Gephyrin Regulates GABAergic and Glutamatergic Synaptic Transmission in Hippocampal Cell Cultures

Stefka H Stancheva,Rocco Pizzarelli,Zeynep Kasap Varley,Roberta Antonelli,Paola Zacchi,Matthias Kneussel,Enrico Cherubini

doi:10.1074/jbc.m111.234641

Abstract

Gephyrin is a scaffold protein essential for stabilizing glycine and GABA(A) receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post- and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.

Highlights

In the present study, scFv-gephyrin fragments were used to characterize further the transynaptic contribution of gephyrin to maintaining and stabilizing GABAergic synapses
Impairing Gephyrin Function with scFv-Gephyrin Reduces the Probability of GABA Release—As recently reported [6], transfecting cultured hippocampal neurons with scFv-gephyrin reduced the number of gephyrin and synaptic ␥2 subunit-containing GABAA receptor clusters
We have demonstrated that disrupting endogenous gephyrin with selective scFv-gephyrin altered the gating properties of GABAA receptors, an effect that was found to be associated with modifications of GABAergic innervation [6]

Summary

EXPERIMENTAL PROCEDURES

Neuronal and Cell Cultures—All experiments were carried out in accordance with the European Community Council Directive of 24 November 1986 (86/609 EEC) and were approved by the local authority veterinary service. Pairs of action potentials (at 50-ms interval) were evoked in nontransfected presynaptic neurons (in current clamp mode) by injecting depolarizing current pulses at a frequency of 0.1 Hz. IPSCs were detected from postsynaptic transfected (scFv-gephyrin) and nontransfected (controls) neurons in voltage clamp mode at a holding potential of 0 mV (near the reversal potential for glutamate). IPSCs were detected from postsynaptic transfected (scFv-gephyrin) and nontransfected (controls) neurons in voltage clamp mode at a holding potential of 0 mV (near the reversal potential for glutamate) In this case, the intracellular solutions contained 135 mM KMeSO4, 10 mM KCl, 10 mM HEPES, 1 mM MgCl2, 2 mM Na2ATP, and 0.4 mM. The differences were considered significant when p Ͻ 0.05

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS