Chen, Tingting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 100-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

La mélatonine est une neuro-hormone secrétée par la glande pinéale pour réguler les rythmes circadiens, le sommeil, la physiologie de la rétine, la reproduction saisonnière et diverses fonctions neuronales. La mélatonine exerce ses fonctions en se liant à deux récepteurs membranaires appelés MT1 et MT2 qui appartiennent à la famille des récepteurs couplés aux protéines G (RCPG). Les RCPG sont connus pour former des homo- et hétérodimères mais la pertinence physiologique de ces complexes reste à démontrer. Plusieurs études montrent que la fonction de ces complexes ne se limite pas à la régulation des protéines G hétérotrimériques, mais inclue également la régulation d'autres protéines comme les transporteurs et les canaux ioniques. Dans ce travail, nous rapportons la formation d'hétérodimères MT1/MT2 dans les photorécepteurs de la rétine de souris et nous montrons que l’augmentation de la sensibilité de ces cellules à la lumière par la mélatonine requiert l'activation de la voie Gq/PLC/PKC qui est spécifique de l’hétéromère. Cette étude confirme alors la pertinence physiologique de l’hétérodimérisation des récepteurs de la mélatonine.Nous avons ensuite cherché à identifier de nouveaux partenaires de MT1 et MT2 en effectuant plusieurs cribles protéomiques et génétiques et un interactome de 378 protéines a pu être construit. L'analyse bioinformatique a révélé la présence de plusieurs protéines présynaptiques (canaux calciques voltage-dépendants Cav2.2, SNAP25, Synapsin et Munc-18) dans l'interactome MT1. Parmi ces partenaires, nous avons montré dans les cellules CHO que le récepteur MT1 interagit avec la protéine Cav2.2 et inhibe l’entrée du calcium d'une manière indépendante de la stimulation par l’agoniste, ce qui suggère un rôle régulateur de MT1 dans la libération des neurotransmetteurs.Un autre partenaire caractérisé est le transporteur de la dopamine DAT. L'interaction physique de DAT avec les récepteurs de la mélatonine diminue l’expression de DAT à la surface cellulaire et diminue l'absorption de la dopamine dans les cellules HEK293. La pertinence physiologique de ces observations a été appuyée par l’augmentation de la recapture de la dopamine dans les synaptosomes du striatum de souris knock-out pour les récepteurs de la mélatonine. En conclusion, ce rapport montre que la construction des interactomes des RCPG offre de nouvelles perspectives pour la découverte de nouvelles fonctions de ces récepteurs, comme les fonctions rétiniennes et neuronales des récepteurs de la mélatonine dans notre étude. La formation de complexes RCPG/RCPG, RCPG/canaux ioniques et RCPG/transporteurs peut avoir un effet fonctionnel réciproque au niveau de l’activité du récepteur et de ces partenaires, mettant ainsi en évidence de nouveaux mécanismes moléculaires de cross-talk cellulaire. / Melatonin is a neurohormone secreated by the pineal gland in a circadian manner. This hormone is involved in the regulation of circadian rhythms, sleep, retinal physiology, seasonal reproduction and various neuronal functions. Melatonin exerts its effects through two G protein-coupled receptors (GPCR) called MT1 and MT2. GPCRs are known to form homo- and heterodimers, but the physiological relevance of these complexes remains a matter of debate. An increasing number of reports show that the function of these GPCR complexes is not restricted to the regulation of heterotrimeric G proteins but include also the regulation of other proteins like transporters and ion channels. Here, we report the formation of MT1/MT2 heterodimers in mouse retinal rod photoreceptors and show that the enhancing effect of melatonin on light sensitivity in these cells requires the activation of the heteromer-specific Gq/PLC/PKC signaling pathway. This study demonstrates the physiological relevance of GPCR heterodimerization.We next searched for new MT1 and MT2 interacting proteins in an unbiased manner by performing several proteomic and genetic screens. An interactome of 378 proteins was built. Bioinformatic analysis revealed the presence of several presynaptic proteins (voltage-gated calcium channel Cav2.2, SNAP25, Synapsin and Munc-18) in the MT1 interactome. Presynaptic localization of MT1 and spatial proximity with presynaptic proteins was confirmed in mouse and rat brains. Among these potential partners, we show that MT1 physically interacts with Cav2.2 in CHO cell line and inhibits Cav2.2-promoted Ca2+ entry in an agonist-independent manner suggesting a regulatory role of MT1 in neurotransmitter release.Another proteins identified in the screens was the dopamine transporter DAT. Physical interaction of DAT with melatonin receptors decreased DAT cell surface expression and diminished dopamine uptake in HEK293 cell. Supporting this result we found using the in vivo model of melatonin receptors knockout mice a respective increase of dopamine uptake in synaptosomal preparations of the striatum of supporting the physiological relevance of these GPCR/transporter complexes. In conclusion, this report shows that GPCR interactome building provides new insights into receptor function, like retinal and neuronal functions of melatonin receptors in our case. Formation of GPCR/GPCR, GPCR/ion channel and GPCR/transporter complexes may have a reciprocal functional impact, on the activity of the receptor and interacting partners thus elucidating new molecular mechanisms cellular cross-talk.

