Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications

Pharmacology & Therapeutics 98 (2003) 299 – 323 www.elsevier.com/locate/pharmthera

Associate editor: A.L. Morrow

Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications Jean-Marc Fritschy*, Ina Bru¨nig1 Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Abstract g-Aminobutyric acidA (GABAA) receptors mediate most of the fast inhibitory neurotransmission in the CNS. They represent a major site of action for clinically relevant drugs, such as benzodiazepines and ethanol, and endogenous modulators, including neuroactive steroids. Alterations in GABAA receptor expression and function are thought to contribute to prevalent neurological and psychiatric diseases. Molecular cloning and immunochemical characterization of GABAA receptor subunits revealed a multiplicity of receptor subtypes with specific functional and pharmacological properties. A major tenet of these studies is that GABAA receptor heterogeneity represents a key factor for fine-tuning of inhibitory transmission under physiological and pathophysiological conditions. The aim of this review is to highlight recent findings on the regulation of GABAA receptor expression and function, focusing on the mechanisms of sorting, targeting, and synaptic clustering of GABAA receptor subtypes and their associated proteins, on trafficking of cell-surface receptors as a means of regulating synaptic (and extrasynaptic) transmission on a short-time basis, on the role of endogenous neurosteroids for GABAA receptor plasticity, and on alterations of GABAA receptor expression and localization in major neurological disorders. Altogether, the findings presented in this review underscore the necessity of considering GABAA receptor-mediated neurotransmission as a dynamic and highly flexible process controlled by multiple mechanisms operating at the molecular, cellular, and systemic level. Furthermore, the selected topics highlight the relevance of concepts derived from experimental studies for understanding GABAA receptor alterations in disease states and for designing improved therapeutic strategies based on subtype-selective drugs. D 2003 Elsevier Science Inc. All rights reserved. Keywords: GABAA receptor; Gephyrin; Synaptic clustering; Neurosteroid; Temporal lobe epilepsy; Benzodiazepine Abbreviations: BDNF, brain-derived neurotrophic factor; DPC, dystrophin-associated protein complex; GABA, g-aminobutyric acid; GABARAP, GABAA receptor-associated protein; GAD, glutamic acid decarboxylase; GAT-1, g-aminobutyric acidA transporter type 1; GRIF-1, GABAA receptor interacting factor-1; GRAMP-1, g-aminobutyric acidA receptor-associated membrane protein; IPSC, inhibitory postsynaptic current; PKC, protein kinase C; RACK, receptor for activated C kinase.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GABAA receptor subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Identification and neuron-specific expression of GABAA receptor subtypes . 2.2. Segregation of GABAA receptor subtypes in distinct neuronal circuits . . . . 2.3. Tonic inhibition mediated by extrasynaptic GABAA receptor subtypes . . . . Trafficking and clustering of postsynaptic GABAA receptors. . . . . . . . . . . . . 3.1. Cell-autonomous sorting and synaptic targeting of GABAA receptor subtypes 3.2. Multiple roles of GABAA receptor-associated proteins . . . . . . . . . . . .

* Corresponding author. Tel.: +41-1-635-5926; fax: +41-1-635-6874. E-mail address: [email protected] (J.-M. Fritschy). 1 Present address: Friedrich-Miescher-Institute, CH-4058 Basel, Switzerland. 0163-7258/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0163-7258(03)00037-8

