Le striatum est composé principalement de neurones épineux de taille moyenne, subdivisés en neurones striatopallidaux et striatonigraux en fonction de leurs projections et de leur expression en récepteurs et neuropeptides. Ces deux populations neuronales sont respectivement à l’origine des voies indirecte (ou inhibitrice) et directe (ou activatrice) des noyaux de la base, présentant des effets opposés à la fois au niveau moteur et motivationnel. Ces deux voies sont également différemment affectées dans différentes pathologies des noyaux de la base, telles que les maladies de Huntington et de Parkinson et les addictions. Les mécanismes moléculaires et cellulaires de régulation des neurones STP et STN ne sont cependant pas encore pleinement compris. Dès lors, l’identification et l’étude de la fonction de gènes spécifiques de l’une ou l’autre de ces sous-populations pourraient constituer une étape importante vers une meilleure compréhension de leur fonctionnement. Dans cette optique, notre laboratoire a précédemment réalisé une étude comparative des profils d’expression de chacune des sous-populations striatales par microarray. Parmi les gènes potentiellement inégalement exprimés dans les neurones STP et STN, nous avons identifié GPRIN3, un membre de la famille G Protein-Regulated Inducer of Neurite outgrowth (GPRIN), comme étant une cible intéressante. Cette famille, bien qu’encore très peu caractérisée, interagit en effet avec les sous-unités Gαi/o des protéines G et joue par conséquent un rôle régulateur sur la fonction et la voie de signalisation de certains GPCRs, tels que le récepteur μ opioïde. De plus, contrairement aux autres membres de la famille GPRIN, nos résultats de microarray suggèrent également un niveau d’expression élevé de GPRIN3 dans les neurones striataux chez l’adulte. Etant donné le rôle crucial des GPCRs au niveau du striatum et plus particulièrement dans le comportement différentiel des neurones STP et STN, GPRIN3 pourrait dès lors constituer un élément important dans le fonctionnement des neurones striataux. Ce travail s'est par conséquent axé sur l’élucidation du rôle de GPRIN3 dans les fonctions striatales. Dans ce but, nous avons dans un premier temps établi le profil d'expression de GPRIN3 chez la souris, au niveau du cerveau adulte et lors de l'embryogénèse. Ceci nous a permis de confirmer, chez l'adulte, l'expression majoritairement striatale de GPRIN3, et l'expression préférentielle dans les neurones STP. Nous avons également généré différents vecteurs d'expression de la protéine GPRIN3 et établi sa localisation subcellulaire en lignée HEK293T. La génération et la caractérisation d'un modèle d'invalidation constitutive ainsi que d'un modèle de répression par interférence ARN ont par la suite mis en évidence une implication, directe ou indirecte, de GPRIN3 dans la régulation fine de la signalisation du D2R. En effet, nous avons montré une modification des comportements liés à la motivation et à la réponse à la cocaïne ainsi qu’une altération de l’état de phosphorylation de DARPP32 et de la réponse à l’halopéridol dans le modèle d’invalidation constitutive. De plus, la réponse au quinpirole est également modifiée dans les deux modèles testés. Pris dans leur ensemble, ces résultats suggèrent dès lors une altération de la voie de signalisation du D2R en l’absence de GPRIN3 fonctionnel. En outre, les neurones STP dépourvus de GPRIN3 fonctionnel présentent des modifications de leur morphologie et de leurs propriétés électrophysiologiques. En conclusion, ce travail de thèse a permis d’apporter les premières pistes quant à la fonction de GPRIN3, une protéine totalement méconnue, dans le striatum, de par la création de modèles d’invalidation constitutive et de répression Cre-dépendante de cette protéine. Divers outils moléculaires ont également été générés et pourront être utilisés dans la suite de la caractérisation des fonctions de GPRIN3. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Les événements vécus peuvent laisser une trace durable au niveau des réseaux cérébraux. Ces réseaux sont constitués de neurones connectés par des synapses, dont l'efficacité de transmission est régulée sur le plan fonctionnel et structural. Les drogues d'abus détournent les circuits neuronaux impliqués dans l'apprentissage régulé par la récompense, induisant une plasticité des neurones striataux de projection (SPN) du noyau Accumbens (NAc), notamment via l'activation de la voie de signalisation Extracellular Regulated Kinase (ERK) et l'augmentation de la densité en épines dendritiques -qui sont les protrusions portant l'élément post-synaptique glutamatergique-. L'objectif de ma thèse était d'étudier l'impact de l'exposition répétée ou unique à la cocaïne sur le mode formation des synapses des SPN du NAc et d'élucider les rôles précis de la voie ERK dans ce phénomène. J'ai pu montrer qu'une ou plusieurs injections de cocaïne chez la souris induisaient la formation de synapses glutamatergiques persistantes au sein des SPN in vivo. Par des expériences d'imagerie en temps-réel sur tranches striatales, j'ai dissocié les phases de pousse et de stabilisation de nouvelles épines dendritiques. J'ai pu mettre en évidence que la voie ERK joue un rôle prépondérant dans ces deux phases via des processus moléculaires distincts. Ainsi, la phase de pousse des épines est directement régulée par ERK, tandis que le maintien est régulé par MNK-1, une kinase cytoplasmique en aval de ERK, et par la synthèse protéique. Ce travail apporte des données nouvelles sur le mode de formation de ces synapses et les mécanismes moléculaires associés. / Brief life occurrences can leave durable changes at the level of neuronal networks. These networks consist of neurons connected by synapses, which transmission efficacy is regulated at the functional and structural levels. Drugs of abuse highjack neuronal circuits involved in reward-driven learning by activating the Extracellular Regulated Kinase (ERK) pathway and induce an increase in the dendritic spines density –protrusions which host the glutamatergic pre-synaptic element- of SPN. The goal of my thesis work was to study the consequences of acute and chronic cocaine exposures on the mode of synapse formation in SPN from the NAc and to decipher the precise roles of ERK pathway in this phenomenon. I demonstrated that acute and chronic cocaine treatments induced the formation of persisting glutamatergic synapses in SPN in vivo. Time-lapse imaging using two-photon microscopy in acute striatal slices allowed me to dissociate the phases of growth and stabilization of the new dendritic spines. I could indeed demonstrate a key role for ERK in those two phases, although through distinct molecular mechanisms. Firstly, the growth phase is dependent on ERK. Secondly, the stabilization of newly grown spines is controlled by MNK-1, a cytosolic kinase downstream ERK, and by protein synthesis. This work brings new results on the mode of synapse formation as well as on the associated molecular mechanisms.