L’IRM apporte des informations morphologiques concernant la tumeur, mais également des informations concernant sa micro-vascularisation. En TEP/TDM, l’accumulation de la 18F-FDopa dans les cellules tumorales résulte de l’activité métabolique plus importante que celle des tissus sains. Nous avons étudié 28 gliomes pour lesquels nous avons analysé les données provenant d’IRM et de TEP/TDM. Une méthode de recalage a été développée afin de combiner les informations issues des deux modalités TEP et IRM et d’extraire des volumes d’intérêt sur la base des données conjointes TEP et IRM. L’analyse du contenu de ces volumes d’intérêt par un modèle de mélange gaussien a permis de différencier, dans ces volumes, les tissus tumoraux et les tissus sains, et d’obtenir ainsi des volumes tumoraux et de référence communs pour les modalités TEP et IRM. Des paramètres issus de la TEP ou de l’IRM ont ensuite été calculés dans ces volumes communs aux deux modalités, pour caractériser les tumeurs et les tissus sains. L’analyse discriminante linéaire (ADL) des données TEP/TDM et d’IRM combinées permet de discriminer les différentes classes tissulaires. Les courbes Receiver Operating Characteristic ROC combinées à l’ADL permettent d’évaluer les critères multiples [SUVmax , rCBV] et [rk1 , rCBV] et conduisent à des AUC respectives de 0,88 et 0,92. En considérant les informations combinées [SUVmax , rCBV], nous avons obtenu une sensibilité de détection des tumeurs de haut grade de 95% pour une spécificité correspondante de 60% ainsi qu’une valeur prédictive négative de 52% pour une valeur prédictive positive de 95%. De même, avec le critère [rk1 , rCBV], nous avons obtenu une spécificité de 60% pour 95% de sensibilité de détection des tumeurs de haut grade ainsi qu’une valeur prédictive négative de 60% pour une valeur prédictive positive de 95%. Nos travaux montrent que la fusion des informations microvasculaires et métaboliques est possible. Dans le cas du diagnostic différentiel des gliomes, l’information microvasculaire n’apporte cependant pas d’information plus discriminante que l’information métabolique seule. / MRI provides morphological information about tumour, but also provides information regarding the micro-vascularization of the tumour. In PET/CT, the accumulation of 18F-FDopa in tumour cells results from the metabolic activity greater than that of healthy tissues. We studied 28 gliomas for which we analysed data from MRI and PET/CT. A registration method has been developed to combine information from both PET and MRI and to extract volumes of interest consistent with the information included in the two modalities. In these volumes, the tumour compartment and normal tissue compartment were identified using a Gaussian mixture model. Parameters from PET or MRI data were then calculated in these compartments. ROC analyses combined with linear discriminant analyses were used to assess whether joint observation of standardized uptake value (SUVmax ) and relative Cerebral Blood Volume (rCBV) or of relative rk1 and rCBV could distinguish between low grade and high grade tumours. We found that using this joint analysis, 82.4% of high-grade tumors and 70.0% of low-grade tumors were correctly classified (AUC of 0.88 for [SUVmax , rCBV] and of 0.92 for [rk1 , rCBV]). Considering the [SUVmax , rCBV] combined information, the sensitivity for detecting high-grade tumors was 95% with a specificity of 60%. The negative predictive value was 52% for a positive predictive value of 95%. Similarly, considering the [rk1 , rCBV] combined information, we also a specificity of 60% associated with a 95% sensitivity for detecting high-grade tumors, with a negative predictive value of 60% and positive predictive value of 95%. Our work shows that joint analysis of microvascular and metabolic information is possible by combining PET and MR imaging data. However, we found that, in our patient population, the microvascular information given by MR did not bring information more discriminating than the metabolic information derived from PET only.

C’est chez un téléostéen qu’il a été démontré pour la première fois que le contrôle stimulateur de l’axe gonadotrope par la GnRH peut être contrebalancé par un contrôle inhibiteur assuré par la dopamine (DA). Ce contrôle dopaminergique inhibiteur a été retrouvé par la suite chez diverses espèces de vertébrés. Cependant l’importance fonctionnelle de cette voie inhibitrice de régulation varie beaucoup d’une espèce à l’autre. Pour approfondir nos connaissances sur ce système dopaminergique inhibiteur de la reproduction, nous avons utilisé le poisson zèbre (Danio rerio), un modèle de vertébrés pour lequel de nombreux outils moléculaires sont disponibles.Nous avons d’abord démontré qu’il existait bien un contrôle dopaminergique de la fonction gonadotrope dans cette espèce: en injectant un antagoniste dopaminergique en même temps qu’un analogue de la GnRH, nous avons pu stimuler l’expression de la LH dans l’hypophyse et réactiver les cycles de ponte chez des femelles âgées sexuellement régressées, un effet qui n’est pas produit par l’agoniste de la GnRH seul. Nous avons ensuite étudié le substrat neuroanatomique de cette action inhibitrice. Après avoir observé l’expression par les cellules à LH des sous-Types de récepteurs D2 qui existent chez le poisson-Zèbre, nous avons mis en évidence de nombreuses terminaisons dopaminergiques sur- ou à proximité- de ces cellules gonadotropes. Nous avons ensuite localisé, par des expériences de traçage rétrograde chez l’adulte, les corps cellulaires des neurones dopaminergiques qui émettent ces projections, dans la partie la plus antéro-Ventrale de l'aire préoptique. Nous avons appelé ces neurones hypophysiotropes: les neurones dopaminergiques préoptico-Hypophysaires (POHDA). Nous nous sommes aussi intéressés au développement des neurones POHDA. Grâce à des repères anatomiques précoces, nous avons pu les répérer précisément au sein de l’aire préoptique et suivre leur apparition sur des embryons de plus en plus jeunes. Nous avons ainsi mis en évidence que les tout premiers neurones POHDA n’apparaissent qu’à partir de 72 