GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro.

ABSTRACT

Synapse formation in CNS neurons requires appropriate sorting and clustering of neurotransmitter receptors and associated proteins at postsynaptic sites. In GABAergic synapses, clustering of GABA(A) receptors requires gephyrin, but it is not known whether presynaptic signals are also involved in this process. To investigate this issue, we analyzed the subcellular distribution of GABA(A) receptors and gephyrin in primary cultures of cerebellar granule cells, by comparing cells receiving GABAergic input with cells devoid of such afferents. Using immunofluorescence staining, we show that the GABA(A) receptor alpha1 and gamma2 subunit, but not alpha6 or delta subunit, form clusters co-localized with gephyrin in granule cell neurites, irrespective of the presence of GABAergic axons. GABAergic terminals typically were surrounded by groups of gephyrin clusters, pointing to the presence of multiple synaptic sites. In contrast, in neurites devoid of GABAergic input, gephyrin clusters were distributed at random and apposed to glutamatergic terminals, suggesting the formation of mismatched synapses. Both populations of gephyrin clusters were co-localized with GABA(A) receptor subunits, indicating that these proteins are associated also in non-GABAergic synapses. To determine whether signaling mediated by GABA(A) receptors is required for the formation of appropriately matched gephyrin clusters, cultures were treated chronically with bicuculline, or with either muscimol or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. All these treatments failed to influence the distribution of gephyrin clusters. We conclude that although GABAergic presynaptic terminals have a preponderant influence on the distribution of gephyrin clusters in dendrites of cerebellar granule cells, GABA transmission is dispensable for postsynaptic clustering of gephyrin and GABA(A) receptors and for the formation of appropriately matched GABAergic synapses.