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3.3. Presynaptic regulation of GABAA receptor postsynaptic clustering . . . . . . . . . 3.4. Trafficking and internalization of GABAA receptors . . . . . . . . . . . . . . . . 4. Regulation of GABAA receptor expression by neurosteroids: physiological and pathophysiological implications . . . . . . . . . . . . . . . . . . . . . 4.1. Interactions between GABAA receptors and neurosteroids in the stress response . . 4.2. Changes in GABAA receptor function in relation to fluctuating progesterone levels 4.3. Interactions between neurosteroids and ethanol: a common mechanism of action at GABAA receptors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Alterations of GABAA receptor expression and function in neurological and psychiatric disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Regulation of GABAA receptor expression following deafferentation: implications for neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . 5.2. GABAA receptor regulation after focal ischemia . . . . . . . . . . . . . . . . . . 5.3. GABAA receptors in epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Synapse-specific alterations of GABAA receptors in schizophrenia . . . . . . . . . 6. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction As the main inhibitory neurotransmitter in the vertebrate CNS, g-aminobutyric acid (GABA) modulates every aspect of brain function. On the molecular level, the action of GABA is mediated by ionotropic (GABAA) (Barnard et al., 1998) and metabotropic (GABAB) (Bowery et al., 2002) receptors, which are ubiquitously expressed, possibly on every single neuron in the CNS. GABAergic function is fine-tuned at multiple levels (Cherubini & Conti, 2001), including transmitter synthesis by two isoforms of glutamic acid decarboxylase (GAD) (Erlander et al., 1991; Esclapez et al., 1994; Soghomonian & Martin, 1998); vesicular storage (Dumoulin et al., 1999; Gasnier, 2000); Ca2 + dependent and independent release (Wall & Usowicz, 1997; Vautrin et al., 2000; Kirischuk et al., 2002); re-uptake in neurons and glial cells (Borden, 1996; Quick et al., 1997); and activation of multiple receptors, localized pre-, post-, and extrasynaptically. All these components are not only molecularly heterogeneous, but they also are regulated both at the transcriptional and post-transcriptional level, allowing for an extremely complex array of interactions at the molecular, cellular, and systemic level. The significance of GABAergic transmission is underscored by the multiple neurological and psychiatric diseases for which an alteration in the GABAergic system has been postulated (Mohler, 2000), including epilepsy (Duncan, 1999; Olsen et al., 1999; Coulter, 2001), anxiety disorders (Malizia, 1999), ethanol dependence (Morrow et al., 2001), Huntington’s disease (Kunig et al., 2000), Angelman syndrome (DeLorey et al., 1998), and schizophrenia (Lewis, 2000; Nutt & Malizia, 2001; Blum & Mann, 2002). In particular, GABAA receptors represent a major site of action for clinically important drugs, including benzodiazepines, barbiturates, and some general anesthetics, as well as drugs of abuse such as

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ethanol (Sieghart, 1995; Grobin et al., 1998; Dilger, 2002; Mohler et al., 2002). The purpose of this review is to highlight recent advances pertaining to the regulation of GABAA receptor expression and function, with a focus on the mechanisms of synapse formation and postsynaptic clustering of GABAA receptors, on dynamic changes of receptor localization and function, on interactions with endogenous modulators, and on alterations associated with major neurological diseases. The selected topics highlight emerging concepts that challenge commonly held views about the establishment, maintenance, and function of GABAergic synapses in the healthy and diseased brain. In particular, it is becoming evident that the number of GABAA receptors available for synaptic transmission is regulated both on a short- and a long-term basis, and that their functional properties can be adjusted very rapidly in response to numerous stimuli. Much of this progress stems from the discovery and characterization during the 1990s of the molecular heterogeneity of GABAA receptors. These studies demonstrated the existence of multiple subtypes of GABAA receptors with differential function, pharmacology, and regulation. These aspects will be reviewed in the next section to highlight the importance of this concept before discussing regulatory mechanisms in the following sections.

2. GABAA receptor subtypes 2.1. Identification and neuron-specific expression of GABAA receptor subtypes GABAA receptors belong to the superfamily of ligandgated ion channels (Unwin, 1993; Barnard, 2001). Along with glycine receptors, they mediate fast inhibitory neuro-