heures post-Fécondation (hpf), soit plus de 24 h après les neurones dopaminergiques du noyau suprachiasmatique (SCDA) voisin. Cette différenciation tardive explique probablement pourquoi les neurones POHDA ont jusque-Là été ignorés dans toutes les études de développement. En outre, nous avons montré que contrairement au nombre des neurones SCDA qui reste constant tout au long de la vie du poisson-Zèbre, celui des neurones POHDA continue d’augmenter tant que le poisson continue à grandir de manière allométrique, grâce à une neurogenèse continue. Enfin, nous avons examiné les profils d'expression de plusieurs gènes en relation avec la régionalisation du cerveau antérieur. Cette étude a permis de montrer que les réseaux génétiques impliqués dans le développement des populations SCDA et POHDA sont au moins en partie différents.Ces travaux démontrent pour la première fois l’existence d’un contrôle dopaminergique inhibiteur de la fonction gonadotrope chez le poisson-Zèbre. Ils décrivent l’anatomie de ce système dopaminergique chez l’adulte, sa mise en place au cours du développement et ses capacités de neurogenèse continue. Ils apportent chez le poisson-Zèbre des bases génétiques sur l’identité régionale de l’aire préoptique qui vont permettre d’aborder des études fonctionnelles sur le développement de ces neurones neuroendocrines mal connus. / It was first demonstrated in a teleost fish that the stimulatory control of the gonadotrope axis by GnRH can be counterbalanced by an inhibitory control exerted by dopamine (DA). Later on, this inhibitory dopaminergic control was found in various vertebrate species. However the functional importance of this regulatory pathway varies according to the species. To deepen our knowledge on this inhibitory dopaminergic system, we used the zebrafish (Danio rerio) as a model, in which numerous molecular tools are available.First we demonstrated that DA indeed plays a role in the neuroendocrine control of zebrafish reproduction. By injecting a dopamine receptor antagonist together with an agonist of the GnRH (GnRHa), we were able to stimulate LH expression in the pituitary, and to reactivate the spawning cycles in sexually regressed old females, an effect of which was not produced by the GnRHa alone.We then studied the neuroanatomical basis of this inhibitory control. After observing the expression of the D2-DA receptors subtypes in LH cells, we highlighted numerous dopaminergic terminals on- or in the vicinity of- these cells. We then localized, by DiI retrograde tracing experiments in adult zebrafish, the dopaminergic cell bodies giving rise to these projections in the most antero-Ventral part of the preoptic area. We have called these hypophysiotropic neurons the preoptico-Hypophysial (POH) DA neurons.We next studied the development of POHDA neurons. Taking advantage of early anatomical landmarks, we followed the embryonic development of these cells. We showed that the first POHDA neurons arise at around 72 hours post fertilization (hpf), more than 24 hours later that the DA neurons in the neighbor suprachiasmatic nucleus (SCDA). This late differentiation would explain why POHDA neurons have not been studied in the developing embryo so far. We showed that contrary to the number of the SCDA neurons, which is constant all along the fish life, that of POHDA neurons increases proportionally to the growth of the fish due to continuous neurogenesis. Finally, we examined the expression profiles of developmental genes related to the regionalization of the anterior forebrain. We showed that the genetic networks involved in the development of POHDA and SCDA populations are at least partly different. To summarize, this work demonstrates for the first time the existence of a dopaminergic inhibitory control of gonadotrope function in zebrafish. It describes the anatomy of the preoptico-Hypophyseal dopaminergic system supporting these DA actions and the setting up of these neurons during embryonic development. We show that these neuroendocrine population displays neurogenesis even during adulthood. Our findings also provide the genetic bases for future functional studies on the development of POHDA, a poorly studied neuroendocrine DA population.

帕金森病（PD）是一種常見的神經退行性疾病，其特徵性的癥狀是運動功能減弱，常伴有認知障礙如工作記憶缺陷。大多數癥狀源於中腦多巴胺神經元的進行性缺失。目前的治療常隨時間進展誘發嚴重的副反應，促使我們進一步研究PD的病理生理學機制。一般認為基底神經節直接和間接通路不平衡的活動導致PD的運動缺陷，但目前關於基底神經節環路突觸特性改變的研究還很少。對PD認知障礙機制的研究則更為少見。 / 本研究中，我們首先關注在對基底神經節提供主要輸入的皮質紋狀體通路。應用全細胞膜片鉗技術結合皮質刺激，并利用在D2受體表達神經元表達綠色螢光蛋白的轉基因小鼠，我們發現PD狀態下，在間接通路表達D2受體的中型多棘神經元（D2 MSN）上記到的皮質紋狀體通路AMPA受體介導電流的成對脉沖比值（PPR）以及NMDA受體介導電流的PPR均降低。此外皮質至D2 MSN突觸間隙的谷氨酸水平也增加而不伴有谷氨酸轉運體的功能異常。這些結果證明PD狀態下皮質至間接通路D2 MSN的谷氨酸釋放選擇性增加。結合基底神經節的功能環路考慮，至間接通路紋狀體投射神經元的皮質谷氨酸釋放增加可能參與了PD的運動癥狀。 / 我們接下來研究了皮質-D2 MSN通路的下游環節即紋狀體至蒼白球通路傳遞的改變。在蒼白球（GP）神經元上應用全細胞膜片鉗記錄結合紋狀體刺激，我們發現在6-羥多巴損毀之後，紋狀體蒼白球通路的PPR降低，GP神經元記到的紋狀體刺激誘發的抑制性突觸后電流（eIPSC）的變異係數降低,以及GP神經元記到的微型IPSC的頻率增加，這些結果證明紋狀體至蒼白球的GABA釋放增加。突觸前III型代謝型谷氨酸受體介導的對紋狀體蒼白球傳遞的抑制作用消失導致了紋狀體蒼白球通路GABA釋放的增加。這一增加，通過影響間接通路的下游環節，也可能參與了PD的運動癥狀。 / 為探討認知障礙的機制，我們研究了參與工作記憶功能的邊緣前皮質至伏核（NAc）的投射。應用與第一部份相似的研究方法，我們發現多巴胺受體對邊緣前皮質NAc通路的傳遞存在高度精確和補償性的調節。在邊緣前皮質-NAc D1 MSN通路，D1和D2受體突觸前分別介導對該傳遞的抑制性和易化性調節。然而，在D2 MSN相關的邊緣前皮質-NAc通路，上述作用發生了反轉。在耗竭NAc多巴胺之後，D2 MSN上誘發到的興奮性突觸后電流增加，提示邊緣前皮質-NAc D2 MSN傳遞增加。此外，在多巴胺損毀的情況下，激活D1和D2受體不再調節邊緣前皮質NAc通路的傳遞。結合邊緣環路考慮，邊緣前皮質至D2 MSN的谷氨酸釋放增加可能參與了PD的認知障礙。 / 綜上所述，PD狀態下，繼多巴胺缺失之後，多條通路發生可塑性改變，這些改變可能參與PD的運動和認知癥狀。 / Parkinson’s disease (PD) is a common neurodegenerative disease with characteristic hypokinetic motor symptoms and cognitive impairments like working memory deficits. Most of the symptoms are derived from progressive loss of dopaminergic neurons in the midbrain. Current therapies often induce severe side effects with time, which promotes us to further investigate the pathophysiological mechanism of PD. It is generally thought that the imbalanced activity between direct and indirect pathways of the basal ganglia underlies the motor deficits in PD, but little is studied about the changes in synaptic properties of the sub-circuits. Even less is known about the mechanism responsible for the cognitive dysfunctions in PD. / In our study, we first focused on the corticostriatal pathway that provides a major input to the basal ganglia. Employing whole-cell patch-clamp recordings with cortical stimulation as well as by taking advantage of transgenic mice with green fluorescent protein co-expressed in the D2 receptor-expressing neurons, we found a selective increase in cortical glutamate release onto indirect-pathway D2 receptor-expressing medium-sized spiny neurons (D2 MSNs), as indicated by reduced corticostriatal AMPA paired-pulse ratios (PPRs) and NMDA PPRs in D2 MSNs as well as increased glutamate level in cortex-D2 MSN synaptic cleft without malfunction in glutamate transporters in parkinsonian states. Considering from the functional organization of the basal ganglia circuits, the increased corticostriatal glutamate release onto indirect-pathway striatal projection neurons may contribute to the motor symptoms of PD. / We next studied whether the striatopallidal transmission, downstream to the cortex-D2 MSNs pathway, is also altered in parkinsonian states. Combining whole-cell patch-clamp recordings in globus pallidus (GP) neurons with striatal stimulation, we demonstrated that the striatopallidal GABA release was increased following 6-hydroxydopamine lesion, as indicated by decreased striatopallidal PPRs, reduced coefficient of variation of striatally evoked inhibitory postsynaptic currents (eIPSCs) and elevated frequency of miniature IPSCs in GP neurons. The loss of tonic presynaptic group III metabotropic glutamate receptors-mediated inhibition on striatopallidal transmission accounted for the increased striatopallidal GABA release. The increase in the striatopallidal GABA release, through affecting the downstream of the indirect pathway, would also contribute to the motor symptoms in PD. / To investigate the underlying mechanism of cognitive deficits, we targeted the prelimbic cortex-nucleus accumbens (NAc) projection that is critical for working memory function. Using similar approaches as the first part, we observed highly precise and complementary modulations by dopamine receptors, with D1 and D2 receptors presynaptically mediating the inhibition and facilitation of the prelimbic cortex-NAc D1 MSN transmission, respectively, and reversed effects in D2 MSN-associated pathway. Following dopamine depletion in NAc, an enhanced prelimbic cortex-NAc D2 MSN transmission was indicated by selectively increased excitatory postsynaptic current evoked in D2 MSNs. Moreover, in the dopamine-depleted state, activating D1 and D2 receptors failed to modulate the prelimbic cortex-NAc transmission. Considering from the information flow in the limbic loop, the increased prelimbic cortical glutamate release onto D2 MSNs may contribute to the cognitive impairments in PD. / In conclusion, in the parkinsonian states, multiple pathways undergo plasticity changes subsequent to dopamine depletion, which may underlie the motor and cognitive symptoms in PD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cui, Qiaoling. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 160-191). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Parkinson’s disease --- p.1 / Chapter 1.1.1 --- Symptoms --- p.1 / Chapter 1.1.2 --- Etiology --- p.1 / Chapter 1.1.3 --- Pathology and pathophysiology --- p.2 / Chapter 1.1.4 --- Therapy --- p.5 / Chapter 1.1.4.1 --- L-DOPA and dopamine receptor agonists treatments --- p.5 / Chapter 1.1.4.2 --- Deep brain stimulation (DBS) and lesional surgery treatments --- p.6 / Chapter 1.1.4.3 --- Neural transplantation --- p.7 / Chapter 1.1.4.4 --- Treatment of nonmotor symptoms --- p.8 / Chapter 1.2 --- Basal ganglia --- p.8 / Chapter 1.2.1 --- Components of basal ganglia --- p.8 / Chapter 1.2.2 --- Pathways in basal ganglia --- p.8 / Chapter 1.2.2.1 --- Anatomical organization of the basal ganglia pathways --- p.8 / Chapter 1.2.2.2 --- Functional consequences of the basal ganglia pathways --- p.10 / Chapter 1.3 --- Striatum --- p.11 / Chapter 1.3.1 --- Anatomy of the striatum --- p.11 / Chapter 1.3.1.1 --- Cellular heterogeneity in the striatum --- p.12 / Chapter 1.3.1.1.1 --- MSNs --- p.12 / Chapter 1.3.1.1.1.1 --- Subpopulations --- p.13 / Chapter 1.3.1.1.1.2 --- Morphology --- p.13 / Chapter 1.3.1.1.1.3 --- Electrophysiological properties --- p.14 / Chapter 1.3.1.1.2 --- Cholinergic interneurons --- p.15 / Chapter 1.3.1.1.3 --- GABAergic interneurons --- p.17 / Chapter 1.3.1.2 --- Innervation of the striatum --- p.18 / Chapter 1.3.1.3 --- Output of the striatum --- p.21 / Chapter 1.3.2 --- Function of the striatum --- p.21 / Chapter 1.3.2.1 --- Function of the associative striatum --- p.22 / Chapter 1.3.2.2 --- Function of the sensorimotor striatum --- p.22 / Chapter 1.3.3 --- The corticostriatal system --- p.23 / Chapter 1.3.3.1 --- Anatomy of the corticostriatal system --- p.24 / Chapter 1.3.3.2 --- Physiology of the corticostriatal system --- p.25 / Chapter 1.3.3.3 --- Function of the corticostriatal system --- p.26 / Chapter 1.3.4 --- Striatum, corticostriatal system and PD --- p.26 / Chapter 1.4 --- GPe --- p.29 / Chapter 1.4.1 --- Anatomy of GPe --- p.29 / Chapter 1.4.1.1 --- Cellular heterogeneity in GPe --- p.29 / Chapter 1.4.1.2 --- Innervation of GPe --- p.31 / Chapter 1.4.1.3 --- Output of GPe --- p.33 / Chapter 1.4.2 --- Neurotransmission in GPe --- p.34 / Chapter 1.4.2.1 --- GABAA receptors in GPe --- p.34 / Chapter 1.4.2.2 --- GABAB receptors in GPe --- p.35 / Chapter 1.4.2.3 --- Evoked responses in GPe from direct striatal and pallidal stimulations --- p.37 / Chapter 1.4.3 --- GPe, striatopallidal system and PD --- p.38 / Chapter 1.5 --- NAc --- p.40 / Chapter 1.5.1 --- Anatomy of NAc --- p.40 / Chapter 1.5.1.1 --- Subregions --- p.40 / Chapter 1.5.1.2 --- Cell heterogeneity in NAc --- p.42 / Chapter 1.5.1.2.1 --- MSNs --- p.42 / Chapter 1.5.1.2.2 --- Interneurons --- p.42 / Chapter 1.5.1.3 --- Inervation of NAc --- p.43 / Chapter 1.5.1.4 --- Output of NAc --- p.43 / Chapter 1.5.2 --- Function of NAc --- p.43 / Chapter 1.5.3 --- The prefrontal cortex (PFC)-NAc system --- p.43 / Chapter 1.5.4 --- NAc, PFC-NAc system and PD --- p.44 / Chapter 1.6 --- Objectives --- p.45 / Chapter Chapter 2 --- General methods --- p.51 / Chapter 2.1 --- Electrophysiological experiments --- p.51 / Chapter 2.1.1 --- Slice preparation --- p.51 / Chapter 2.1.2 --- Whole-cell patch-clamp recordings --- p.52 / Chapter 2.1.3 --- Uncaging experiment --- p.54 / Chapter 2.1.4 --- Data analysis and statistics --- p.54 / Chapter 2.2 --- Dopamine depletion --- p.55 / Chapter 2.2.1 --- 6-hydroxydopamine (6-OHDA) injection into medial forebrain bundle (MFB) --- p.55 / Chapter 2.2.2 --- 6-OHDA injection into NAc --- p.56 / Chapter 2.2.3 --- Reserpine treatment --- p.56 / Chapter 2.3 --- Limb-use asymmetry test (cylinder test) --- p.57 / Chapter 2.4 --- Tyrosine hydroxylase (TH) immunohistochemistry and analysis --- p.57 / Chapter 2.4.1 --- TH immunohistochemistry of SNc and striatal slices --- p.57 / Chapter 2.4.2 --- TH immunohistochemistry and analysis of NAc slices --- p.58 / Chapter 2.5 --- Tracing study --- p.59 / Chapter 2.6 --- Genotyping and quantitative polymerase chain reaction (qPCR) --- p.60 / Chapter Chapter 3 --- Alteration of corticostriatal glutamatergic transmission onto D2 MSNs in PD models --- p.62 / Chapter 3.1 --- Summary --- p.62 / Chapter 3.2 --- Introduction --- p.63 / Chapter 3.3 --- Materials --- p.65 / Chapter 3.3.1 --- Animals --- p.65 / Chapter 3.3.2 --- Chemicals --- p.66 / Chapter 3.4 --- Results --- p.66 / Chapter 3.4.1 --- Comparison of corticostriatal paired-pulse ratios (PPRs) between hemizygotes and homozygotes of D2-EGFP BAC transgenic mice --- p.66 / Chapter 3.4.2 --- Corticostriatal AMPA PPR was specifically decreased in D2 MSNs following dopamine depletion --- p.67 / Chapter 3.4.2.1 --- Corticostriatal AMPA PPR was specifically decreased in D2 MSNs following reserpine treatment --- p.67 / Chapter 3.4.2.2 --- Corticostriatal AMPA PPR was specifically decreased in D2 MSNs following 6-OHDA lesion --- p.68 / Chapter 3.4.3 --- Increased glutamate release underlying reduction of corticostriatal PPR in D2 MSNs in parkinsonian states --- p.69 / Chapter 3.4.3.1 --- Effect of γ-DGG on the corticostriatal eEPSCs of D2 MSNs --- p.70 / Chapter 3.4.3.2 --- Effect of γ-DGG on the corticostriatal eEPSCs of D2 MSNs in the presence of CTZ --- p.70 / Chapter 3.4.3.3 --- Decay kinetics of eEPSCs of D2 MSNs in the presence of CTZ or PEPA were not consistently altered following dopamine depletion --- p.71 / Chapter 3.4.3.4 --- Corticostriatal NMDA PPR was decreased in D2 MSNs following dopamine depletion --- p.72 / Chapter 3.4.4 --- AMPA receptor occupancy was increased in D2 MSNs following dopamine depletion --- p.73 / Chapter 3.4.5 --- Increased postsynaptic AMPA receptor desensitization contributing to the reduction of corticostriatal PPR in D2 MSNs of parkinsonian states --- p.74 / Chapter 3.4.5.1 --- Effect of CTZ on the corticostriatal AMPA PPR of D2 MSNs --- p.74 / Chapter 3.4.5.2 --- Effect of PEPA on the corticostriatal AMPA PPR of D2 MSNs --- p.75 / Chapter 3.4.6 --- Loss of dopamine D2 receptor activation did not contribute to the increased corticostriatal glutamate release onto D2 MSNs in the parkinsonian states --- p.75 / Chapter 3.4.7 --- Postsynaptic Ca2+ involved in the modification of the corticostriatal transmission in D2 MSNs of parkinsonian state --- p.77 / Chapter 3.5 --- Discussion --- p.78 / Chapter 3.5.1 --- Corticostriatal glutamate release onto D2 MSNs was increased in the parkinsonian states --- p.78 / Chapter 3.5.2 --- AMPA receptor occupancy was increased in D2 MSNs following dopamine depletion --- p.80 / Chapter 3.5.3 --- Postsynaptic AMPA receptor desensitization was increased in D2 MSNs following dopamine depletion --- p.81 / Chapter 3.5.4 --- Loss of dopamine D2 receptor activation did not contribute to the increased corticostriatal glutamate release onto D2 MSNs in the parkinsonian states --- p.81 / Chapter 3.5.5 --- Postsynaptic Ca2+ involved in the modification of the corticostriatal transmission in D2 MSNs of parkinsonian state --- p.82 / Chapter 3.5.6 --- The increased corticostriatal glutamate release onto D2 MSNs and PD --- p.83 / Chapter Chapter 4 --- Alteration of striatopallidal GABAergic transmission in 6-OHDA lesioned PD model --- p.98 / Chapter 4.1 --- Summary --- p.98 / Chapter 4.2 --- Introduction --- p.99 / Chapter 4.3 --- Materials --- p.101 / Chapter 4.3.1 --- Animals --- p.101 / Chapter 4.3.2 --- Chemicals --- p.101 / Chapter 4.4 --- Results --- p.101 / Chapter 4.4.1 --- Striatopallidal paired-pulse ratio (PPR) was decreased following 6-OHDA lesion --- p.101 / Chapter 4.4.1.1 --- Striatopallidal PPR was unchanged following reserpine treatment --- p.102 / Chapter 4.4.1.2 --- Striatopallidal PPR was decreased following 6-OHDA lesion --- p.102 / Chapter 4.4.2 --- Increased striatopallidal GABA release underlying the reduction of striatopallidal PPR following 6-OHDA lesion --- p.103 / Chapter 4.4.2.1 --- CV of eIPSC1 in GP neurons was reduced following 6-OHDA lesion --- p.103 / Chapter 4.4.2.2 --- mIPSCs frequency was increased in GP neurons following 6-OHDA lesion --- p.104 / Chapter 4.4.3 --- Mechanism for the increased striatopallidal GABA release following 6-OHDA lesion --- p.105 / Chapter 4.4.3.1 --- Loss of dopamine D2 receptor activation did not contribute to the increased striatopallidal GABA release following 6-OHDA lesion --- p.105 / Chapter 4.4.3.2 --- GABAB receptor modulation did not contribute to the increased striatopallidal GABA release following 6-OHDA lesion --- p.106 / Chapter 4.4.3.3 --- Loss of presynaptic tonic group III mGluR inhibition accounted for the increased striatopallidal GABA release following 6-OHDA lesion --- p.107 / Chapter 4.5 --- Discussion --- p.109 / Chapter 4.5.1 --- Striatopallidal GABA release was increased in the parkinsonian state --- p.109 / Chapter 4.5.2 --- Mechanism underlying the increased striatopallidal GABA release in the parkinsonian state --- p.111 / Chapter 4.5.2.1 --- Loss of dopamine D2 receptor activation did not contribute to the increased striatopallidal GABA release following 6-OHDA lesion --- p.111 / Chapter 4.5.2.2 --- GABAB receptor modulation did not contribute to the increased striatopallidal GABA release following 6-OHDA lesion --- p.112 / Chapter 4.5.2.3 --- Loss