Dendritic spines undergo synaptic pruning in the medial prefrontal cortex (mPFC) during adolescence, a process which is excessive in schizophrenia, believed to underlie cognitive impairment. Despite its importance, the mechanism underlying synaptic pruning in the mPFC is not known. Our preliminary findings suggest that extrasynaptic α4βδ GABAA receptors (GABARs), which transiently emerge on the spine at puberty onset (~PND 35) for 10d, trigger adolescent synaptic pruning in the mPFC. These receptors generate a shunting inhibition which impairs NMDA receptor activation. Our previous findings in CA1 hippocampus established that the inhibition generated by these receptors reduces expression of the Rho-GEF spine protein Kalirin-7, necessary for spine maintenance, via this reduced NMDA receptor activity. We will test the hypothesis that similar effects on spine proteins play a role in adolescent pruning in mPFC. The proposed aims will use multiple techniques, including in vivo two-photon imaging, electrophysiological, pharmacological, and immunocytochemical assays. To this end, we will chronically manipulate α4βδ function during adolescence (~PND 35-44) and quantify spine density at 8 and 12 wks of age. Preliminary data shows that spine density is decreased in both layer 3 and 5 post- pubertally in +/+ but not α4-/- mice, implicating α4βδ GABARs. Because synaptic GABAergic input targets the spine in the cortex, the role of α1β2γ2 GABARs will also be examined. These experiments will use acute pharmacological (agonists, modulators) and genetic manipulation (global/conditional/local knock-down) of α4βδ and α1β2γ2 to explore the role of these receptors in regulating spine density and morphology assessed using Golgi and electrophysiological techniques. To explore the effect of this pruning on circuit and behavioral plasticity, in vivo two-photon techniques in combination with multi- neuron recording will be used to image the Ca++ signals and record activity generated by neuronal ensembles in response to visual stimuli in the awake mouse across adolescence and after GABAR modulation/knock-down. We will thus assess the effect of pubertal α4βδ GABARs and GABAR- generated pruning on circuit function, including the power and synchronization of theta and gamma oscillations as well as on circuit activity/synchronization of ensembles and mPFC-dependent learning tasks. The findings from the proposed studies will directly address the functional role of GABAergic inhibition on dendritic spines at puberty, and will also provide mechanisms for the process of synaptic pruning, as well as explore functional outcomes of alterations in this process. These results are relevant for schizophrenia where excessive synaptic pruning and impaired working memory are reported in association reduced power/sychronization of visually-evoked gamma oscillations, and where single nucleotide polymorphisms of the δ gene are reported, suggesting a genetic link.