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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.