of presynaptic tonic group III mGluR inhibition accounted for the increased striatopallidal GABA release following 6-OHDA lesion --- p.113 / Chapter 4.5.3 --- The increased striatopallidal GABA release and PD --- p.114 / Chapter 4.5.4 --- The striatopallidal group III mGluR system and PD --- p.116 / Chapter Chapter 5 --- Role of D1 and D2 receptors in prelimbic cortex-nucleus acumbens transmission in normal and parkinsonian states --- p.128 / Chapter 5.1 --- Summary --- p.128 / Chapter 5.2 --- Introduction --- p.129 / Chapter 5.3 --- Materials --- p.131 / Chapter 5.3.1 --- Animals --- p.131 / Chapter 5.3.2 --- Chemicals --- p.131 / Chapter 5.4 --- Results --- p.132 / Chapter 5.4.1 --- Prelimbic cortex innervated both D1 MSNs and D2 MSNs in core subregion of NAc- --- p.132 / Chapter 5.4.2 --- D1 and D2 receptors presynaptically modulated the D1 MSN-associated prelimbic cortex-NAc transmission in opposite manner --- p.133 / Chapter 5.4.3 --- D1 and D2 receptors presynaptically modulated the D2 MSN-associated prelimbic cortex-NAc transmission in a reverse manner --- p.134 / Chapter 5.4.4 --- Effects of D1 and D2 receptor antagonists on the prelimbic cortex-Nac transmission --- p.135 / Chapter 5.4.5 --- Basal synaptic transmission was enhanced in D2 MSN-associated prelimbic cortex-NAc pathway following NAc dopamine depletion --- p.136 / Chapter 5.4.6 --- D1 and D2 receptor modulation of the prelimbic cortex-NAc transmission disappeared following dopamine depletion --- p.137 / Chapter 5.5 --- Discussion --- p.138 / Chapter 5.5.1 --- Prelimbic cortex innervated both D1 MSNs and D2 MSNs in core subregion of NAc- --- p.138 / Chapter 5.5.2 --- Prelimbic cortex-NAc projections were presynaptically modulated by D1 and D2 receptors in a highly precise and complementary pattern --- p.138 / Chapter 5.5.3 --- Glutamatergic transmission was selectively enhanced in D2 MSN-associated prelimbic cortex-NAc pathway following NAc dopamine depletion --- p.140 / Chapter 5.5.4 --- D1 and D2 receptor modulation of the prelimbic cortex-NAc transmission was lost following dopamine depletion --- p.142 / Chapter Chapter 6 --- General discussion --- p.154 / Chapter 6.1 --- Enhanced corticostriatal glutamate release, enhanced striatopallidal GABA release and motor deficits in PD --- p.154 / Chapter 6.2 --- Enhanced prelimbic cortical glutamate release onto accumbal D2 MSNs and cognitive deficits in PD --- p.155 / Abbreviations --- p.158 / References --- p.160

Dans la maladie de Parkinson, un déficit d'activité électrique serait impliqué dans le processus de dégénérescence des neurones dopaminergiques (DA) de la substance noire. Or des structures non-DA, en particulier les neurones cholinergiques du noyau pédonculopontin (PPN) et orexinergiques de l'hypothalamus, sont affectés dans la pathologie. Ces neurones étant la source d'une innervation excitatrice pour les neurones DA, un déficit de ces inputs chez les patients pourrait jouer un rôle clé dans la progression de la perte DA. Le premier but de ce projet était d'évaluer l'impact d'une lésion des neurones cholinergiques du PPN sur la survie des neurones DA nigraux de rats et de macaques sains et parkinsoniens. Nous avons montré que 1) une déafférentation cholinergique induit une atrophie neuronale voire une dégénérescence DA, 2) une lésion DA induit une perte de neurones cholinergiques du PPN, et 3) combiner une lésion cholinergique du PPN à une lésion DA induit une exacerbation des pertes neuronales dans les deux systèmes. Enfin, le taux de dégénérescence DA est corrélé à l'intensité de la perte des neurones cholinergiques. Nos résultats confirment que le PPN joue un rôle clé dans la physiopathologie de la maladie de Parkinson et démontrent la forte interrelation existant entre ces deux systèmes. Concernant le système orexinergique, nous montrons que les fibres sont en position de moduler l'activité des neurones DA, mais qu'une lésion sélective du système DA chez le macaque n'est pas suffisante pour induire la mort des neurones à orexine. Ces résultats suggèrent que l'atteinte du système orexinergique chez des patients parkinsoniens pourrait résulter de lésions non-DA. / In Parkinson’s disease, there is evidence that dopaminergic (DA) neurons of the substantia nigra degenerate when they become electrically less active. Many non-DA structures including cholinergic neurons of the pedunculopontine nucleus (PPN) and orexinergic neurons of the hypothalamus, are also degenerating. Since these non-DA neurons are sources of excitatory inputs to the nigral DA neurons, their lesion in parkinsonian patients might play a key role in the progression of DA neuronal death. The first goal of this study was to evaluate the effect of a cholinergic PPN lesion on the survival of nigral DA neurons in healthy and parkinsonian rats and macaques. We found that 1) a PPN cholinergic lesion induced neuronal atrophy and death; 2) a DA lesion alone resulted in a loss of PPN cholinergic neurons; and 3) adding a PPN cholinergic lesion to a DA lesion in rats when the process of DA degeneration was in progress exacerbated neuronal losses in both systems. Last, the rate of DA degeneration was highly correlated to the level of cholinergic loss. Our results highlight the key role of the PPN in the physiopathology of Parkinson’s disease and clearly demonstrate strong reciprocal interactions with nigral DA neurons. The second aim of our study was to focus on the hypothalamic orexinergic system using a morphological approach in macaques. We show that orexinergic fibers are in position to modulate DA neurons activity. However, a relatively selective DA lesion in macaques was not sufficient to induce death of the orexinergic neurons. These data suggest that the loss of orexinergic neurons observed in parkinsonian patients likely results from non-DA lesions.