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transmission in the vertebrate CNS by gating Cl ions through an integral membrane channel. GABAA receptors form multimeric complexes assembled from a family of at least 21 constituent subunits (a1 – 6, b1 –4, g1– 4, d, r1 –3, q, p) (Barnard et al., 1998; Whiting, 1999). The molecular heterogeneity of GABAA receptors is much larger than that of any other ligand-gated ion channel, which renders their functional analysis technically challenging. The subunit composition and stoichiometry of native GABAA receptors have not been elucidated. The available evidence favors the existence of pentameric complexes containing 2a/2b/1g-subunit variants (Farrar et al., 1999; Knight et al., 2000; Baumann et al., 2001; Klausberger et al., 2001). Immunochemical, pharmacological, and functional analyses of GABAA receptors give convergent results that the majority of GABAA receptors contain a single type of a- and b-subunit variant, with the a1b2g2 combination representing the largest population of GABAA receptors, followed by a2b3g2 and a3b3g2. Receptors containing the a4-, a5-, or a6-subunit, as well as the b1-, g1-, g3-, d-, p-, and q-subunits, form minor receptor populations. The rsubunits are expressed primarily in the retina, and correspond to the so-called GABAC receptors (Bormann, 2000). Pharmacological analysis of GABAA receptors immunoprecipitated with antibodies against specific a-subunit variants allows differentiating between GABA A receptor subtypes. The a1-, a2-, a3-, and a5-GABAA receptors correspond to diazepam-sensitive receptors, whereas the a4and a6-GABAA receptors are insensitive to diazepam (Benson et al., 1998; Wingrove et al., 2002). The former are distinguished further by their affinity to zolpidem (a1 > a2 = a3 >> g5) and various b-carbolines (a1 > a2= a3) (Sieghart, 1995; Mohler et al., 1996). Beyond this classical pharmacological distinction, neurosteroids, which are positive allosteric modulators of recombinant and native GABAA receptors (Lambert et al., 2001), are most potent on receptors containing the d-subunit (Adkins et al., 2001; Wohlfarth et al., 2002). Furthermore, novel ligands are being introduced, which exhibit intrinsic activity only on specific GABAA receptor subtypes (Sigel & Dodd, 2001; Collins et al., 2002). Functionally, distinct subunit-specific properties have been identified in both recombinant and native receptors, supporting the concept that GABAA receptor heterogeneity is a major facet determining the functional properties of GABAergic inhibitory circuits (reviewed in Mohler, 2000; Sieghart, 2000). In particular, the type of asubunit determines the kinetics of receptor deactivation (Verdoorn et al., 1990; Hutcheon et al., 2000; Devor et al., 2001), and the presence of the d-subunit results in markedly increased agonist affinity and apparent lack of desensitization (Burgard et al., 1996; Fisher & Macdonald, 1997; Adkins et al., 2001). Immunohistochemical analyses of the distribution of GABAA receptor subtypes based on the visualization of a-subunit variants revealed a region- and neuron-specific distribution pattern (Fig. 1a; Table 1) that is largely con-

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served across species (Fritschy & Mohler, 1995; Waldvogel et al., 1998; Pirker et al., 2000; Schwarzer et al., 2001). These findings also underscored the fact that the functional relevance of a given GABAA receptor subtype is not necessarily correlated with its relative abundance. For example, a3-GABAA receptors are the main GABAA receptor subtype expressed by monoaminergic and basal forebrain cholinergic neurons (Gao et al., 1993) (Table 1), which innervate most of the brain by dense and widespread axonal arbors. a3-GABAA receptors are also uniquely located to regulate the activity of neurons in the thalamic reticular nucleus (Huntsmann et al., 1999), thereby potentially modulating the entire thalamo-cortical network. Yet, a3-GABAA receptors represent only  10% of all GABAA receptors. On the other hand, morphological studies also revealed that the apparent simplicity of certain circuits is masked by an unsuspected abundance of GABAA receptor subtypes. For instance, cerebellar granule cells receive their GABAergic innervation exclusively on distal dendrites from a single source, the Type II Golgi cells of the cerebellum. Nevertheless, these cells express probably more than 12 GABAA receptor subtypes (Sieghart et al., 1999). From these two extreme examples, one might postulate the existence of cell-specific mechanisms regulating the expression GABAA receptor subtypes and their precise subcellular localization. These aspects will be discussed in the next section. 2.2. Segregation of GABAA receptor subtypes in distinct neuronal circuits In the adult brain, the expression of GABAA receptor subtypes exhibits a remarkable region- and neuron-specificity, suggesting that individual subtypes are present in distinct neuronal circuits (Fig. 1a; Table 1). Although the functional relevance at the systemic level of having, for instance, an a1-instead of an a2-GABAA receptor in a given type of neuron remains speculative, the specificity of subtype expression is underscored by the remarkable selectivity of action of diazepam in knock-in mutant mice carrying ‘‘custom-made’’ diazepam-insensitive GABAA receptor subtypes (Rudolph et al., 2001). Thus, abolition of diazepam binding on a2-GABAA receptors in vivo by exchanging the conserved His101 residue with an Arg residue results in selective suppression of the anxiolytic action of this drug, whereas anxiolytic drugs acting by other mechanisms are unaffected (Lo¨w et al., 2000). Although the a2-subunit has a widespread distribution in numerous brain areas, this finding indicates that a2-GABAA receptors are strategically located in circuits mediating the anxiolytic action of diazepam. Likewise, the sedative effects of diazepam are abolished, even at a high dose, in mice carrying a1H101R-GABAA receptors, while its anxiolytic action is fully retained in these mice (Rudolph et al., 1999; McKernan et al., 2000). Again, one concludes that brain circuits containing a1-GABAA receptors mediate sedation,

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and that activation of these receptors by diazepam is ineffective for relieving anxiety. The cellular corollary of the specificity of diazepam action is the synapse-specific distribution of GABAA receptor subtypes, in particular in neurons expressing multiple GABAA receptor subtypes such as hippocampal pyramidal neurons (Figs. 1b and 2). In these cells, a high level of a1-, a2-, and a5-subunit expression has been reported, along with b1-3- and g2-subunit expression (Fritschy & Mohler, 1995; Pirker et al., 2000), suggesting that they express at least three main GABAA receptor subtypes. a1-GABAA receptors are located postsynaptically in a majority of somatodendritic synapses, and to a lesser extent in the axon initial segment. In contrast, a2-GABAA receptors are particularly abundant in the axon initial segment, and are only few in somatodendritic synapses (Nusser et al., 1996a; Fritschy et al., 1998) (Fig. 1b). Finally, a5-GABAA receptors have an extrasynaptic localization, being distributed throughout the somatodendritic compartment of hippocampal pyramidal cells, without being aggregated at postsynaptic sites (Bru¨nig et al., 2002a; Crestani et al., 2002). Their functional role is discussed in Section 2.3. Strikingly, in the soma of hippocampal pyramidal cells, a1- and a2-GABAA receptor subtypes are segregated to distinct synapses formed by two separate populations of basket cells (Nyiri et al., 2001; Klausberger et al., 2002) (Fig. 2). These interneurons, which selectively innervate the somatic region of pyramidal cells, are subdivided into two groups based on differential expression of the neurochemical markers parvalbumin and cholecystokinin (Freund & Buzsaki, 1996) and functional properties, fast-spiking versus regular spiking basket cells, respectively (Pawelzik et al., 2002). Thus, parvalbumin-positive basket cells form GABAergic synapses containing the a1-subunit (Klausberger et al., 2002), whereas cholecystokinin-positive basket cells form GABAergic synapses containing the a2-subunit (Nyiri et al., 2001), suggesting that presynaptic afferents

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modulate the targeting of receptor subtypes to specific types of synapses. It is of note that the CB1-type of cannabinoid receptors are localized presynaptically, selectively on the terminals of CCK-positive basket cells in the hippocampus and amygdala (Katona et al., 1999, 2001) (Fig. 2). These receptors have been shown to mediate depolarizationinduced suppression of inhibition (Wilson et al., 2001), a form of short-term GABAergic plasticity first described in cerebellar Purkinje cells and hippocampal pyramidal neurons (Llano et al., 1991; Pitler & Alger, 1992; Wilson & Nicoll, 2002). Therefore, endogenous cannabinoids potentially modulate hippocampal circuits containing a2-GABAA receptors, while they will have no effect on circuits containing a1-GABAA receptors. Although it is not known whether a2-GABAA receptors involved in the anxiolytic action of diazepam are located in the hippocampus, these observations underscore the existence of multiple, parallel, functionally specialized neuronal circuits within the same brain structure. The segregated distribution of a1- and a2-GABAA receptors in synapses formed by fast- and regular-spiking basket cells on the soma, or in somatic synapses versus axo-axonic synapses, correlates with the differential kinetics of deactivation of these receptor subtypes. Indeed, a1-GABAA receptors are characterized by faster kinetics of deactivation and/or desensitization than a2-GABAA receptors (Brussaard et al., 1997; Hutcheon et al., 2000; Ju¨ttner et al., 2001; Vicini et al., 2001). In line with the results of Pouille and Scanziani (2001), who showed that somatic inhibition in hippocampal pyramidal cells is an important determinant ensuring precise coincidence detection of excitatory inputs, one might hypothesize that GABAA receptors with fast kinetics (i.e., containing the a1-subunit) are involved in precise discrimination of high-frequency signals, whereas channels with slower kinetics (i.e., containing the a2-subunit) might be most efficient for acting as an on/off switch of neuronal activity, in particular on the axon initial segment.

Fig. 1. Distribution of postsynaptic GABAA receptor subunit clusters in vivo and in vitro. a: Cell type-specific expression of GABAA receptor subtypes in hippocampal neurons, shown by double-immunofluorescence for the a1-subunit (green) and the a2-subunit (red). The a1-subunit is most prominent on the interneurons, and exhibits both a clustered distribution on the soma and dendrites, representing presumptive postsynaptic receptors, and a diffuse staining of extrasynaptic receptors. The a2-subunit immunoreactivity appears as brightly stained puncta, surrounding the soma (stars) and the axon-initial segment of pyramidal cells (arrows), representing postsynaptic receptors. b: Selective targeting of clusters of a2-subunit immunoreactivity (red; arrowheads) on the axon initial segment of a cortical pyramidal cell. The cytoskeleton of this neuron is visualized by staining of neurofilaments, using the monoclonal antibody SMI-32 (cyan). Note that only a few a2-subunit-positive clusters are present around the soma. c: ‘‘Matched’’ and ‘‘mismatched’’ GABAA receptor subunit clusters on a cultured hippocampal neuron obtained from embryonic rat hippocampus and maintained in vitro for 21 days, shown by double-staining for GAD (green), labeling GABAergic presynaptic terminals, and the a2-subunit (red). Note that while the majority of GAD-positive terminals are apposed to one or several a2subunit puncta, representing ‘‘matched’’ clusters (arrowheads), numerous a2-subunit puncta are not facing a GABAergic terminal. These puncta most likely represent ‘‘mismatched’’ clusters. d: Identification of mismatched clusters by triple-immunofluorescence staining for the a2-subunit (red), GAD (green), and the Type 1 glutamate vesicular transporter (vGlut1; blue; a selective marker of glutamatergic terminals) on dendrites of cultured hippocampal neurons. Note that all of the a2-subunit clusters are apposed either to a GAD-positive terminal (‘‘matched’’ clusters in a GABAergic synapse) or to a vGlut1-positive terminal (‘‘mismatched’’ clusters). e: Selective co-localization of dystrophin (panel e3; green) with a2-subunit-positive clusters (panel e2; red) in GABAergic synapses (panel e1; GAD staining; blue), and not in mismatched synapses, as shown by triple immunofluorescence staining. Panels e1 – e3 represent the separate signals for each marker; panel e4 represents the superposition of the three markers, with GABAergic synapses containing dystrophin appearing white. f: Colocalization of dystrophin clusters (green) and a2-subunit clusters (red) in vivo, as depicted in a section of the dentate gyrus granule cell layer (gcl) of an adult rat. Individual granule cell somata are surrounded by double-labeled clusters, which are also abundant in the molecular layer (ml) and in the hilus. Two dystrophin-positive blood vessels (arrowheads) are also visible. Scale bars: a, 10 mm; b, 10 mm; c, 5 mm; d, 2 mm; e, 2 mm; f, 10 mm.

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Table 1 Cellular and subcellular localization of GABAA receptor subtypes Main subunit

Proposed subunit repertoire

Regional distribution

Identified neurons

Subcellular localization (synaptic is determined by co-localization with gephyrin)

Major sites of expression

Regions of low or absent expression

a1

a1b2g2

Cerebral cortex (layers I – VI), hippocampus, amygdala, olfactory bulb, thalamus, basal forebrain, globus pallidus, substantia nigra pars reticulata, inferior colliculus, cerebellum, brainstem

Olfactory bulb granule cells, striatum, thalamic reticular nucleus, inferior olive, motoneurons

Mitral cells and short-axon cells (olfactory bulb); principal cells and selected interneurons in cerebral cortex and hippocampus; GABAergic neurons in pallidum and substantia nigra; thalamic relay neurons; Purkinje cells and granule cells.

Synaptic (soma and dendrites) and extrasynaptic in all neurons with high expression

a2

a2b3g2

Cerebral cortex (layers I – IV), hippocampal formation, amygdala, striatum, olfactory bulb, hypothalamus, superior colliculus, inferior olive, motor nuclei

Deep cortical layers, thalamus, pallidum, substantia nigra, inferior colliculus, cerebellum, most of brainstem

Principal cells in hippocampal formation and amygdala; spiny stellate striatal neurons; olfactory bulb granule cells; motoneurons.

Mainly synaptic, enriched in axon initial segment of cortical and hippocampal pyramidal cells

a3

a3bxg2, a3g2, a3q

Cerebral cortex (layers V – VI), amygdala, olfactory bulb, thalamic reticular and intralaminar nuclei, superior colliculus, brainstem, spinal cord, locus coeruleus, raphe, medial septum

Superficial cortical layers, striatum, pallidum, most of thalamus, cerebellum, motoneurons

Tufted cells (olfactory bulb); reticular thalamic neurons; cerebellar Golgi Type II cells; serotonergic and catecholaminergic neurons; basal forebrain cholinergic neurons.

