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ABSTRACT: “Mechanism of GABAergic synapse formation: an in vivo study in the rodent Cerebellar cortex”. The clustering of neurotransmitter receptors at postsynaptic sites is an essential feature of synapse formation and is critical for fast signal transmission between neurons. Different studies indicate that scaffolding or anchoring molecules are crucial for the accumulation and dynamic regulation of receptors in the postsynaptic membrane. Gephyrin is a peripheral membrane protein that copurifies with the glycine receptor and is localized at postsynaptic sites of GABAergic and glycinergic synapses throughout the brain and spinal cord. Gephyrin interacts with a cytoplasmic loop of the glycine receptor subunit and is strictly required for the accumulation of glycine receptors in the postsynaptic plasma membrane. The function of gephyrin at GABAergic synapses is still controversial. Distruption of postsynaptic GABAA receptor clusters in knockout mice lacking either the 2 or the 1 subunits causes a loss of gephyrin clusters, indicating that gephyrin depends on GABAA receptors for proper synaptic localization. Conversely, antisense experiments in cultured neurons have demonstrated that gephyrin is required for postsynaptic clustering of GABA A receptors. However, a substantial number of GABAA receptor clusters is retained in the spinal cord of gephyrin knockout mice, as well as in cultured hippocampal neurons and in organotypic retina cultures of these mutant mice, suggesting gephyrin-independent formation of GABAA receptor clusters. Our understanding of the processes underlying the assembly of GABAergic synapses is mainly derived from studies on cell cultures. Therefore a major aim of this study is to investigate the development of GABAergic synapses in the cerebellar cortex, a neural center in which synaptogenesis is entirely postnatal. Specifically, I addressed three major questions. First, I analyzed synapse development to establish whether the synaptic aggregation of gephyrin occurs in parallel with that of GABAA receptors. Next, I investigated the importance of GABAA receptors for postsynaptic clustering of gephyrin, using conditional knockout mice in which major GABAA receptor subunits have been deleted from cerebellar Purkinje cells. Finally, I investigated the regional and synaptic localization of collybistin, a GDP/GTP exchang factor (GEF) that is believed to regulate the subcellular distribution of gephyrin. I found that, as soon as GABAA receptors aggregate at postsynaptic sites during development, they associate with gephyrin, suggesting that this scaffolding molecule is involved in the initial phases of the differentiation of postsynaptic specializations. In addition, gephyrin and GABAA receptors clustered selectively at postsynaptic sites facing VIAAT-positive boutons, with no evidence of ectopic expression. Together, these findings demonstrate that gephyrin and GABAA receptors co-aggregate during cerebellar development and that the large majority of such molecular complexes are found at synapses capable of releasing GABA. Surprisingly, I found that gephyrin is expressed only transiently at perisomatic synapses established by basket cells with Purkinje cells, and that the loss of gephyrin was accompanied by a remodelling of postsynaptic specializations and a reduction in the size of GABAA receptor clusters. Having established that gephyrin and GABAA receptors aggregate at postsynaptic sites with a similar time course during cerebellar development, I investigated what are the effects of a selective deletion of GABAA receptors on inhibitory synaptogenesis. For this aim, I analyzed conditional knockout mice in which either the 1 or the 2 subunit has been ablated selectively from Purkinje cells by expression of Cre-recombinase under the control of the L7 gene promoter. In both mutant mice, there was a reduction in the density of GABAA receptor clusters in the molecular layer, as assessed by immunofluorescence and confocal microscopy. Postsynaptic clustering of gephyrin was also affected, however some gephyrin clusters persisted at postsynaptic sites despite the loss of GABAA receptors. These data confirm previous studies indicating that GABAA receptors are important for stabilizing gephyrin at postsynaptic specializations of GABA synapses. In addition, they indicate that clustering of gephyrin is not critically dependent on GABA A receptors and that there could be alternative mechanisms that control the synaptic localization of this molecule. Collybistin is a GDP/GTP exchange factor that is required for postsynaptic clustering of gephyrin through activation of a Rho/Rac GTPase. Recent studies on collybistin deficient mice have indicated that collybistin is essential for gephyrin dependent clustering of a specific set of GABA A receptors, but is not required for glycine receptor postsynaptic localization. These data suggest that collybistin may be present in only a subset of the gephyrin expressing synapses, or that other GEFs may contribute to regulate gephyrin clustering at glycinergic synapses. To better understand the role of collybistin, I have started to investigate the synapse-specific localization of this molecule, using a panel of collybistin-specific antibodies. The results indicate that collybistin colocalizes with gephyrin at postsynaptic sites of inhibitory synapses. However, collybistin is present in only a specific subset of gephyrin-positive synapses, raising the possibility that other proteins are involved in regulating the postsynaptic localization of this scaffolding molecule. These findings provide the first in vivo evidence that collybistin is localized at inhibitory postsynaptic sites in the intact brain.