La maladie de Parkinson (MP) est caractérisée par une perte progressive des terminaisons présynaptiques dopaminergiques et reste actuellement incurable. Néanmoins, dans les études épidémiologiques, il a été montré que l’utilisation des statines, médicaments hypocholestérolémiants, diminue le risque de développer une MP. Les statines sont également capables d'inhiber les effets neurodégénératifs dans les modèles précliniques in-vitro et in-vivo de la MP. Cependant, les mécanismes moléculaires à l’origine de ces effets neuroprotecteurs ne sont pas encore complétement élucidés. Ainsi, nous avons étudié les effets potentiels des statines sur l'expression des marqueurs synaptiques et sur le transport de la dopamine. Dans nos études, les statines induisent la croissance des neurites dans les cellules dopaminergiques et déclenchent une augmentation de l’expression des protéines synaptiques dopaminergiques telles que le transporteur vésiculaire des monoamines (VMAT2) et le transporteur de la dopamine. Les statines induisent une diminution de la recapture de la dopamine cellulaire et des changements d’affinités aux niveaux des sites de liaison des inhibiteurs sélectifs du VMAT2. L’activation du facteur de transcription nucléaire protéine-1 se liant à l'élément de régulation des stérols (SREBP-1), cholestérol-dépendent, serait l’élément inducteur de la surexpression des marqueurs dopaminergiques présynaptiques induite par les statines. En outre, ces résultats soutiennent un potentiel thérapeutique neuroprotecteur et/ou neurorestaurateur des statines précédemment proposées dans la MP et permettent de mettre en évidence de nouvelles cibles thérapeutiques comme le facteur SREBP. / Parkinson disease (PD) is characterized by a progressive loss of dopaminergic presynaptic terminals and remains incurable. However in epidemiological studies, it has been shown that the use of statins, which are hypocholesterolemic drugs, diminishes the risk to develop a PD. Statins are able to inhibit the neurodegenerative effects in in-vitro and in-vivo models of PD. However, the molecular mechanisms driving neuroprotective effects are not yet fully understood. Consequently, we investigated the potential effects of statins on the synaptic expression and dopamine transport function in the dopaminergic system. In our studies, statins enhance the neurite outgrowth in the dopaminergic cells and trigger an increase in the expression levels of presynaptic dopaminergic proteins such as vesicular monoamine transporter 2 (VMAT2) and dopamine transporter. Statins induce a reduction of dopamine cellular uptake and modulate the binding-affinity of the specific inhibitors for VMAT2. The activation of the nuclear transcriptional factor sterol regulatory element-binding protein 1 (SREBP-1), cholesterol-dependent, could be the key element of the overexpression of dopaminergic presynaptic markers induced by the statins. Furthermore, these findings highlight the therapeutic neuroprotective and/or neurorestorative potentials of statins previously proposed in PD and allow to bring out new potential therapeutic targets such as SREBP factor.

Dans ce travail de thèse, nous avons modélisé le rôle de la dopamine dans l'apprentissage et dans les processus de sélection de l'action en lien avec les ganglions de la base. L'activité des neurones dopaminergiques présente de nombreuses similarités avec l'erreur de prédiction de la récompense utilisée par les algorithmes d'apprentissage par renforcement. Ainsi, ces neurones sont supposés guider le processus de sélection de l'action.Dans une première partie, nous avons analysé l'information encodée par les neurones dopaminergiques dans une tâche à choix multiples en la comparant à différentes informations utilisées par les modèles d'apprentissage par renforcement. Nos résultats suggèrent que l'information encodée par les neurones dopaminergiques enregistrer dans la tâche n'est que partiellement compatible avec une erreur de prédiction et semble en partie dissociée du comportement.Dans une deuxième partie, nous avons simulé l'effet de la dopamine sur un modèle des ganglions de la base prenant en compte des connections existant chez le primate, souvent négligées dans la littérature. La plupart des modèles actuels font en effet l'hypothèse d'une séparation stricte de deux chemins dans les ganglions de la base : le chemin direct lié à la récompense et le chemin indirect lié à la punition. Cependant des études anatomiques remettent en question cette dissociation, en particulier chez le primate. Nous proposons ainsi d'étudier comment différents niveaux de dopamine, dans le contexte de la maladie de Parkinson, affectent l'apprentissage et la sélection de l'action dans ce modèle / In this thesis work, we modelled the role of dopamine in learning and in the processes of action selection through its interaction with the basal ganglia. During the 90’s, the work of Schultz and colleagues has led to major progress in understanding the neural mechanisms underlying the influence of feedback on learning. The activity of dopaminergic neurons exhibited properties of the reward prediction error signal used in so-called Temporal Difference (TD) machine learning algorithms. Thus, DA has been thought to be the neural signal that help us to adapt our behavior. In the first part of my PhD, we analyze the information encoded by dopaminergic neurons recorded during a multi-choice task. In this purpose, we modeled the task and simulated different TD learning algorithms to quantitatively compare their ability to reproduce dopamine neurons activity. Our results show that the information carried out by dopamine neurons is only partly consistent with a reward prediction error and seems to be dissociated from behavioral adaptation.In the second part of my PhD, we study the effect of different levels of dopamine in a biologically plausible model of primates basal ganglia that considers existing connections often neglected in the literature. Indeed, most of current models of basal ganglia assume the existence of two segregated pathway: the direct pathway associated with reward and the indirect pathway associated with punishment. However, anatomical studies in primates revealed that these two pathways are not dissociated. We study the ability of such a model to reproduce beta oscillations observed in Parkinsonian and the differences in reward and punishment sensitivity, with high or low-level of dopamine.