Mainly synaptic, including some axon initial segments; extrasynaptic in inferior olivary neurons

a4

a4bxd

Dentate gyrus, thalamus

Rest of the brain

Dentate gyrus granule cells.

Extrasynaptic (no direct morphological evidence)

a5

a5b3g2

Hippocampus, deep cortical layers, amygdala, olfactor bulb, hypothalamus, superior colliculus, superior olivary nucleus, spinal trigeminal nucleus, spinal cord

Cerebellum, thalamus, inferior colliculus, midbrain tegmentum, brainstem

Pyramidal cells (hippocampus); granule cells and periglomerular cells (olfactory bulb); superior olivary neurons; spinal trigeminal neurons.

Extrasynaptic in hippocampus, cerebral cortex, and olfactory bulb; synaptic and extrasynaptic in spinal trigeminal nucleus and superior olivary nucleus

a6

a6b2,3g2; a6b2,3d; a1a6b2,3g2

Cerebellum, dorsal cochlear nucleus

Rest of the brain

Granule cells (cerebellum)

Synaptic (cerebellar glomeruli) and extrasynaptic on granule cell dendrites and soma

2.3. Tonic inhibition mediated by extrasynaptic GABAA receptor subtypes Unlike glycine receptors, which, for the most part, are localized postsynaptically, a substantial proportion of GABAA receptors is found extrasynaptically, diffusely distributed in somato-dendritic membranes. In addition, the g2subunit exhibits a widespread extrasynaptic localization (Fritschy et al., 1998), indicating that its requirement for postsynaptic clustering (Essrich et al., 1998) (see Section 3.2) does not preclude the existence of extrasynaptic GABAA receptors containing the g2-subunit. It is of note

that the detection of postsynaptic GABAA receptor clusters by immunohistochemistry requires either weakly fixed tissue for light microscopy (Koulen et al., 1996; Fritschy et al., 1998; Giustetto et al., 1998) or the use of post-embedding methods for electron microscopy (Nusser et al., 1994), through which access of antibodies in the synaptic cleft is facilitated. The prominent staining obtained with classical immunohistochemical methods in strongly fixed tissue (which precludes detection of postsynaptic receptors) underscores the abundance of extrasynaptic (and intracellular) GABAA receptors in all CNS areas (Fritschy & Mohler, 1995).

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Fig. 2. Diagrammatic representation of the subcellular distribution of GABAA receptor subtypes in relation to synaptic inputs in a CA1 hippocampal pyramidal cell. A segregation of a1- and a2-GABAA receptor clusters is evident on the soma and axon initial segment according to different populations of GABAergic interneurons. Extrasynaptic a5-GABAA receptors are distributed on the entire somato-dendritic compartment. CCK, cholecystokinin; PV, parvalbumin. See text for details.

In addition to the a5-GABAA receptors in hippocampal pyramidal cells (see Section 2.2), at least two other GABAA receptor subtypes appear to be localized selectively at extrasynaptic sites (Table 1). In cerebellar granule cells, receptors containing the subunit combination a6bd form a population of extrasynaptic receptors providing tonic inhibition, as shown by light and electron microscopy (Nusser et al., 1996b, 1998; Sassoe`-Pognetto et al., 2000), and electrophysiological recording in slices (Brickley et al., 1996). The assembly of these receptors is dependent on the presence of the a6-subunit, as shown in a6-subunit-deficient mice, in which the d-subunit protein becomes undetectable in the cerebellum (Jones et al., 1997). The a6-subunit seems to direct GABAA receptors mainly to non-synaptic sites, since its ectopic expression in hippocampal pyramidal cells leads to the formation of extrasynaptic receptors mediating tonic inhibition (Wisden et al., 2002). In dentate gyrus granule

cells, a4bd receptors likewise mediate tonic inhibition, and are most likely extrasynaptic (Nusser & Mody, 2002), although there is no morphological evidence for their precise subcellular localization. In these cells, the mean tonic current measured in acute slices is about 4 times larger than the mean phasic currents generated by postsynaptic receptors, underscoring the potential importance of extrasynaptic receptors for the control of neuronal excitability (Nusser & Mody, 2002). This tonic current was not responsive to modulation by benzodiazepines, in line with the lack of diazepam sensitivity of a4-GABAA receptors. In heterologous expression systems, GABAA receptors containing the a-subunit are characterized by high-agonist affinity and slow desensitization, making them suitable for sensing low ambient GABA concentrations (Saxena & Macdonald, 1994). Likewise, GABAA receptors mediating tonic inhibition in dentate granule cells have a higher affinity for

