Seul 1,2% du génome code des protéines :98,8% est non-codant,cependant 93% du génome est transcrit, principalement en longs ARN non-codants (lncRNA). Or ces lncRNA constituent une nouvelle classe de régulateurs génomique agissant à tous les niveaux d’expression des gènes et ils sont fortement spécifiques du tissu,modulés au cours du temps et en conditions physiopathologiques.Ainsi,nous proposons que chaque cellule spécifiée exprime son répertoire de lncRNA spécifique avec une carte des zones de chromatines ouvertes renseignant son identité cellulaire.Dans cette perspective,nous avons isolé par FACS 2types cellulaires impliqués dans des pathologies: i) des neurones dopaminergiques humains(nDA) différenciés à partir d’hiPS et ii) des neurones DA et sérotoninergiques (n5-HT)murins.Sur ces 2types neuraux isolés,nous avons identifié 1363 lncRNA exprimés dans les nDA (dont 989nouveaux) constituant le répertoire des neurones DA et 1257 lncRNA dans les n5-HT (719nouveaux) constituant le répertoire des n5-HT.Or leur comparaison a montré que seuls 194 lncRNA sont communs aux 2types cellulaires:la majorité des lncRNA est exprimée soit dans les nDA soit dans les n5-HT,attestant leur spécificité cellulaire.De plus,39%des zones de chromatines ouvertes/potentiellement régulatrices des nDA ne sont pas non plus retrouvées dans les n5-HT.Ainsi, nous avons généré un catalogue d’éléments non codants constituant des signatures moléculaires spécifiques des nDA et n5-HT,ouvrant de nouvelles pistes physiopathologiques:Dans cette optique,les signatures non codantes DA ont été comparées avec les SNP associés à la maladie de Parkinson et des études de fonction sur des lncRNA candidats ont été réalisées. / Only 1.2% of the genome codes for proteins; 98.8% is thus non-coding, despite 93% of the human genome being actively transcribed, mostly in long non-coding RNA (lncRNA).These lncRNA constitute a new class of genomic regulator capable of acting at all levels of gene expression and their expression is highly tissue-specific,modulated during the time and under normal/pathological conditions.Thus, we propose that each specified cell expresses a specific repertoire of lncRNA correlated to open/active chromatin regions specifying its cellular identity.In this context, we isolated by FACS 2neural types involved in many pathologies: i) human dopaminergic neurons (nDA) differentiated from hiPS and ii) DA and serotoninergic (n5-HT) neurons. From these 2neural types, we identified 1,363 lncRNA in nDA (among which 989 new, whether 73%) constituting the repertoire of nDA, and 1,257 lncRNA (among which 719 new) constituting the repertoire of n5-HT. Moreover,their comparison has shown that only 194 lncRNA are common to both neural types:thus the majority of lncRNA is expressed either in nDA or in n5-HT, indicating a high degree of cell-specificity.In addition, 39% of open chromatin regions, potentially regulatory, were also not detected in the n5-HT.Thus, we have generated DA and 5-HT specific catalogues of non-coding elements of the genome, which constitute DA and 5-HT specific molecular signatures, that could participate in deepening our knowledge regarding nDA or n5-HT development and dysfunctions. With this in mind,these DA specific elements have been compared with the SNP described as Parkinson Disease risk variants and candidate lncRNA were selected to perform studies of function.

No description available.

