Development of -Aminobutyric Acidergic Synapses in Cultured Hippocampal Neurons

The formation and maturation of -aminobutyric acid (GABA)-ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19–90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the 5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These 5 clusters were mostly extrasynaptic. The 1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these 1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after preand postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs. J. Comp. Neurol. 495:497–510, 2006. © 2005 Wiley-Liss, Inc. Indexing terms: synaptogenesis; GABAA receptor; GABA transporter; mIPSC Three basic stages of synaptogenesis have been described: 1) initial contact between presynaptic and postsynaptic elements, 2) maturation of the synapse, and 3) emergence of adult isoforms (Sanes et al., 2000). Most studies of synaptogenesis have concentrated on the neuromuscular junction and glutamatergic synapses (Sanes and Lichtman, 2001; Goda and Davis, 2003). Although the formation and organization of -aminobutyric acid (GABA)-ergic synapses are thought generally to follow the same process (Moss and Smart, 2001; Meier, 2003), the development of GABAergic synapses has not been fully