Regular article
Association of gephyrin with synaptic and extrasynaptic GABAa receptors varies during development in cultured hippocampal neurons

https://doi.org/10.1016/S1044-7431(03)00069-1Get rights and content

Abstract

Several studies have reported extrasynaptic clusters of GABAa receptors in hippocampal neurons. Yet their functional relevance as well as their evolution in relation with gephyrin during synaptogenesis remain unknown. We have analyzed the expression pattern of the main proteins of the GABAergic synapses during synaptogenesis in cultured hippocampal neurons. We found that GABAergic terminals, characterized by VIAAT and GAD-65 expression, differentiated 3 to 7 days after the glutamatergic endings. At the postsynaptic side, the GABAaR- β3 subunit was first diffuse and then clustered when GABAergic terminals differentiated and gephyrin formed large clusters. Colocalization of these proteins was high and increased with development. At later stages, GABAaR β3 clusters colocalized with gephyrin at synaptic but also at extrasynaptic sites. GABAaR γ2 subunits were directly expressed as clusters which were first extrasynaptic and not associated with gephyrin. Subsequently, the GABAaR γ2 subunits associated with gephyrin at synaptic and/or extrasynaptic sites. Our data indicate that formation of GABAaR γ2 subunit clusters is gephyrin independent.

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

References (57)

  • S.E. Ahmari et al.

    Assembly of presynaptic active zones from cytoplasmic transport packets

    Nature Neurosci.

    (2000)
  • F.J. Alvarez et al.

    Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord

    J. Comp. Neurol.

    (1997)
  • G.A. Banker et al.

    Rat hippocampal neurons in dispersed cell culture

    Brain Res.

    (1977)
  • M.I. Banks et al.

    Kinetic differences between synaptic and extrasynaptic GABA(A) receptors in CA1 pyramidal cells

    J. Neurosci.

    (2000)
  • E.E. Bellocchio et al.

    Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter

    Science

    (2000)
  • Y. Ben-Ari

    Excitatory actions of GABA during developmentthe nature of the narture

    Nature Rev. Neurosci.

    (2002)
  • D.L. Benson et al.

    Characterization of GABAergic neurons in hippocampal cell cultures

    J. Neurocytol.

    (1994)
  • D.L. Benson et al.

    Activity-independent segregation of excitatory and inhibitory synaptic terminals in cultured hippocampal neurons

    J. Neurosci.

    (1996)
  • I. Brunig et al.

    Intact sorting, targeting, and clustering of gamma-aminobutyric acid A receptor subtypes in hippocampal neurons in vitro

    J. Comp. Neurol.

    (2002)
  • I. Brunig et al.

    GABAergic terminals are required for postsynaptic clustering of dystrophin but not of GABA(A) receptors and gephyrin

    J. Neurosci.

    (2002)
  • S.B. Christie et al.

    GABAergic innervation organizes synaptic and extrasynaptic GABAA receptor clustering in cultured hippocampal neurons

    J. Neurosci.

    (2002)
  • I. Colin et al.

    Localization of components of glycinergic synapses during rat spinal cord development

    J. Comp. Neurol.

    (1998)
  • A.M. Craig et al.

    Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons

    J. Neurosci.

    (1996)
  • A. Dumoulin et al.

    Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons

    Eur. J. Neurosci.

    (2000)
  • C. Essrich et al.

    Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin

    Nature Neurosci.

    (1998)
  • G. Feng et al.

    Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity

    Science

    (1998)
  • F. Fischer et al.

    Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse

    J. Comp. Neurol.

    (2000)
  • T.L. Fletcher et al.

    The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture

    J. Neurosci.

    (1991)
  • Cited by (0)

    View full text