Neuronal communication in the mammalian brain relies on the presynaptic release of neurotransmitters which bind to ligand-gated ion channels found on postsynaptic neurons to modulate neuronal excitability. One such neurotransmitter is acetylcholine (ACh), a small molecule that is the signalling messenger of the cholinergic system. The cholinergic system is involved in a variety of behavioural functions including motor activity, sensory function, and higher executive commands. Dopaminergic neurons in the substantia nigra pars compacta (SNc) and the basal ganglia in general have long been implicated in initiation and completion of voluntary movement. Studies have shown that cholinergic neurons from two brainstem nuclei, the laterodorsal tegmental nucleus and the pedunculopontine nucleus, project onto substantia nigra dopaminergic (DA) neurons in the midbrain and release ACh, GABA or both to modulate motor behaviours. However, with prior research primarily focused on demonstrating the phenomenon of co-transmission itself, the subcellular distribution and dynamics of ACh and GABA release onto SN DA neurons receiving co-transmitted inputs largely remains to be investigated. The present study investigates the spatial and physiological properties of ACh/GABA co- transmission from brainstem cholinergic axons synapsing onto medial SN DA neurons to understand its role in tuning the neuron’s excitatory-inhibitory balance. To that end, we developed a channelrhodopsin (ChR2)-based functional input mapping technique with high spatial resolution to probe the dendritic distribution of ACh and GABA synaptic inputs onto DA neurons in ChATcre::ChR2 mice. Using this technique, we discovered three different types of monosynaptic inputs from cholinergic axons onto DA cells: co-transmitted ACh/GABA, GABA only, and ACh only. Furthermore, we revealed a somatodendritic patterning of cholinergic input distribution onto DA cells with a predominant GABA conductance along the lateral dendrites and a soma-centered mix ACh/GABA transmission. Physiological findings were corroborated using immunolabeling against VGAT and VAChT, which showed many closely spatially clustered ACh and GABA- specific cholinergic terminals and few truly colocalized VAChT and VGAT terminals. This result revealed that true co-transmission represents a minority of the presynaptic mode of release from cholinergic axons onto medial SN DA neurons, and that the majority actually share closely spatially clustered ACh and GABA-specific cholinergic terminals. To investigate the dynamic properties of soma-centered ACh/GABA transmission, we restricted our stimulation field to the cell body to measure the contribution of nAChR and GABAR-mediated conductances without recruiting the lateralized population of primary GABA inputs. We then employed a deconvolution method to understand the relative plasticity of contributions of nAChRs and GABARs to ACh/GABA transmission onto DA cells. We confirmed an initial dominant GABAergic component of ACh/GABA transmission that was previously reported. However, we found that the GABAergic contribution had a greater decay compared to the ACh component with repeated stimulations. As such the predominant initial inhibition is followed by a subsequent equalization of excitatory and inhibitory conductances. Finally, we performed similar experiments to compare the short-term plasticity of the isolated GABA conductance during 15 Hz stimulation between the populations of mix ACh/GABA inputs proximally and the population of primary GABA inputs found on the lateral dendrites 160 μm from the cell body. Interestingly, the lateral GABA component was more sustained across repeated stimulations compared to the proximal GABA conductance, suggesting a differential contribution to excitation/inhibition balance by spatially distributed populations of ACh and GABA inputs from cholinergic axons onto the dendrites of medial SN DA neurons. To our knowledge, this is the first study to examine the distribution and dynamics of ACh/GABA transmission onto midbrain DA system using fine-scale ChR2-assisted subcellular input mapping and conductance deconvolution. / Graduate / 2022-04-12

No description available.

