Gephyrin is a scaffold protein that ensures the correct localization and clustering of glycine and GABA-A receptors (GABA-ARs) at postsynaptic sites. In this study, gephyrin-specific single chain antibody fragments (scFv) containing nuclear localization signals were used to perturb endogenous gephyrin clustering in rat hippocampal neurons in culture (Zacchi et al., J. Mol. Neurosci. 34: 141-148, 2008). Immunocytochemical experiments on scFv-transfected cells revealed a significant reduction in the density of gephyrin clusters, as compared to EGFP transfected controls (9.7 ± 1.5 in scFv vs 15.9 ± 1.4 in EGFP clusters/100mm²; p<0.01; n=22). The density of g2-containing GABA-AR clusters co-localizing with the vesicular GABA transporter VGAT (expressed as percentage of the total g2 immunoreactivity) was also reduced (4.8 ± 1.7 % in scFv vs 17.0 ± 2.8 % in EGFP; p<0.01; n=11). Electrophysiological experiments demonstrated that these effects were associated with a reduction in frequency (0.18 ± 0.03 Hz in scFv vs 0.28 ± 0.04 Hz in EGFP; p<0.05; n=23) and amplitude (29.8 ± 1.7 pA in scFv vs 42.6 ± 3.0 pA in EGFP; p<0.01; n=23) of spontaneous GABA-A-mediated miniature inhibitory postsynaptic currents. In addition, hampering gephyrin function with scFv produced a significant decrease in the frequency of AMPA-mediated glutamatergic events (0.26 ± 0.06 Hz in scFv vs 1.2 ± 0.34 Hz in EGFP; p<0.01; n=14), without any modification in their amplitude (32.8 ± 4.5 pA in scFv vs 34.3 ± 4.6 pA in EGFP; p>0.05; n=14). Immunocytochemical analysis of vesicular glutamate transporter VGLUT resulted in a two-fold reduction in immunoreactive puncta in scFv as compared to controls (n=5) indicating a loss of glutamatergic innervation. This may be due to a homeostatic compensatory mechanism and may involve specialized cell adhesion molecules such as neuroligins and neurexins which, bridging the cleft, provide a direct link between the pre and the postsynaptic site of the synapse. Preliminary experiments on scFv-transfected neurons using antibodies against Neuroligin-2 which is predominantly localized at GABAergic synapses revealed a three-fold decrease in Neuroligin-2 immunoreactivity as compared to controls (n=4). It is likely that gephyrin interacts with neuroligin-neurexin complexes to regulate both GABAergic and glutamatergic transmission in the hippocampus. This would ensure the appropriate inhibitory-excitatory balance critical for the correct functioning of neuronal networks.