Gephyrin regulates trans-synaptic signaling at GABAergic connections in the hippocampus

Gephyrin is the central component of the postsynaptic scaffold at inhibitory synapses, ensuring receptor accumulation in precise apposition to presynaptic release sites. Synapses are highly dynamic structures, with receptors constantly moving in and out of postsynaptic sites. The mechanisms regulating synaptic organization are thus crucial for an efficient and reliable synaptic transmission. My thesis focuses on the role of gephyrin in regulating GABAergic transmission. To study gephyrin function, I used intracellular single chain antibody fragments against gephyrin (scFv-gephyrin) which could remove endogenous gephyrin from its subcellular location, leading to an overall loss of gephyrin clusters. Transfecting hippocampal neurons in culture with scFv-gephyrin led to a reduced density of synaptic g2-subunit containing GABAA receptors. This effect was associated with a decrease in the amplitude and frequency of mIPSCs, and a slow-down in their onset kinetics. Using an ultrafast agonist application system which mimics synaptic events, I found that the slow onset of GABA-evoked currents was due to a slower entry of the receptors into the desensitized state. Hence, hampering gephyrin function affects the gating properties of GABAA receptors. Disruption of gephyrin clusters also altered the GABAA mediated tonic conductance, an effect that could be attributed to a reduced GABAergic innervation. Gephyrin ablation led to a reduction in the density of the vesicular GABA transporter, VGAT. Moreover, pair recordings from interconnected neurons revealed a reduction in amplitude and in the number of successes as well as an increase in paired-pulse ratio and in the coefficient of variation of GABAA-mediated synaptic currents in scFv-gephyrin transfected neurons, indicating a reduced probability of GABA release. Gephyrin may exert this trans-synaptic action through the neuroligin/neurexin complex. Consistent with this hypothesis, I found that upon scFv-transfection, the neuroligin isoform known to be preferentially localized at GABAergic synapses (neuroligin 2) was significantly reduced. Furthermore, in molecular biology experiments, gephyrin was found to immunoprecipitate neuroligin 2 from rat brain lysates, indicating the formation of a complex between these two proteins. Co-expression of neuroligin 2 with scFv-gephyrin was able to rescue the reduction in GABAergic innervation, suggesting that gephyrin can regulate GABA release through neuroligin 2. Neuroligins can localize at both GABAergic and glutamatergic synapses, and modulate the excitatory/inhibitory (E/I) balance within the neuronal network. Interestingly, scFvgephyrin transfection resulted in a significant reduction in glutamatergic innervation, as revealed by the decrease in the density of the vesicular glutamate transporter, VGLUT, as well as by the reduction in the frequency of mEPSCs. Rescue experiments with the coexpression of neuroligin 2 and scFv-gephyrin did not reverse the effect of scFv-gephyrin on glutamatergic innervation, suggesting that it was not due to a homeostatic compensatory mechanism. Based on the observation that gephyrin can coimmunoprecipitate neuroligin 1 (the isoform enriched at glutamatergic synapses), it is possible that gephyrin modulates both GABAergic and glutamatergic synapses via neuroligins. However, whether gephyrin regulates glutamate release via neuroligin 1 remains to be elucidated. Overall, interfering with gephyrin clustering at the post-translational level has revealed new insights on the role of this scaffold protein in GABAergic synapses and has prompted further investigation into the function of gephyrin in regulating the E/I balance possibly through neuroligins.