Elsevier

Neuropharmacology

Volume 88, January 2015, Pages 199-208
Neuropharmacology

Benzodiazepine ligands rapidly influence GABAA receptor diffusion and clustering at hippocampal inhibitory synapses

https://doi.org/10.1016/j.neuropharm.2014.06.002Get rights and content

Highlights

  • Positive and negative BZD modulators have opposing effects on GABAAR channel gating.

  • DMCM also increases GABAAR diffusion and decreases clustering and synaptic function.

  • Diazepam on the contrary stabilizes GABAARs at synapses.

  • BZD ligands influence both channel gating and receptor dynamics.

Abstract

Benzodiazepines (BZDs) are widely used in the treatment of a variety of neurological and psychiatric conditions including anxiety, insomnia and epilepsy. BZDs are thought to act predominantly by affecting the gating of GABAA receptor channels, resulting in enhanced GABA-mediated currents in neurons. However, mutations mimicking the effect of BZDs on GABAAR channel gating have been shown to also impact the membrane dynamics and synaptic anchoring of the receptors. Here, using single molecule tracking combined with electrophysiological recordings, we show that BZD ligands rapidly influence the dynamic behavior of GABAARs in hippocampal neurons. Application of the inverse BZD agonist DMCM rapidly increased the diffusion and reduced the clustering of GABAARs at synapses, resulting in reduced postsynaptic currents. Conversely, the BZD full agonist diazepam had little effect at rest but reduced lateral diffusion and increased synaptic stabilization and clustering of GABAARs upon sustained neuronal activity, resulting in enhanced potency of inhibitory synapses. These effects occurred in the absence of detectable changes in gephyrin clusters, suggesting they did not reflect a rapid dispersion of the synaptic scaffold. Thus, alterations of the diffusion and synaptic anchoring of GABAARs represent a novel, unsuspected mechanism through which BZDs rapidly modulate GABA signaling in central neurons.

This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.

Introduction

GABAA receptors (GABAARs) are heteropentameric receptors of the Cys-loop ligand-gated ion channel family and mediate both tonic and phasic inhibition in the CNS. Whereas most synaptic receptors are composed of α(1–3), β2/3 and γ2 subunits (Brunig et al., 2002), extrasynaptic receptor composition differs in different brain structures and synapses and may predominantly include α5, β3 and γ2 (Prenosil et al., 2006, Sur et al., 1998) or α4/6, β1-3 and δ subunits (Mangan et al., 2005). These receptors are the targets of a variety of widely used drugs such as several general anesthetics as well as barbiturates and benzodiazepines (BZDs) used in the treatment of anxiety, insomnia and some forms of epilepsy (Rudolph and Knoflach, 2011).

These drugs all act by potentiating GABAAR activity through mechanisms which are only partially understood at the single channel level (e.g. (Loscher and Rogawski, 2012)). In particular, BZDs bind GABAARs at a high-affinity binding site located between the α and γ subunit (Sigel and Buhr, 1997). This results in an apparent increased affinity of the receptor for GABA (Rogers et al., 1994). However, detailed analysis of current kinetics using partial receptor agonists recently demonstrated that BZDs may act primarily by favoring a pre-activated state before channel opening rather than directly affecting the receptor affinity for GABA (Gielen et al., 2012). Thus, BZDs may induce a conformational change of GABAARs to promote preactivation of the receptor.

In addition to modulating channel gating and/or ligand binding, the efficacy of GABAAR-mediated transmission may be rapidly adjusted by altering the aggregation of GABAARs at synapses, thereby modulating the number of available postsynaptic receptors and, consequently, synaptic strength (Nusser et al., 1997, Nusser et al., 1998). GABAARs are anchored at synaptic sites by scaffolding molecules including gephyrin (Kneussel and Betz, 2000, Sassoe-Pognetto and Fritschy, 2000) which acts to trap receptors diffusing in the plasma membrane (Jacob et al., 2005, Levi et al., 2008, Triller and Choquet, 2008), possibly through a direct interaction with α subunits (Tretter et al., 2008). This interaction may be modulated by neuronal activity. Thus, activity-dependent regulation of synaptic inhibition has been shown to involve changes in the diffusive properties and synaptic anchoring of GABAARs (Bannai et al., 2009, Muir et al., 2010). These changes occur before any detectable change in synaptic gephyrin clusters (Niwa et al., 2012) and instead may rely at least in part on calcineurin-dependent dephosphorylation of S327 residue on the γ2 subunit (Bannai et al., 2009, Muir et al., 2010).

We recently reported that a point mutation in GABAAR γ2 subunit associated with generalized epilepsy (Baulac et al., 2001) affects both the gating kinetics (Bianchi et al., 2002, Eugene et al., 2007) and the membrane dynamics and synaptic aggregation of the receptor (Bouthour et al., 2011). Since this mutation is located in the second extracellular domain of γ2 (Baulac et al., 2001), it is unlikely to directly affect GABAAR interaction with submembrane gephyrin scaffold. Instead, this interaction might be influenced by changes in GABAAR conformation. Here, we tested whether allosteric modulators such as BZDs may induce similar changes in GABAAR diffusion and clustering. Using single particle tracking (SPT) and electrophysiology, we show that application of BZD ligands is sufficient to rapidly affect GABAAR diffusion and synaptic aggregation, leading to significant changes in the amplitude of synaptic currents. The effect is bi-directional, depending on whether BZD agonist or inverse agonist is used. Thus, our results reveal that in addition to modulating the gating of GABAAR channels, BZD ligands also act on a rapid yet slower time scale, to indirectly modulate the mobilization of GABAARs at synapses, thereby affecting synaptic potency.