La nicotine est considérée comme étant la « molécule » addictogène de la cigarette et du tabac. Mais différentes études cliniques, utilisant notamment des substituts nicotiniques, débouchent pratiquement toutes sur une même conclusion : efficacité ne dépassant que de peu celle d’un placebo, et très limitée dans le temps, contrastant avec le pouvoir hautement addictif du tabac, qu'il soit chiqué, prisé ou fumé. Dans ce travail de thèse, nous avons essayé de mettre en évidence le rôle que pourraient avoir certains des autres composés présents dans le tabac ou produits par pyrolyse. Nous avons d’abord utilisé des extraits aqueux de fumée et de tabac pour approcher un aspect global de ce que les fumeurs absorbent chaque fois qu’ils fument une cigarette, nous rapprochant ainsi des conditions physiologiques du fumeur. Puis nous avons choisi un certain nombre de substances. La cotinine, métabolite de la nicotine. L’harmane, une ß-carboline, synthétisée au cours de la combustion et dans l’organisme des fumeurs. La norharmane, une ß-carboline, présente en partie dans le tabac et synthétisée dans la fumée par pyrolyse. La technique utilisée tout au long de ce travail est l’enregistrement électrophysiologique. Cette technique s’applique très bien à l’étude in vivo de différents systèmes neuronaux y compris le système dopaminergique. Nous l’avons utilisée chez le rat, la souris WT et la souris Knockout ß2 (ß2KO). Nous nous sommes intéressés à deux aspects de l’activité cellulaire des neurones dopaminergiques de l’aire tegmentale ventrale : la fréquence de décharge (le firing) et les bouffées (bursts). En parallèle, nous avons conduit des expériences de liaison (binding) sur des cultures de cellules exprimant le récepteur nicotinique α4ß2. Nos résultats les plus significatifs ont montré que : Les bursts sont le plus souvent absents après les injections d’extraits de tabac et de fumée. Cela pourrait, entre autres, impliquer qu’il existe dans le tabac et la fumée des composés autres que la nicotine qui bloquent les effets de la nicotine sur les bursts. Les effets des extraits de tabac et de fumée sur le firing et les bursts ne sont plus présents chez les souris ß2 KO, ce qui implique que l’ensemble des composés du tabac agit essentiellement sur les récepteurs nicotiniques porteurs de la chaine ß2, même si des hypothèses alternatives existent. L’harmane a des effets activateurs très puissants sur le firing des neurones dopaminergiques, et ces effets sont bloqués à 80% par la mécamylamine, ce qui démontre qu’un des principaux composés du tabac et de la fumée autre que la nicotine agit par un mécanisme essentiellement nicotinique. Les expériences de binding confirment que les effets du tabac et de la fumée impliquent les récepteurs nicotiniques d’une façon majeure, mais d’une façon qui diffère légèrement de celle de la nicotine.Les résultats que nous avons obtenus montrent que les effets pharmacologiques du tabac ne se résument pas à ceux de la seule nicotine. Ils peuvent constituer un point de départ pour d’autres travaux, notamment pour étudier de plus près les effets des ß-carbolines. Il est nécessaire d’identifier les types de récepteurs sur lesquels elles se fixent, en utilisant des agonistes et antagonistes de récepteurs aux neurotransmetteurs contrôlant l’activité des neurones dopaminergiques. Des expériences sur des souris transgéniques chez lesquelles différents types de sous-unités de récepteurs nicotiniques ont été supprimés doivent également être envisagées, pour déterminer les mécanismes d’action des composants autres que la nicotine contenus dans le tabac et sa fumée sur les neurones dopaminergiques / Nicotine is generally considered as the sole tobacco addictive compound. However, nicotine replacement therapy studies almost all end with the same conclusion: the effectiveness of nicotine replacement is very limited on the short-term, and hardly exceeds that of placebo on the long-term. In addition, studies dealing with the effects of denicotinized cigarettes have provided evidence that these cigarettes have an addictive potential. In the present work, we tried to determine the behavioral role of some tobacco or smoke compounds other than nicotine at the neuronal level. We first compared the effects of nicotine with those of whole tobacco and smoke extracts, given that these preparations closer mimic the smoking situation than nicotine alone. We then examined the effects of a number of selected tobacco or smoke compounds. Cotinine, a major nicotine metabolite. Harmane and norharmane, two ß-carbolines synthesized in smoke as well as in the body of smokers. The technique used consists in the in vivo recording of the firing rate and bursts of dopamine neurons in the ventral tegmental area after intravenous injections of compounds in rats and mice. This electrophysiological technique is known to be a useful way to investigate the properties of selected compounds. In the case of mice, we used wild type and ß2 KO mice. We also made a series of in vitro experiments investigating the binding properties of the compounds on cells expressing high densities of α4ß2 nicotinic receptors. The main results of our studies are the following: Bursts are absent most of the times after the injection of the extracts. These results suggest that tobacco and smoke extracts contain compounds that inhibit the burst-promoting effects of nicotine. Increased firing is no longer present in ß2 KO mice treated with tobacco or smoke extracts, indicating that tobacco and smoke components, as a whole, primarily acts on nicotinic receptors that carry the ß2 chain, although alternative hypotheses