Le striatum est une région du cerveau impliquée dans d'importantes fonctions physiologiques telles que l'apprentissage par renforcement ou le contrôle du mouvement, mais aussi dans des pathologies comme l'addiction. Le fonctionnement de ce système s'appuie sur deux types de neurones de projection, appelés " neurones épineux de taille moyenne ". Les uns expriment le récepteur de la dopamine de type 1 (D1R) et les autres expriment le récepteur de type 2 (D2R). L'objectif de cette thèse est de caractériser ces deux types de neurones au niveau épigénétique, en conditions basales et après traitement à la cocaïne. Il a été nécessaire de développer de nouvelles méthodes utilisant la cytométrie de flux pour distinguer les populations de neurones exprimant D1R ou D2R. La première méthode utilise des souris transgéniques L10a-GFP et du tissu non fixé, la seconde répond aux limitations de la précédente et utilise du tissu fixé. Nous avons montré que la cocaïne régule de nombreuses modifications post-traductionnelles d'histones, de façon spécifique de populations neuronales. Par ailleurs, nous avons identifié plus d'une centaine de gènes différemment méthylés ou hydroxyméthylés entre les deux types neuronaux. Certains gènes sont déjà connus pour avoir un rôle fonctionnel important dans l'une des populations. La comparaison des neurones exprimant D1R ou D2R est un bon modèle pour explorer les liens entre méthylation de l'ADN, hydroxyméthylation et transcription. Par exemple, nous observons une association très claire entre l'augmentation de la méthylation de l'ADN et la répression de la transcription, ainsi qu'une corrélation entre modifications de méthylation et d'hydroxyméthylation. / The striatum is a brain region implicated in physiological functions such as reinforcement learning or movement selection but also in pathologies such as addiction or Parkinson’s disease. It relies on two types of projecting neurons, named “medium spiny neurons” because of their morphology. They are very similar but have a complementary and opposite role. One type expresses the dopamine receptor type 1 (D1R) and the other type expresses the dopamine receptor type 2 (D2R). The aim of this work was to characterize this two neuronal types epigenetically, in basal conditions and after cocaine treatment. We have developed new flow cytometry techniques to be able to distinguish the two cell types. The first method uses transgenic L10-eGFP mice and fresh tissue, the second one goes beyond the limitations of the first one and uses fixed tissue. We have shown that cocaine regulates many post-translational histone modifications, dynamically, and differently between the two populations. Moreover, we have identified more than 100 genes differentially methylated or hydroxymethylated between the two neuronal types. Some of these genes are already known for having a functional role in one of the populations. The comparison between D1R and D2R neurons is a good model to explore the links between DNA methylation, hydroxymethylation and transcription. For example, we have observed a strong association between an increase in DNA methylation and a transcriptional repression, as well as a correlation between DNA methylation and hydroxymethylation.

