GABA(A) receptors can initiate the formation of functional inhibitory GABAergic synapses

The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABA(A) receptors (GABA(A)Rs) themselves – the essential functional postsynaptic components of GABAergic synapses – can be sufficient to initiate formation of synaptic contacts, a novel co-culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. α1/β2/γ2-GABA(A)Rs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse-like contacts in these co-cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle-luminal domain-specific antibody; and they supported spontaneous and action potential-driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N-methyl-d-aspartate receptor-expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin-2 was co-expressed with GABA(A)Rs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABA(A)Rs can promote the adhesion of inhibitory axons and the development of functional synapses.