may exist. Harmane very strongly activates the firing of dopaminergic neurons. Up to 80% of this effect is blocked by mecamylamine, demonstrating that that a major component of tobacco and smoke other than nicotine acts primarily through a nicotinic mechanism. The binding experiments confirm that the effects of tobacco and smoke involve nicotinic receptors in a major way, but in a way that slightly differs from that of nicotine. Our results may constitute a new starting point for further work, especially for a closer look at the effects of ß-carbolines. Attempts to identify the types of receptors involved in these effects are needed, using agonists and antagonists of neurotransmitter receptors that control the activity of dopamine neurons. Experiments on transgenic mice with deletion of different types of subunits of nicotinic receptors should also be made, to determine the different mechanisms of action of tobacco and smoke compounds other than nicotine on dopaminergic neurons

Parkinson’s disease (PD) is an incurable, chronically progressive neurodegenerative disease leading to premature invalidity and death. The locomotor disability of PD patients is mainly rooted in the gradual and insidious degeneration of dopaminergic neurons (DA) projecting from the midbrain substantia nigra (SN) to the basal ganglia striatum, a pathological process highlighted microscopically by the formation of insoluble cytosolic protein aggregates, known as Lewy bodies and Lewy neurites. The pathogenic mechanisms leading to PD remain poorly understood, arguably owing to the lack of suitable animal and cellular experimental models of the disease. Therefore, there is an urgent need for developing reliable experimental models that recapitulate the key features of PD. The recent development of induced pluripotent stem cell (iPSC) technology has enabled the generation of patient-specific iPSC and their use to model human diseases, although it is currently unclear whether this approach could be useful to successfully model age-related conditions. Importantly, disease modeling using iPSC largely relies on the existence of efficient protocols for the differentiation of disease-relevant cell types. Here, we first developed an efficient protocol for the differentiation of iPSC to authentic midbrain-specific DA neurons with SN properties by forced expression of LMX1A using a lentivirus-mediated gene delivery system. Next, we generated an iPSC-based cellular model of PD that recapitulates key phenotypic features of PD, such as DA neuron loss and α-synuclein accumulation in DA neurons from PD patients. Overall, our results demonstrate that we have developed a valuable tool for elucidating the pathogenic mechanisms leading to PD, as well as an experimental platform for screening new drugs that may prevent or rescue neurodegeneration in PD. / La malaltia de Parkinson (MP) és una malaltia neurodegenerativa incurable que causa invalidesa i mort prematura. Els pacients de la malaltia de Parkinson presenten alteracions motores degudes a una degeneració gradual de les neurones dopaminèrgiques que projecten des de la substància nigra fins a l’estriat. A nivell microscòpic s’observa la presència d’agregats proteics insolubles en el citosol de les neurones coneguts com cossos o neurites de Lewy. Els mecanismes patològics responsables de la MP no es coneixen bé, possiblement a causa de la manca de models animals i cel•lulars adequats. Per tant, existeix una gran necessitat de desenvolupar models experimentals fiables que recapitulin les característiques bàsiques de la MP. El recent desenvolupament de les cèl•lules mare pluripotents induïdes (iPSC) ha permès la generació de iPSC específiques de pacient i el seu ús per modelar malalties humanes, ara bé, no és clar si aquesta estratègia es pot utilitzar per modelar exitosament malalties d’origen tardà, com ara la MP. És important destacar que el modelatge de malalties utilitzant iPSC, es basa, en gran mesura en l'existència de protocols eficients per a la diferenciació de les iPSC cap al tipus cel•lular rellevant per a la malaltia. Durant aquest període, per primera vegada, s’ha desenvolupat un protocol per a l’eficient diferenciació de les iPSC cap a neurones dopaminèrgiques amb les propietats característiques de neurones dopaminèrgiques nigrostriatals, mitjançant l’expressió forçada de LMX1A utilitzant vectors lentivirals. A continuació, s’ha generat un model cel•lular usant iPSC derivades de pacients de MP que recapitula les principals característiques fenotípiques de la malaltia, com ara la pèrdua de neurones dopaminèrgiques i l'acumulació de α-sinucleïna en les neurones dopaminèrgiques. En general, els nostres resultats demostren que hem desenvolupat una eina valuosa per a l’estudi dels mecanismes patològics que condueixen a la MP, així com una nova plataforma pel descobriment de nous fàrmacs encaminats a prevenir o evitar la neurodegeneració.

