em In vivo /em , NL2 would then recruit further postsynaptic density proteins (Poulopoulos em et?al /em ., 2009), and perhaps this, together with the increased synaptic activity, would further strengthen and stabilize the connection (Hartman em et?al /em ., 2006; Varoqueaux em et?al /em ., 2006; Chubykin em et?al /em ., 2007). 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. 1/2/2-GABAARs, 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 (Gross co-culture model system, is supported by results emerging from the analysis of mutant mice lacking specific GABAAR subunits. For example, in 1 subunit knockout mice, the function and synaptic localization of gephyrin, a major postsynaptic scaffold protein, at inhibitory synapses, is disrupted (Fritschy evidence for a role for GABAARs in synapse assembly has yet to emerge. The multiplicity of GABAAR subtypes expressed in neurones (Schofield and are subject to all of IkB alpha antibody the caveats that should surround any study in a reduced system, these co-cultures have allowed the potential for GABAARs to participate directly in synapse formation to be demonstrated. In agreement with studies of synapse formation in NL2 knockout mice (Varoqueaux studies could perhaps Fosdagrocorat be explained, at least in part, by the different combinations of neuronal cell types Fosdagrocorat and postsynaptic GABAAR subtypes tested. This, in addition, to the high level and consistency of cell surface expression of GABAAR subunits in the stably transfected HEK293 cell line used in our study, and in contrast to the transiently expressed GABAARs in previous studies, may have been crucial for the reliable detection of synapse formation and activity across the population of cells in co-culture. The number of functional contacts was enhanced significantly by concomitant overexpression of NL2, as seen in neurones (Fu & Vicini, 2009). Stable connections, involving several synapse-like contacts per axon, do occur in the absence of NL2. However, comparison of sIPSC, AP-IPSC and mIPSC amplitudes indicates that single axon connections may involve more presynaptic terminals, and that each terminal elicits a stronger postsynaptic response when NL2 is co-expressed together with GABAARs. NL2 may also be important for the rigid membrane appositions typical of synapses (Varoqueaux em et?al /em ., 2006; Blundell em et?al /em ., 2009; Gibson em et?al /em ., 2009) or em in vitro /em . That these 1/2/2-GABAARs were sufficient alone to support and stabilize functional synapse-like contacts is interesting in the light of a study by Gibson em et?al /em . (2009). In this study, the synapses innervated by fast-spiking, parvalbumin-containing interneurones in the hippocampus, which are mediated by 1-GABAARs (Thomson em et?al /em ., 2000; Nyiri em et?al /em ., 2001), were found to be the most powerfully affected in NL2 knockouts. Both quantal amplitude and quantal content (i.e. the number of quanta, or synapses, contributing to each event) were lower than at wild-type connections. These findings in NL2 knockout mice have a striking parallel in the present study, where the absence of NL2 coincided with decreases in both the number of functional synapses and the quantal amplitude, in a much more reduced system employing a different class of presynaptic neurone. A larger mIPSC, or quantal amplitude, is typically explained either by a larger number of postsynaptic receptors, or by an increase in their single channel conductance. HEK293-GABAAR-NL2 cells received a large number of synapse-like contacts, which were often very close neighbours (Fig.?5A; Fig. S2A), whereas HEK293-GABAAR cells received more sparse innervation (Figs?3A and D). If such a finding were obtained in a neuronal system, it might suggest that the larger quantal amplitudes seen in HEK293-GABAAR-NL2 cells are attributable to spill-over from one terminal to receptors lying under one or more neighbouring terminals. However, although these cultures did Fosdagrocorat not contain glial cells, whose active re-uptake of GABA might otherwise have curtailed its diffusion, the extracellular space in the co-cultures is very large, and the released GABA can be expected to have diffused rapidly away from the HEK293 cell. There was, moreover, little evidence Fosdagrocorat for clustering of receptors in these HEK293-GABAAR cells (Fig.?1C; Fig. S1A), and there was no evidence that the surface expression.