Numerous social and educational psychologists propose that intrinsic motivation generated by personal interests and spontaneous satisfactions is qualitatively different from extrinsic types of motivation generated by external compensations and also that intrinsic motivation is more beneficial to learning than extrinsic types of motivation. However, in the field of neuroscience, intrinsic motivation has been little studied while extrinsic types of motivation (e.g., incentive motivation) have been thoroughly studied. The purpose of the present studies was to expand the neural understanding of motivation to include intrinsic motivational processes. To do so, a series of three event-related functional magnetic resonance imaging (fMRI) studies were conducted. Study 1 and Study 2 compared the neural activities when participants decided to act for intrinsic reasons (i.e., self-determined volitional and agentic behavior) versus when they decided to act for extrinsic reasons (i.e., non-self-determined volitional and agentic behavior). Both studies showed that the anterior insular cortex, known to be related to a sense of agency, was more activated during self-determined behavior associated with intrinsic reasons for acting while the posterior parietal regions (e.g., posterior cingulate cortex, angular gyrus), known to be related to a sense of a loss of agency, were more activated during non-self-determined behavior associated with extrinsic reasons for acting. These findings confirm the existence of neural-based intrinsic motivational processes, differentiate intrinsic motivation from incentive motivation, and document the important neural activities which function for generating self-determined agentic action. Study 3 examined these same neural activities as participants engaged in interesting and uninteresting versions of two experimental tasks. Results confirmed the results of the earlier two studies, as the anterior insular cortex was more recruited when participants performed the interesting, but not the uninteresting, version of the tasks. Results also extended the findings from Studies 1 and 2 in an important way in that the ventral striatum, a well-known brain region for reward processing, was more activated when participants performed the interesting, but not the uninteresting, version of the experimental tasks. These findings suggest that intrinsic motivation is generated based on the feeling of intrinsic need satisfaction (from anterior insular cortex activations) and the feeling of reward (from ventral striatum activations). Overall, the present research established three new findings: (1) the neural bases of intrinsic motivation lies largely in increased anterior insular cortical activities; (2) when people made decisions about self-determined intrinsically-motivated behavior, they show enhanced insular cortical activities and suppressed posterior parietal cortical activities; and (3) when people engaged in actual self-determined intrinsically-motivated behavior, they show enhanced insular cortical and ventral striatal activities. In establishing these new findings, the paper introduces a new area of study for motivational neuroscience--namely, intrinsic motivation.

Clustering of single-cell RNA sequencing data is often used to show what states and subtypes cells have. Using this technique, striatal cells were clustered into subtypes using different clustering algorithms. Previously known subtypes were confirmed and new subtypes were found. One of them is a third medium spiny neuron subtype. Using the observed heterogeneity, as a second task, this project questions whether or not differences in individual neurons have an impact on the network dynamics. By clustering spiking activity from a neural network model, inconclusive results were found. Both algorithms indicating low heterogeneity, but by altering the quantity of a subtype between a low and high number, and clustering the network activity in each case, results indicate that there is an increase in the heterogeneity. This project shows a list of potential striatal subtypes and gives reasons to keep giving attention to biologically observed heterogeneity.

Long-term postnatal treatment of rats with SCH 23390 is associated with a reduction in the development of dopamine D1 receptors in the striatum. Because the tripeptide, l-prolyl-l-leucylglycinamide (PLG) attenuates the neuroleptic-induced increase in D2 receptors in the striatum in adult rats, this study was undertaken with the objective of determining whether PLG could modulate a developmental alteration in the D1 subtype of receptor. Rats were treated with the dopamine D1 receptor antagonist, SCH 23390 (R[+]-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1-H-3benzazepine) (0.30 mg/kg/d i.p.) for 32 successive days from birth, while D1 receptors in the striatum were assessed at 5 and 8 weeks from birth. Postnatal treatment with SCH 23390 reduced in vitro binding of [3H]SCH 23390 to homogenates in the striatum by 70% at 8 weeks. Scatchard analysis at 5 weeks determined that the Bmax for the binding of [3H]SCH 23390 was reduced by 78%, while the Kd was unaltered. When PLG (1.0 mg/kg/d i.p.) was administered together with SCH 23390 for the initial 32 days from birth, the binding of [3H]SCH 23390 to homogenates of the striatum was unchanged from that of the control group at 8 weeks. Also, at 5 weeks the Bmax and Kd were unaltered from control in the group that was treated with both SCH 23390 and PLG. The binding of [3H]SCH 23390 was not altered from control in the group treated with PLG alone. Also, PLG given in vitro did not alter the binding of [3H]SCH 23390 to control homogenates of the striatum. These findings indicate that PLG is able to attenuate neuroleptic-induced alterations in dopamine d1 receptors in the striatum.