Parkinson’s disease (PD) is a debilitating movement disorder. The cardinal symptoms of PD are bradykinesia, resting tremors and rigidity. PD is characterized by degeneration of dopaminergic neurons of A9 region, substantia nigra pars compacta (SNpc) and loss of dopaminergic terminals in striatum while the dopaminergic neurons of A10 region, ventral tegmental area (VTA) are relatively protected. Putative mechanisms, such as mitochondrial dysfunction, dysregulation of the ubiquitin proteasome system and increased oxidative stress have been hypothesized to mediate PD pathology. However, precise mechanisms that underlie selective vulnerability of SNpc dopaminergic neurons to degeneration are unknown. The aim of this thesis was to evaluate the pathological mechanisms that may contribute to degeneration of SNpc dopaminergic neurons in PD. Dopaminergic neurons of SNpc are pacemakers and constant calcium entry through L-type calcium channel, Cav1.3 has been reported in these neurons during pacemaking. In addition, these neurons have poor calcium buffering capacity. Together, this leads to dysregulation of calcium homeostasis in the SNpc dopaminergic neurons leading to increased oxidative stress. Gene expression of the full length channel and the variant was investigated in the mouse midbrain and further their presence was verified in mouse SNpc and VTA and also in SNpc and VTA in the MPTP mouse model of PD. Gene expression of Cav1.3 -42 and its variant was also studied in SNpc from autopsy tissue from PD patients and age matched controls. Having studied differential expression of the calcium channels, global changes in gene expression in SNpc from the MPTP mouse model of PD and PD autopsy tissues were next examined. This is the first report of transcriptome profile alterations from SNpc in mouse model and PD tissue performed using RNA-seq. Gene expression profiles were examined from SNpc 1 day post single exposure to MPTP, in which case there is no neuronal death and 14 days after daily MPTP treatment where SNpc has undergone ~50% cell death. Further, RNA- seq was performed to study gene expression alterations in SNpc from human PD patients and age- matched controls. The RNA-seq data was taken through extensive analyses; analysed for differential gene expression, gene-set enrichment analysis, pathway analysis and network analysis. Glutaredoxin 1 (Grx1) is a thiol disulfide oxidoreductase that catalyses the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis. Down-regulation of Grx1 has been established to exacerbate neurodegeneration through impairment of cell survival signalling. Previous work from our laboratory has demonstrated that perturbation of protein thiol redox homeostasis through diamide injection into SNpc leads to development of PD pathology and motor deficits. It was therefore investigated if Grx1 down-regulation in vivo, leading to increased glutathionylation and protein thiol oxidation, could result in PD pathology. This work is thus the first study of RNA-seq based transcriptomic profile alterations in SNpc from human PD patients. This work also highlights several differences between mouse model and human PD tissue indicating that the underlying mechanisms of PD pathogenesis differ from mouse to humans in addition to developing a novel model for PD.

<div><p>Parkinson’s disease (PD) is a devastating neurodegenerative disorder that affects 10 million people worldwide and is characterized by pronounced motor symptoms. Dementia with Lewy Bodies (DLB) involves both cognitive and motor deficits and affects ~1 million people in the United States. To date there is no cure for PD or DLB, and current treatments address only a subset of the symptoms that define these diseases. PD and DLB are ‘synucleinopathies’, defined as disorders involving the accumulation in patients’ brains of Lewy bodies. Lewy bodies are cellular inclusions that consist largely of aggregated species of alpha-synuclein (aSyn), a presynaptic protein that exists as both cytosolic and membrane-bound forms. Pathophysiological findings suggest that aggregated aSyn is involved in neurodegeneration in PD and DLB. However, mechanisms by which aSyn forms neurotoxic aggregates, and neurotoxic processes that distinguish different synucleinopathies such as PD and DLB, are poorly understood. To address these gaps, we have (i) designed a protocol to establish a primary cell culture model that can recapitulate key neuropathological features of PD, (ii) examined effects of expressing aSyn variants in a rat model of PD, and (iii) examined the expression profiles of neuroprotective genes in PD and DLB brain specimens.</p><p> </p><p>In the first part of my thesis, I describe the development of an optimized protocol to prepare primary midbrain and cortical cultures from rat embryonic brains for the study of PD and other synucleinopathies. The establishment of cellular models that simulate specific aspects of neuropathology can enable the characterization of molecular perturbations that lead to dopaminergic (DA) neuronal death. Our primary midbrain mixed culture model provides an outstanding opportunity to explore therapeutic strategies to rescue DA neurons from toxicity elicited by a range of PD-related insults. In addition, our primary cortical mixed cultures can be used to model cortical neuropathology in various CNS disorders including synucleinopathies.