS 67: 156, (Ottawa).