Psychedelics are a subset of hallucinogenic drugs that exert their characteristic effects through agonist activity at the serotonin receptor 2A (5-HT2A). In this study, I aimed to characterize the modulatory role of the metabotropic glutamate subtype 2 receptor (mGluR2) in the 5-HT2A-specific rodent model of hallucinogenic action, head-twitch response (HTR). Secondly, I aimed to explore if 5-HT2A agonist-induced deficits in prepulse inhibition (PPI) of the startle response, an additional model of hallucinogenic action, could be produced in mice. Though 5-HT2A agonist-induced PPI deficits, which represent interruptions in normal sensorimotor gating, have been described in both rats and humans, attempts to translate this behavior to mice are rare. In contrast to prior gene knockout studies suggesting the mGluR2 is necessary for 5-HT2A agonist-induced HTR, mGluR2 knockout (Grm2-/-) mice still displayed HTR upon administration of the psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI). Additionally, DOI and lysergic acid diethylamide (LSD) produced unexpected improvements in PPI in male 126S6/Sv wild-type mice, depending on the experimental protocol used and the origin of the animals. Sex differences were observed as DOI-induced improvements in PPI were present in female 129S6/Sv mice of the same origin and tested with the same protocol as their male counterparts; this effect in females was absent in 5-HT2A knockout (Htr2a-/-) mice. The results of this study shed light on issues with replicability and reproducibility in science, the importance of highlighting the origin and background of animal subjects, and potential sex differences in hallucinogenic drug action.

Les récepteurs opioïde delta (DORs) sont des récepteurs couplés aux protéines G et sont fortement exprimés au niveau du bulbe olfactif, du cortex, du striatum, du noyau basolateral de l'amygdala et des noyaux du pons (Mansour et al., 1995; Le Merrer et al., 2009). Les souris mutantes de première génération (souris knockout, délétion totale du gène) ont déjà permis de démontrer que DOR joue un rôle critique dans le contrôle de la douleur chronique (Gavériaux-Ruff et al., 2011), la régulation de l’activité motrice et des réponses émotionnelles (Filliol et al ., 2000) et l’association drogue-contexte (Le Merrer et al., 2011). Le but de notre étude est d’identifier les circuits neuronaux dans lesquels les DORs contrôlent les processus émotionnels et cognitifs. Nous avons développé une lignée de souris de deuxième génération, dans laquelle les récepteurs sont supprimés spécifiquement dans les neurones GABAergiques du cerveau antérieur. Nous avons ensuite étudié le rôle des DORs exprimés par ces neurones dans les réponses émotionnelles, locomotrices et la sensibilité aux crises épileptiques. / Delta opioid receptors (DORs) are G-protein coupled receptors belonging to the opioid system, which play a central role in chronic pain and emotional responses. DORs are strongly expressed in olfactory bulb, cortex, striatum, basolateral nucleus of the amygdala and pons nuclei. Using constitutive gene knockout, we have previously demonstrated the role of DORs in reducingchronic pain (Gaveriaux-Ruff, Nozaki et al. 2011), anxiety-related behaviors and impulsivity(Olmstead,Ouagazzal et al. 2009), regulating locomotor activity (Filliol, Ghozland et al. 2000) and facilitating context learning (Le Merrer, Faget et al. 2012; Le Merrer, Rezai et al. 2013), Although these functions are well-established, neuronal networks and mechanisms underlying DOR-regulated behaviors remain poorly understood. The aim of this thesis work was to identify neuronal populations and brain circuits that support DOR functions. Recent evidence showed that DOR is highly expressed in GABAergic neurons (Scherrer et al.. 2006;Erbs et al., 2012; Rezai et al.. 2012). We therefore developed a conditional knockout mouse line (Dlx-DOR)by breeding floxed DOR gene (Oprd1) with a transgenic Dlx-5/6-Cre mouse line (Monorv et al., 2006) in order to produce a specific deletion of DOR in GABAergic neurons of the forebrain. We first determined brain distribution of delta receptors in Dlx-DOR at mRNA and protein levels. Then, behavioral analysis were performed to assess whether DORs expressed in forebrain GABAergic neurons contribute to the regulation of emotional contrai, locomotor activity as well as epileptogenic effect of SNC80, the prototypal DOR agonist. Finally, we initiated a project focused on DORs detected at the level of BLA.

<p>Circulating immune complexes play an important role in the modulation of antibody responses and in the pathogenesis of immune diseases. This thesis deals with the <i>in vivo </i>regulatory properties of antibodies and their specific Fc receptors.</p><p>The immunosuppressive function of IgG is used clinically, to prevent rhesus-negative women from becoming sensitized to rhesus-positive erythrocytes from the fetus. The mechanism behind this regulation is poorly understood but involvement of a receptor for IgG, FcγRII, has been suggested. It is shown in this thesis that IgG and also IgE induce immunosuppression against sheep erythrocytes to a similar extent both in mice lacking all the known Fc receptors as in wild-type animals. These findings imply that antibody-mediated suppression of humoral responses against particulate antigens is Fc-independent and that the major operating mechanism is masking of epitopes.</p><p>Immunization with soluble antigens in complex with specific IgG leads to an augmentation of antibody production. The cellular mechanism behind this control is examined here and it is found that the capture of IgG2a immune complexes by a bone marrow-derived cell expressing FcγRI (and FcγRIII) is essential. An analysis of the ability of IgG3 to mediate this regulation indicated that, in contrast, this subclass of IgG augments antibody responses independently of FcγRI (and FcγRIII). These findings suggest that distinct mechanisms mediate the enhancing effect of different subclasses of antibodies.</p><p>Finally, the contribution of FcγRIII was studied in the development of collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis in humans. It was discovered that while DBA/1 wild-type control mice frequently developed severe CIA, with high incidence, FcγRIII-deficient mice were almost completely protected, indicating a crucial role for FcγRIII in CIA.</p><p>The results presented here help to understand how immune complexes regulate immune responses <i>in vivo</i> and show that Fc receptors for IgG, if involved, could be new targets for the treatment of immune complex-related disorders.</p>