</p><p> </p><p>A number of mutations in the gene that codes for aSyn are associated with familial, early-onset forms of PD. A major goal of my thesis research is to characterize neurotoxic effects of a recently discovered familial substitution, A53E. This mutant was chosen based on the rationale that the introduction of a negatively charged residue at position 53 could potentially interfere with aSyn-membrane interactions and favor A53E aggregation, as we described for other familial aSyn mutants. For the first time, we have reproduced the neurotoxicity of A53E seen in human patients by expressing the mutant protein in rat midbrain. Rats injected unilaterally in the substantia nigra (SN) with rAAV encoding A53E and another familial mutant, A53T, but not rAAV encoding WT aSyn or a vector-control (‘stuffer’) virus, exhibited a significant motor impairment. Immunohistochemical analysis at 14 weeks after the viral injection revealed that brain sections from aSyn-expressing rats exhibit key features reminiscent of neuropathology in human PD, including nigral dopaminergic neuron loss (confirmed by unbiased stereology), striatal terminal depletion, and aSyn inclusion formation. In addition, it was determined that WT aSyn and the A53E and A53T mutants invaded the non-injected substantia nigra, implying that expressed aSyn protein can spread throughout the brain in the rat rAAV-aSyn model. These results yield insights into the molecular basis for the neurotoxicity of A53E and shed light on a potential role for membrane-induced aSyn aggregation in PD pathogenesis in vivo, thus setting the stage for developing therapies to slow neurodegeneration in the brains of familial and idiopathic PD patients. </p><p> </p><p>aSyn neurotoxicity varies with the expression of neuroprotective proteins, and misfolded aSyn affects cellular functions and gene expression. These observations suggest that differential gene expression patterns can inform us about similarities and differences in pathogenic mechanisms of different synucleinopathy disorders. A third phase of my thesis research was aimed at determining the expression levels of a panel of candidate neuroprotective genes in post-mortem brain samples from DLB and PD patients and age-matched controls (5 individuals in each group). mRNAs encoding the following proteins were quantified via qRT-PCR in homogenates prepared from the frontal cortex and the BA24 region encompassing the cingulate gyrus: DJ-1, a protein with antioxidant and chaperone activities; PGC1α, a master regulator of mitochondrial biogenesis and oxidative metabolism; MsrA, an antioxidant enzyme responsible for repairing oxidatively damaged proteins; and ATP13A2, a lysosomal protein involved in autophagy. In addition to yielding new insights into differential gene expression patterns in cortex versus cingulate gyrus, the data revealed differences in mRNA expression levels in DLB versus non-DLB cortical tissue. Although levels of all four neuroprotective mRNAs were increased (or showed a trend towards being increased) in DLB cortex, Western blot analysis revealed that only the DJ-1 and PGC1α proteins showed a trend towards being up-regulated, whereas levels of ATP13A2 and MsrA were unchanged. These findings suggest that there is a failure to induce cellular antioxidant responses and lysosomal autophagy at the protein level in DLB cortex, and in turn this failure could contribute to neuropathology. Interestingly, analysis of the same panel of neuroprotective genes in PD cortical samples did not show significant differences in mRNA or protein levels compared to control samples, suggesting that different neuroprotective mechanisms are induced in DLB versus PD cortex. These studies shed light on brain-region specific changes in gene expression associated with different synucleinopathy disorders, and they set the stage for developing new diagnostic tests and therapeutic strategies.</p></div><br>

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that most notably influence dopamine (DA) release. In this thesis, I examine the role of nAChRs in mediating DA-related behaviors such as movement and drug dependence. To accomplish this, I utilized a “gain-offunction” knock-in mouse (the Leu9’Ala line) containing agonist-hypersensitive α4* nAChRs (* indicates other nAChR subunits in addition to α4 are within the receptor complex) that renders receptors 50-fold more sensitive to nicotine and acetylcholine than wild-type (WT) receptors. I found that DHβE, a selective antagonist for α4β2* nAChRs, induced reversible and robust motor dysfunction characterized by hypolocomotion, akinesia, catalepsy, tremor, and clasping in Leu9’Ala but not WT mice. Reversal of the phenotype was achieved by targeting dopamine signaling. Blockade of mutant α4* nAChRs elicited activation of brain regions in the basal ganglia including dorsal striatum and substantia nigra pars reticulata indicated by c-Fos immunoreactivity. These data indicate that blocking α4* nAChRs in Leu9’Ala mice activates the indirect motor pathway resulting in a motor deficit. We also determined that α4* nAChRs involved in motor behaviors did not contain the α6 subunit, a nAChR subunit highly expressed in DAergic neurons suggesting that different nAChR subtypes modulating striatal DA release have separate functions in motor output. Conditioned place aversion and hypolocomotion, behaviors elicited during nicotine withdrawal, were also induced by DHβE in nicotine-naïve Leu9’Ala but not WT mice. Together these data suggest that DHβE globally reduces DA release in the CNS. In a separate project, I determined that α4* and α6* nAChRs modulate drug-induced hypnosis. Activation of nAChRs increased sensitivity to ketamine-induced hypnosis; whereas antagonizing nAChRs had the opposite effect. Additionally, α4 knockout (KO) mice were less sensitive to the hypnotic effects of ketamine, but α6 KO were more sensitive. High doses of ethanol induce an anesthesia-like state characterized by immobility, analgesia, and hypnosis. Testing the effects of ethanol hypnosis in α4 KO revealed that α4* nAChR do not play a large role in the acute effects of ethanol-induced hypnosis, but are involved in tolerance to this ethanol-induced behavior. The mechanisms of anesthetic-induced hypnosis are still largely unclear, despite the wide use of anesthesia. Future work on these receptors and their involvement in the anesthetic response will help to define a mechanism for hypnosis and improve the use of anesthetic drugs.