La dopamine (DA) contrôle l'apprentissage lié à la récompense en régulant l'activité et la plasticité de la transmission corticostrialale. Les effets à long terme de la DA impliquent des changements dans la transcription des gènes. Le but de ce travail de thèse est d'étudier les changements transcriptionnels produit dans le striatum ventral, dorsal et cortex préfrontal, par un protocole d'apprentissage opérant ou après une activation du système de la récompense par des injections de cocaïne. Les neurones épineux moyens du striatum peuvent être séparé en deux populations fonctionnelles sur la base de l'expression du récepteur de type 1 de la DA (D1R) ou de type 2 (D2R). Des souris transgéniques expriment une protéine ribosomale étiqueté avec la GFP sous le contrôle du promoteur de D1R ou D2R ont été utilisés afin d'isoler les ARN messagers (ARNm) des neurones D1 ou D2. La première partie de ce travail est centré sur la comparaison de l'expression des gènes dans les différentes populations neuronales exprimant le D1R ou D2R appartenant aux différentes régions d'intérêt, ce qui apporte une caractérisation précise nouvelle des neurones cibles de la DA. Dans un deuxième temps, nous avons caractérisé les modifications produites dans chaque population neuronale par une stimulation passive du système de récompense (traitement chronique à la cocaïne) ou un recrutement actif (apprentissage opérant pour la nourriture). / Dopamine (DA) controls movement execution, action selection, and incentive learning by regulating the activity and plasticity of corticostriatal transmission. Long-term modifications require changes in gene transcription. The aim of this work is to study the changes in transcriptions following an operant learning protocol or mimicking stimulation of the reward system with cocaine in the dorsal striatum and the nucleus accumbens in the striatum, and in the prefrontal cortex. The medium-size spiny striatal projection neurons (SPNs) can be divided into 2 different populations based on the expression of the D1 or D2 DA receptor that participate in distinct pathways, which have opposite functional effects on their target regions. We used transgenic mice that express a tagged ribosomal protein (L10a-EGFP) under control of the D1 or D2 receptor promoter to isolate currently translated mRNA and nuclei from each population of SPNs, as well as from D1 neurons of the prefrontal cortex following passive stimulation of the reward system (chronic treatment with cocaine) and active recruitment of the reward system (operant learning for food). Firstly we compared the basal gene expression in the different neuronal populations characterized by the expression of D1 or D2 receptors and their regional localization. We identified hundreds of differentially expressed mRNA providing a precise characterization of the cellular and regional differences. In the second part, we characterized the changes induced in each neuronal population by a 1-week exposure to cocaine or after operant training for food.

Parkinson's disease (PD) is a progressive neurodegenerative and movement disorder that involves specific loss of dopaminergic neurons in the substantia nigra of the brain. Exact causes of PD are unknown. However, cells affected in PD are centers of dopamine (DA) synthesis, storage, and metabolism, which implicate DA as an endogenous neurotoxin that contributes to PD. Furthermore, DA is known to undergo oxidation to radicals and quinones. These reactive species exert deleterious effects on cells through a variety of mechanisms that are relevant to the pathogenesis of PD. Another potential mechanism of toxicity for DA is metabolism to 3,4-dihydroxyphenylacetaldehyde (DOPAL). This reactive metabolite is significantly more toxic than the parent DA. DOPAL has several demonstrated mechanisms of toxicity, including formation of protein-adducts via reaction with amine-type cellular nucleophiles. However, known toxicity mechanisms do not fully account for DOPAL's high toxicity. Oxidation of DOPAL to a reactive quinone or radical could help explain its high toxicity. Therefore, the hypothesis of this work is that DOPAL is capable of undergoing oxidation that leads to increased protein modification and nucleophilic reactivity. Experimentally, oxidation of DOPAL results in formation of a semi-quinone radical and an ortho-quinone, as confirmed by electron paramagnetic resonance spectroscopy and nuclear magnetic resonance spectroscopy, respectively. In agreement with the stated hypothesis, oxidation of DOPAL enhanced its ability to induce protein cross-linking of a model protein (glyceraldehyde 3-phosphate dehydrogenase) as indicated by polyacrylamide gel-electrophoresis. Also, the presence of anti-oxidants (ascorbate, N-acetyl cysteine) attenuated the reactivity of DOPAL with the model aminenucleophile N-acetyl lysine. These results indicate that DOPAL oxidation enhances both protein cross-linking and nucleophilic reactivity. This work resulted in several other important findings. DOPAL is shown to undergo carbonyl-hydration in aqueous media, and spontaneous oxidation of DOPAL results in formation of superoxide. Furthermore, DOPAL is shown to be susceptible to oxidation by cyclooxygenase-2, an enzyme known to be involved in PD. This provides a potential mechanism for formation of the oxidized products identified here. As DA metabolism and oxidation occur in cells affected by PD, the experimental results demonstrated here are likely relevant for understanding the pathogenesis of PD.