<p>Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. </p><p>In order to study the BMP role for catecholaminergic neurons <i>in vivo</i>, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. </p><p>Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia.</p><p>The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor.</p><p>Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. </p><p>These results demonstrate a positive role of BMPs for MDN fate<i> in vivo</i>. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.</p>

Circulating immune complexes play an important role in the modulation of antibody responses and in the pathogenesis of immune diseases. This thesis deals with the in vivo regulatory properties of antibodies and their specific Fc receptors. The immunosuppressive function of IgG is used clinically, to prevent rhesus-negative women from becoming sensitized to rhesus-positive erythrocytes from the fetus. The mechanism behind this regulation is poorly understood but involvement of a receptor for IgG, FcγRII, has been suggested. It is shown in this thesis that IgG and also IgE induce immunosuppression against sheep erythrocytes to a similar extent both in mice lacking all the known Fc receptors as in wild-type animals. These findings imply that antibody-mediated suppression of humoral responses against particulate antigens is Fc-independent and that the major operating mechanism is masking of epitopes. Immunization with soluble antigens in complex with specific IgG leads to an augmentation of antibody production. The cellular mechanism behind this control is examined here and it is found that the capture of IgG2a immune complexes by a bone marrow-derived cell expressing FcγRI (and FcγRIII) is essential. An analysis of the ability of IgG3 to mediate this regulation indicated that, in contrast, this subclass of IgG augments antibody responses independently of FcγRI (and FcγRIII). These findings suggest that distinct mechanisms mediate the enhancing effect of different subclasses of antibodies. Finally, the contribution of FcγRIII was studied in the development of collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis in humans. It was discovered that while DBA/1 wild-type control mice frequently developed severe CIA, with high incidence, FcγRIII-deficient mice were almost completely protected, indicating a crucial role for FcγRIII in CIA. The results presented here help to understand how immune complexes regulate immune responses in vivo and show that Fc receptors for IgG, if involved, could be new targets for the treatment of immune complex-related disorders.

Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. In order to study the BMP role for catecholaminergic neurons in vivo, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia. The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor. Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. These results demonstrate a positive role of BMPs for MDN fate in vivo. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.

Cannabinoids have been shown to alter synaptic plasticity but the mechanism by which this occurs at hippocampal CA3-CA1 synapses in vivo is not yet known. Utilizing in vivo electrophysiological recordings of field excitatory postsynaptic potentials (fEPSP) on anesthetized rats and mice as well as three lines of conditional knockout mouse models, the objective was to show a two-part mechanistic breakdown of cannabinoid-evoked CA3-CA1 long-term depression (LTD) in its induction as well as early and later-phase expression stages. It was determined that this cannabinoid-induced in vivo LTD requires cannabinoid type-1 receptors (CB1Rs) on astrocytes, but not CB1Rs on glutamatergic or GABAergic neuronal axons/terminals. Pharmacological testing determined that cannabinoid-induced in vivo LTD also requires activation of NMDA receptors (NMDAR) and subsequent postsynaptic endocytosis of AMPA receptors (AMPAR). There exists a clear role for NR2B-containing NMDARs in a persistent, transitory form, potentially related to prolonged or delayed glutamate release (possibly as a result of the astrocytic network). A key determination of the expression phase is the involvement of new protein synthesis (using translation and transcription inhibitors) – further evidence of the long-term action of the synaptic plasticity from a single cannabinoid dose.

No description available.

No description available.

The control of cellular tyrosine phosphorylation levels is of great importance in many biological systems. Among the kinases and phosphatases that modulate these levels, the LAR-RPTPs have been suggested to act in several key aspects of neural development, and in a dysfunctional manner in various pathologies from diabetes to cancer. The aim of this thesis is to describe the physiological functions of one of the members of this subfamily of RPTPs, namely RPTPsigma. First, we showed that glucose homeostasis is altered in RPTPsigma null mice. They are hypoglycemic and more sensitive to exogenous insulin and we proposed that the insulin hypersensitivity observed in RPTPsigma-null mice is likely secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency. In addition to regulating nervous system development, RPTPsigma was previously shown to regulate axonal regeneration after injury. In the absence of RPTPsigma, axonal regeneration in the sciatic, facial and optical nerves was enhanced following nerve crush. However, myelin-associated growth inhibitory proteins and components of the glial scar such as CSPGs (chondroitin sulfate proteoglycans) have long been known to inhibit axonal regeneration in the CNS, making spinal cord injury irreversible. In collaboration with Dr Samuel David, we unveiled that RPTPsigma null mice are able to regenerate their corticospinal tract following spinal cord hemisections as opposed to their WT littermates. We then isolated primary neurons from both sets of animals and found that the absence of RPTPsigma promotes the ability of the neurons to adhere to certain inhibitory substrates. Finally, in order to better understand the physiological role of RPTPsigma, we used a yeast substrate-trapping approach, to screen a murine embryonic library for new substrates. This screen identified the RhoGAP p250GAP as a new substrate, suggesting a downstream role for RPTPsigma in RhoGTPase signaling. We also identified p130Cas and Fyn as new binding partners. All these proteins have clear functional links to neurite extension. The characterization of RPTPsigma and its signaling partners is essential for understanding its role in neurological development and may one day translate into treatments of neural diseases and injuries.