Les neurones dopaminergiques (DAergiques) de l’aire tegmentaire ventrale (ATV) et de la substance noire compacte (SNc) sont impliqués dans de nombreuses fonctions physiologiques telles que la motivation, la récompense, l’apprentissage ou encore le contrôle du mouvement volontaire. Ces neurones sont également connus pour être perturbés dans plusieurs grandes maladies du cerveau telles que la schizophrénie, les maladies associées aux drogues d’abus ou dans la maladie de Parkinson. Des études in vivo ont démontré que la structure des terminaisons axonales DAergiques pouvait être de type « synaptique » et « non-synaptique ». Ces terminaisons dites « non-synaptiques », dépourvues de toute apposition avec un domaine membranaire postsynaptique, semblent représenter la grande majorité des terminaisons axonales établies par les neurones DAergiques. De façon intéressante, certaines des terminaisons synaptiques ont quant à elles, la capacité de co-libérer du glutamate ou du GABA. D’une façon générale, la formation et le maintien des synapses fait intervenir des protéines d’adhésion cellulaire dont les plus courantes sont les neurexines (Nrxn) et les neuroligines (Nlgn). Au niveau présynaptique, ces molécules d’adhésion interagissent avec des protéines de la zone active qui sont impliquées dans la régulation de l’exocytose. Parmi elles, on retrouve RIM1/2, Piccolo/Bassoon, ELKS ou encore Munc-13. Du côté postsynaptique, ces protéines d’adhésion cellulaire interagissent directement avec les protéines d’échafaudages telles que PSD95 ou Géphyrine. Mes travaux de doctorat ont consisté dans un premier temps à caractériser de façon exhaustive les terminaisons axonales établies par les neurones DAergiques. La proportion et la structure moléculaire des terminaisons synaptiques et non-synaptiques ont ainsi été évaluées. Dans un premier article, via l’utilisation d’un système in vitro, nous avons démontré qu’une minorité des terminaisons DAergiques (20%) était de nature synaptique, une proportion totalement différente lorsque comparée avec les neurones glutamatergique ou GABAergiques, dont les terminaisons synaptiques sont très fortement majoritaires (80%). De façon intéressante, la protéine de ZA Bassoon a été retrouvée majoritairement au sein des terminaisons synaptiques suggérant une différence de structure avec les terminaisons non-synaptiques. Finalement, au niveau du mécanisme de formation des synapses, nous avons mis en évidence que la surexpression de la protéine présynaptique Nrxn-1SS4- dans les neurones DAergiques permet d’augmenter la proportion de terminaisons synaptiques alors que la surexpression de la Nlgn-1AB est, quant à elle, capable d’induire une différentiation présynaptique DAergique. Dans un second article, nous avons voulu investiguer plus en détails le rôle des Nrxn dans la synaptogénèse DAergique. Pour ce faire, nous avons pris avantage d’un modèle animal totalement inédit mais absolument fascinant où nous avons évalué l’impact d’une délétion conditionnelle de l’ensemble des Nrxn sur la connectivité DAergique. Dans cette étude nous avons démontré que la densité de synapses excitatrices et inhibitrices établies par les neurones DAergiques n’était pas affectée chez les souris DAT::NrxnsKO et ce en comparaison des animaux de souche sauvage. Dans un deuxième temps, via des enregistrements électrophysiologiques, nous avons évalué la neurotransmission excitatrice et inhibitrice établie par les neurones DAergiques. Les résultats n’ont pas révélé de changement dans la neurotransmission excitatrice mais ont curieusement révélé un renforcement de l’activité synaptique inhibitrice chez les animaux Nrxn DAT::NrxnsKO et ce par rapport aux animaux du groupe contrôle. Finalement, via la mise en place de tests comportementaux, nous avons pu observer que les animaux DAT::NrxnsKO avaient une capacité d’apprentissage et de locomotion identique aux animaux de souche sauvage, cependant, une stimulation pharmacologique du système DAergique par l’amphétamine a révélé d’une diminution significative de la locomotion chez les souris DAT::NrxnsKO, pouvant refléter une baisse de la neurotransmission DAergique en condition non physiologique. Ces travaux de doctorat amènent pour la première fois une nouvelle vision sur la capacité des neurones DAergiques à établir une arborisation axonale majoritairement non-synaptique. Cette thèse démontre que les neurones DA du mésencéphale ont un programme intrinsèque de développement de leur arborisation axonale qui est différent des neurones GABAergiques du striatum et glutamatergiques