The effect of chronic postnatal treatment of rats with selective D1- and/or D2-receptor antagonists on the development of D1- and D2-receptors in the striatum was studied. When neonatal rats were treated postnatally from the day of birth for 32 successive days with the D1-receptor antagonist, SCH-23390 (0.30 mg/kg i.p.), the development of striatal dopamine D1-receptors was markedly impaired, and the development of striatal D2-receptors was slightly impaired. Alternatively, chronic treatment with the D2-receptor antagonist, spiroperidol (1.0 mg/kg i.p.), resulted in a markedly impaired development of striatal dopamine D2-receptors, and a slightly impaired development of striatal D1-receptors. Scatchard analysis revealed that chronic SCH-23390 treatment during development resulted in a 78% decrease in the Bmax for in vitro binding of [3H]SCH-23390 to striatal homogenates, while the Kd was unaltered. Similarly, chronic postnatal treatment with spiroperidol was associated with a 74% reduction in the Bmax, while the Kd for in vitro binding of [3H]spiroperidol to striatal homogenates was unchanged. These findings demonstrate that chronic selective dopamine receptor antagonism affects development of both striatal D1- and D2-receptor types. The critical period during which striatal dopamine receptor ontogeny can be altered is not restricted to prenatal periods, since suitable postnatal challenge will alter striatal dopamine-receptor development.

Following a cortical injury, neurons in areas near and connected to the site of injury begin to degenerate. The observed neuronal death may contribute to the severity of the observed behavioral impairments. The purpose of the present study was to examine if progesterone, a hormone known for its effectiveness at reducing cerebral edema, could protect against secondary neuronal death and facilitate the acquisition of an avoidance learning task in an ablation model of cortical injury. Rats served as sham controls or received bilateral ablation of the medial prefrontal cortex followed by a 10-day regimen of progesterone (4 mg/kg) or oil vehicle (1 ml/kg) beginning 1 h after cortical lesions. Progesterone-treated lesion rats showed a significant facilitation of avoidance learning compared to oil- treated lesion controls. In addition, progesterone-treated lesion animals did not differ from either progesterone- or oil-treated sham controls in avoidance learning. Anatomical analysis revealed that progesterone treatment decreased the amount of neuronal death seen in the striatum and the mediodorsal nucleus of the thalamus. The findings are consistent with the notion that progesterone is an effective neuroprotective agent and suggest that the hormone can reduce the behavioral impairments associated with frontal cortical ablation injury.

Long-term postnatal treatment of rats with the dopamine D2 receptor antagonist, spiroperidol, results in the impaired development of striatal D2 receptors. Because the tripeptide prolyl-leucyl-glycinamide (MIF-1) attenuates haloperidol-induced up-regulation of striatal dopamine D2 receptors in adult rats, we studied the effect of MIF-1 on the spiroperidol-induced alteration of striatal D2 ontogeny. Postnatal treatment of rats with spiroperidol (1.0 mg/kg/day, IP, ×32 days from birth) resulted in a 74% decrease in the Bmax for [3H]spiroperidol binding with no change in the Kd at 5 weeks. When rats were studied at 8 weeks, in the absence of additional treatment, total specific [3H]spiroperidol binding was reduced by 59%. While MIF-1 alone (1.0 mg/kg/day, IP, ×32 days from birth) had no effect on [3H]spiroperidol binding, MIF-1 completely attenuated the ontogenic impairment of striatal D2 receptors that was produced by spiroperidol treatment. At 5 weeks the Bmax for [3H]spiroperidol binding was at the saline control level in the group of rats cotreated with spiroperidol and MIF-1. At 8 weeks, with no additional treatments, the specific binding of [3H]spiroperidol to striatum was also at control levels in the group cotreated with spiroperidol and MIF-1. These findings demonstrate that MIF-1 attenuates spiroperidol-induced impairment of development of striatal dopamine D2 receptors in rats.

The effect of chronic treatment with vitamin E (VE) on acute l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity, as assessed by striatal dopamine (DA) depletion, was studied. Male C57B1/6J mice were fed VE (48 mg kg-1 per day, intragastric) for 4, 8, or 12 weeks prior to administration of MPTP (20 mg kg-1, i.p. X 3, 2 h intervals) or its diluent. Brain VE concentration was increased by exogenous supplements for 12 weeks. Striatal DA content was reduced by 85% to 90% after MPTP in control and VE-treated mice. Mice with elevated cerebral VE were not protected from MPTP toxicity, with DA content as an indicator. In conclusion, these findings indicate that moderate elevation of brain VE is not adequate for protecting DA-containing neurons against the toxic actions of a high dose of MPTP.