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GABA compared with those responsible for phasic inhibition (Stell & Mody, 2002), in line with their need to be activated by low GABA concentration in the extracellular space. Extrasynaptic GABAA receptors likely contribute to normal GABAergic inhibition in vivo and are involved in the expression of specific behaviors. This was shown by functional and behavioral analysis of mice with targeted mutations of the a5- and d-subunit genes. Thus, a5-subunitdeficient mice exhibit enhanced learning of a hippocampaldependent task (spatial learning), but no change in anxiety responses (Collinson et al., 2002). The amplitude of spontaneous inhibitory postsynaptic potentials recorded in hippocampal pyramidal cells in a slice preparation was slightly reduced compared with wild-type mice, but it was not determined whether this change corresponds to loss of a5GABAA receptors or to compensatory alterations of remaining subunits (Collinson et al., 2002). Tonic inhibition was not tested in these mice. A selective role of a5-GABAA receptors in hippocampal-dependent tasks was also reported in a mouse line carrying an H105R point-mutation in the a5-subunit gene (Crestani et al., 2002). Unexpectedly, this mutation caused a profound reduction of a5-GABAA receptors selectively in the hippocampal formation, and was correlated with altered performance in a delay trace-conditioning task. In contrast, learning of non-hippocampus-dependent behavioral tasks was not affected, pointing to a selective role of extrasynaptic GABAA receptors for proper hippocampal function (Crestani et al., 2002). d-Subunit-deficient mice exhibit spontaneous seizures and a selective reduction of responses to neuroactive steroids (Mihalek et al., 2001) (see Section 4.2). They are also more sensitive to pentylenetetrazole-induced seizures, and this phenotype was correlated with a faster decay of evoked and miniature inhibitory postsynaptic potentials in dentate gyrus granule cells (Spigelman et al., 2002), a major site of expression of the d-subunit. Morphologically, the absence of the d-subunit was paralleled with a strong reduction of a4subunit labeling and an increased expression of the g2subunit (Korpi et al., 2002; Peng et al., 2002), indicating an obligatory association between the a4- and d-subunits for receptor formation and replacement of missing a4bg receptors by receptors containing the g2-subunit. The subcellular localization of these new receptors in relation to synaptic sites has not been established. These and other compensatory regulatory mechanisms activated in mutant mice (Brickley et al., 2001; Tretter et al., 2001) limit the use of the knockout gene strategy to investigate the functional role of individual GABAA receptor subtypes in vivo.

3. Trafficking and clustering of postsynaptic GABAA receptors Synapses are highly specialized subcellular compartments containing a vast array of proteins that are required presynap-

tically for transmitter synthesis, storage, release, and reuptake, and postsynaptically for signal reception and transduction. A probably even larger number of proteins provide a structural scaffold ensuring appropriate assembly, location, and function of the pre- and postsynaptic specializations. For instance, an ensemble of  70 proteins has been identified in the postsynaptic densities of excitatory synapses, based on proteomics analysis of biochemically purified synaptic preparations, including glutamate receptor subunits, signaling molecules, and scaffolding proteins (Husi et al., 2000; Walikonis et al., 2000). In addition to this structural complexity, it is becoming increasingly evident that receptors and signaling proteins are not statically anchored in the postsynaptic density, but that they shuffle constantly between the postsynaptic membrane domain and the adjacent extrasynaptic or intracellular domains. In particular, internalization and recycling of receptors seems to represent an important mechanism for short-term plasticity of synaptic transmission (Kittler & Moss, 2001; Kneussel, 2002) (see Section 3.4). Postsynaptic densities of inhibitory synapses cannot be isolated selectively in biochemical preparations, and much less is known about their molecular constituents. The available data indicate, however, that they are markedly distinct from excitatory postsynaptic densities, the main difference being the apparent absence of proteins with PDZ domains mediating protein-protein interactions (Sheng & Sala, 2001). Attempts to isolate GABAA receptor- or glycine receptorinteracting proteins using the yeast-two-hybrid system have proven rather deceiving, although the number of candidates is increasing steadily (for reviews, see Kneussel & Betz, 2000; Luscher & Fritschy, 2001; Moss & Smart, 2001). In this section, we will review current knowledge about the mechanisms governing the sorting, targeting, and clustering of GABAA receptors and associated-proteins, and the dynamic regulation of GABAA receptor subtypes in GABAergic synapses. Sorting and targeting mechanisms determine the subcellular compartment in which receptors are localized, and clustering refers to the aggregation at synaptic sites. 3.1. Cell-autonomous sorting and synaptic targeting of GABAA receptor subtypes The mechanisms underlying the cell-specific expression of GABAA receptor subtypes have been investigated in primary cultures of dissociated hippocampal neurons (Bru¨nig et al., 2002a). Under these conditions, the precise, layerand cell-specific innervation pattern formed by GABAergic interneurons is largely disrupted. If presynaptic terminals carry important information governing the sorting of postsynaptic GABAA receptor subtypes, specific cellular and subcellular organization of GABAA receptor subtypes would be altered in vitro. Yet, the results of these experiments show that the cell-type and synapse-specific expression of GABAA receptor subtypes is largely preserved in these cultures, notably with regard to localization of the a2-subunit on the axon initial segment and extrasynaptic distribution of the

