Regular articleAssociation of gephyrin with synaptic and extrasynaptic GABAa receptors varies during development in cultured hippocampal neurons
Introduction
Extrasynaptic clusters of GABAa receptors (GABAaRs) have been reported in hippocampal neurons (Banks and Pearce, 2000, in situ; Christie et al 2002, Kannenberg et al 1999, Nusser et al 1995, in situ; Scotti and Reuter, 2001) but their involvement in the construction of the postsynaptic domains remained unexplored. It is established that inhibition of gephyrin expression leads to a dramatic decrease in GABAaR clusters Essrich et al 1998, Feng et al 1998, Kneussel et al 1999. Conversely, mice knocked-out for the GABAaR γ2 subunits show significant reduction of gephyrin clustering (Essrich et al., 1998). As a direct binding of gephyrin to the GABAaR could not be demonstrated (Meyer et al., 1995), it has been proposed that gephyrin mediates clustering and/or synaptic anchoring Craig et al 1996, Kneussel et al 1999 of GABAaR through indirect binding to its γ2 and maybe β3 subunits (Kirsch et al., 1995). Surprisingly, however, the association of GABAaR and gephyrin at extrasynaptic sites remains poorly studied. Moreover, only few data concerning the association of these proteins during development are available (Dumoulin et al., 2000). The comparison of the expression pattern of gephyrin with that of GABAaR γ2 and β3 subunits during synaptogenesis is necessary to understand the role of each partner. Detailed study of GABAergic synaptogenesis has been performed in spinal interneurons (Dumoulin et al., 2000), but in this system, only few extrasynaptic clusters of GABAaRs are detected. In addition, inhibitory synapse maturation in spinal interneurons is achieved within 10–12 days (Dumoulin et al., 2000), while it needs about 21 days in cultured hippocampal neurons Craig et al 1996, Levi et al 2002. Christie et al. (2002) have studied the effect of GABAergic innervation on GABAaRs clustering in cultured hippocampal neurons, but the developmental aspects were not investigated, and the relationships between synaptic localization of GABAaR clusters and gephyrin association have not been assessed. In contrast, in the same system, Scotti and Reuter (2001) have analyzed the localization of GABAaR clusters at some developmental stages, but the relation of these clusters with gephyrin was not investigated.
We have now analyzed the development of inhibitory synapse components in cultured hippocampal neurons. The presynaptic inhibitory elements were identified by the presence of the GABA synthesizing enzyme GAD-65 and/or the vesicular inhibitory amino acid transporter immunoreactivity (VIAAT-IR, characterized in Caenorhabditis elegans as Unc47/VGAT) McIntire et al 1997, Sagne et al 1997. On the postsynaptic side, we have followed the GABAaR γ2 and β3 subunits and gephyrin-IR. This allowed us to unravel unexpected stages in the sequence of GABAergic synaptogenesis. We observed that during synaptic maturation GABAaR γ2 and β3 subunits behave differently and that these subunits are able to associate with synaptic and extrasynaptic clusters of gephyrin. Our data are compatible with a role of gephyrin in GABAaR synaptic stabilization rather than in clusters formation.
Section snippets
Inhibitory boutons differentiate after excitatory ones
In cultured hippocampal neurons, 6 to 7% of neurons are GABAergic interneurons, whereas the remainder are pyramidal glutamatergic cells (Benson et al., 1994). To investigate the formation of inhibitory presynaptic elements, we double-labeled rat hippocampal neurons cultured from 3 up to 21 days in vitro (DIV) with antibodies against VIAAT and synapsin. The time course and pattern of synapsin expression were comparable to those described in other studies (e.g., Fletcher et al., 1991). A diffuse
Discussion
In this study, we have found that: (1) the GABAergic presynaptic elements were generated 3 to 7 days after the glutamatergic boutons; (2) the GABAaR subunits became mainly synaptic about 4 days after initial differentiation of the presynaptic elements; and (3) GABAaR and gephyrin clusters were first expressed as independent extrasynaptic clusters and were associated prior to being detected at synaptic sites.
Cell cultures
Hippocampal cultures were prepared as described Banker and Cowan 1977, Goslin and Banker 1998. Briefly, hippocampi were dissected from 18-day-old, fetal Sprague–Dawley rats. Cells were dissociated by treatment with 0.25% trypsin for 15 min at 37°C, and triturated through a fire-constricted Pasteur pipette in DNase (0.1 mg/ml; Sigma). Neurons were plated onto polyornithine-coated glass coverslips (12-mm diameter) at a density of 5000 cells/cm2 in minimal essential medium (MEM; Gibco BRL)
Acknowledgements
We thank Drs. B. Gasnier (Paris), W. Sieghart (Vienna), and B. Giros (Paris) for generously supplying antibodies against VIAAT, GABAaR β3, and VGLUT1, respectively, and Dr. J.-M. Fritschy (Zurich) for GABAaR γ2 antibody and helpful advice on the staining protocol. We thank Drs. C. Vannier, A. Dumoulin, and S. Lévi for critical reading of the manuscript. This work was funded by the Institut National de la Santé et de la Recherche Médicale and supported by grants from the Institut de la Recherche
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