Section snippets

Neuronal culture and transfection

Primary cultures of hippocampal neurons were prepared as described (Goslin et al., 1998) from embryonic day 18 or 19 Sprague–Dawley rats. Tissue was trypsinized (0.25% v/v), and mechanically dissociated in HBSS (Invitrogen) containing 10 mM HEPES (Invitrogen). Neurons were plated at a density of 2.3 × 104 cells/cm2 onto 18-mm diameter glass coverslips coated with poly-d,l-ornithine (80 μg/ml, Sigma–Aldrich) in plating medium composed of minimum essential medium (MEM, Sigma) with horse serum

Opposing control of synaptic GABAAR gating by benzodiazepine ligands in hippocampal neurons

We sought to evaluate the impact of allosteric modulation of GABAARs on their membrane dynamics and synaptic stabilization. BZDs ligands are known to specifically modulate the gating of GABAAR without directly affecting their unitary conductance or ligand affinity (Gielen et al., 2012). We compared the effects of two BZD ligands on the properties of miniature IPSCs recorded from primary hippocampal neurons: the inverse agonist DMCM (1 μM) and the agonist diazepam (1 μM). mIPSCs were

Discussion

We have shown that benzodiazepine ligands have multiple and opposing effects on GABAAR-mediated synaptic transmission. Acute application of a BZD agonist or inverse agonist acts primarily by affecting the decay kinetics of IPSCs with no detectable change in peak amplitude. However, more prolonged applications lead to an additional change in IPSC amplitude reflecting bi-directional changes in GABAAR diffusion and synaptic clustering. Thus BZDs, in addition to their well-documented effect on

Funding

This work was funded by INSERM (Avenir program to JCP) and grants from the Fondation Electricité de France, Fondation pour la Recherche Médicale and the City of Paris (to JCP). NLR was supported by the Agence Nationale de la Recherche (ANR EPISOM grant to JCP).

Author's contribution

SL and JCP designed research; SL, NLR, EE and JCP performed research and analyzed data; SL and JCP wrote the paper.

Acknowledgments

We thank Michèle Carnaud (IFM) for preparing and maintaining hippocampal cultures, Eric Schwartz and Pierre-Jean Corringer for stimulating discussions and critical reading of the manuscript.

References (64)

  • J.C. Poncer et al.

    Modulation of synaptic GABAA receptor function by benzodiazepines in area CA3 of rat hippocampal slice cultures

    Neuropharmacology

    (1996)
  • E. Sigel et al.

    The benzodiazepine binding site of GABAA receptors

    Trends Pharmacol. Sci.

    (1997)
  • V. Tretter et al.

    Molecular basis of the gamma-aminobutyric acid A receptor alpha3 subunit interaction with the clustering protein gephyrin

    J. Biol. Chem.

    (2011)
  • A. Triller et al.

    New concepts in synaptic biology derived from single-molecule imaging

    Neuron

    (2008)
  • K.A. Wafford

    GABAA receptor subtypes: any clues to the mechanism of benzodiazepine dependence?

    Curr. Opin. Pharmacol.

    (2005)
  • A.P. Alivisatos et al.

    Quantum dots as cellular probes

    Annu. Rev. Biomed. Eng.

    (2005)
  • H. Bannai et al.

    Imaging the lateral diffusion of membrane molecules with quantum dots

    Nat. Protoc.

    (2006)
  • S. Baulac et al.

    First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene

    Nat. Genet.

    (2001)
  • M.T. Bianchi et al.

    Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans

    J. Neurosci.

    (2002)
  • S. Bonneau et al.

    Single quantum dot tracking based on perceptual grouping using minimal paths in a spatiotemporal volume

    IEEE Trans. Image Process.

    (2005)
  • W. Bouthour et al.

    A human mutation in Gabrg2 associated with generalized epilepsy alters the membrane dynamics of GABAA receptors

    Cereb. Cortex

    (2011)
  • 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)
  • M. Calamai et al.

    Gephyrin oligomerization controls GlyR mobility and synaptic clustering

    J. Neurosci.

    (2009)
  • V.B. Caraiscos et al.

    Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A receptors

    Proc. Natl. Acad. Sci. U. S. A.

    (2004)
  • C. Charrier et al.

    Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane

    J. Neurosci.

    (2006)
  • C. Charrier et al.

    A crosstalk between beta1 and beta3 integrins controls glycine receptor and gephyrin trafficking at synapses

    Nat. Neurosci.

    (2010)
  • M. Dahan et al.

    Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking

    Science

    (2003)
  • Y. De Koninck et al.

    Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABAA receptor channels

    J. Neurophysiol.

    (1994)
  • E. Eugene et al.

    GABA(A) receptor gamma 2 subunit mutations linked to human epileptic syndromes differentially affect phasic and tonic inhibition

    J. Neurosci.

    (2007)
  • G. Gauvain et al.

    The neuronal K–Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines

    Proc. Natl. Acad. Sci. U. S. A.

    (2011)
  • M.C. Gielen et al.

    Benzodiazepines modulate GABAA receptors by regulating the preactivation step after GABA binding

    J. Neurosci.

    (2012)
  • K. Goslin et al.

    Rat Hippocampal Neurons in Low-density Culture

    (1998)
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