du cortex. Aussi, au travers de ces travaux, nous montrons clairement que des protéines aussi fondamentales que les Nrxn et les Nlgn ont un impact limité dans la formation, le maintien et le fonctionnement des synapses établies par les neurones DAergiques. / Dopamine (DA) neurons from the substantia nigra compacta (SNc) and ventral tegmental area (VTA) are key players of the neuronal circuitry regulating movement initiation, reward and learning. Their functioning and survival are also perturbed in diseases such as schizophrenia, drug abuse and Parkinson’s. The axonal connectivity of DA neurons is particularly intriguing due to the hyperdense nature of the axonal arbor of these neurons, containing a very large number of neurotransmitter release sites. Ultrastructural examination of the axon terminals established by DA neurons failed to identify a tight pre- and postsynaptic coupling at most of release sites, giving rise to the concept of non-synaptic terminals and “diffuse” or volume transmission. Furthermore, it is now well established that a subset of terminals established by DA terminals has the capacity to release other neurotransmitters such a glutamate and GABA. A large literature implicates trans-synaptic proteins including neurexins (Nrxns) and neuroligins (Nlgns) in the development of synaptic contacts. In the presynaptic compartment, these cell adhesion molecules interact with active zone proteins like RIM1/2, Bassoon, ELKS and Munc-13 involved in regulating exocytosis. In the postsynaptic compartment, these cell adhesion molecules closely interact with scaffolding proteins like PSD95 or Gephyrin. In this thesis work, we first performed an exhaustive characterization of axon terminals established by DA neurons in primary co-culture system. We evaluated the proportion and the molecular structure of synaptic and non-synaptic terminals established by VTA and SNc DA neurons. In our first article, using and efficient in vitro system, we demonstrated that DA neurons develop a small proportion of synaptic terminals that is strikingly lower compared to glutamatergic and GABAergic neurons. Interestingly, we discovered that the active zone protein Bassoon is mainly expressed in DA terminals that are in contact or in close proximity to a target cell, and less expressed in non-synaptic DA terminals. Finally, we found that overexpression of Nrxn-1SS4- in DA neurons leads to an increase in the proportion of synapses whereas, overexpression of Nlgn-1A+B is able to trigger a DA presynaptic differentiation of DA neurons, suggesting a key role for these transsynaptic proteins in synapse formation by DA neurons. In a second article, we studied more globally the role of Nrxns proteins in the formation of synapses by DA neurons. We took advantage of a recently introduced triple conditional Nrxn mouse line to selectively delete Nrxns in DA neurons and examine the impact of this gene inactivation on the connectivity of DA neurons. In this part we demonstrated that the density of excitatory and inhibitory synapses density established by DA neurons is not affected by the deletion of all Nrxns, in comparison to the wild type group and does not impair the basic development and axonal connectivity of DA neurons at least, in vitro. In a second set of experiments, using patch-clamp recordings, we evaluated the function of excitatory and inhibitory synapses established by DA neurons in the striatum. GABA and glutamate synaptic currents evoked in the striatal medium spiny neurons by optogenetic stimulation of DA neuron axons revealed that glutamate release was unchanged, but identified a strong tendency for enhanced GABA co-release. Furthermore, using fast scan cyclic voltammetry, we found that the loss of Nrxns was associated with impaired DA transmission in the brain of adult mice, revealed by a reduced rate of DA reuptake after electrically-evoked DA release and with impaired amphetamine-induced locomotion. With this thesis, we bring a new perspective on the capacity of DA neurons to develop an axonal arborization that is mainly non-synaptic. With this work, we provide strong evidence arguing that mesencephalic DA neurons are endowed with an intrinsic developmental program leading them to develop an axonal connectivity that is very different from striatal GABA neurons or cortical glutamate neurons. Ours findings suggest that although Nrxns and Nlgns are unlikely to be the main determinants of on the formation of synapses by DA neurons, that are likely to act as key regulators of DA and GABA signaling by these.