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a5-subunit in pyramidal cells (Bru¨nig et al., 2002a). These observations suggest that the cell-specific expression and the subcellular localization of GABAA receptor subtypes are governed by largely cell-autonomous mechanisms. We postulate the existence of protein-protein interaction mechanisms that depend on sequence motifs present in particular GABAA receptor subunits to ensure appropriate sorting and synaptic targeting. These mechanisms are expected to be neuron-specific, as shown for the a5-subunit, which is extrasynaptic in hippocampal pyramidal cells and olfactory bulb granule cells, but postsynaptic in neurons of the spinal trigeminal nucleus (Crestani et al., 2002). It should be emphasized that the site(s) of GABAA receptor sorting and membrane insertion has not been identified. In the case of glycine receptors, there is evidence that newly synthesized receptors are inserted in the membrane mainly at non-synaptic sites on the cell body and proximal dendrites (Rosenberg et al., 2001). The receptors appear as small clusters that are transported to distal dendrites by lateral diffusion in the plasma membrane. If such a process applies for GABAA receptors, it is hard to envision how the targeting to specific synaptic sites occurs. However, this process could represent a simple mechanism for cell surface expression of extrasynaptic receptors. 3.2. Multiple roles of GABAA receptor-associated proteins The appropriate subcellular and synaptic localization of GABAA receptors depends on sorting, targeting, and clustering mechanisms that probably are mediated by distinct proteins (Table 2). The first GABAA receptor-associated protein identified was gephyrin, a 93-kDa peripheral protein initially co-purified with glycine receptors (Pfeiffer et al., 1984). Ultrastructurally, gephyrin is selectively restricted to the postsynaptic density of symmetric synapses (Triller et al., 1985; Sassoe`-Pognetto et al., 1995, 2000; Giustetto et al., 1998; Lim et al., 1999). As shown by high-resolution fluorescence microscopy in brain sections and in primary neuron cultures (Fig. 1e), GABAA receptor subunits form postsynaptic clusters precisely co-localized with gephyrin (Rao et al., 2000a; Sassoe`-Pognetto et al., 2000; Bru¨nig et al., 2002a, 2002b). The importance of this interaction has been demonstrated in g2-subunit-deficient mice, in which postsynaptic clustering of both gephyrin and major GABAA receptor subtypes is severely impaired (Essrich et al., 1998). The same effect is observed in gephyrin-deficient mice (Kneussel et al., 1999), indicating that GABAA receptors and gephyrin are interdependent for postsynaptic clustering and that their functional interaction requires the g2-subunit. However, since a direct binding between gephyrin and GABAA receptor subunits has never been demonstrated biochemically (Meyer et al., 1995), additional, as yet unidentified, partners are likely to be involved. In g2subunit-deficient mice, clustering of gephyrin and GABAA receptors can be re-established by transgenic expression of the g3-subunit (Baer et al., 1999), suggesting that the

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required interaction motifs are shared by the g2- and g3subunits. Finally, gephyrin-independent clustering of GABAA receptors has been described in vivo and in vitro, notably in the retina and spinal cord of gephyrin-deficient mice (Fischer et al., 2000; Kneussel et al., 2001), but direct evidence for the postsynaptic localization of these receptors is lacking. Several proteins interacting with gephyrin have been identified, including collybistin, profilin, GABARAP (see below) and dynein light-chain 1 and 2 (for reviews, see Luscher & Fritschy, 2001; Moss & Smart, 2001; Kneussel, 2002). However, none of these proteins is directly involved in the postsynaptic clustering of GABAA receptors. Gephyrin can also interact with itself and with microtubules (for a review, see Kirsch, 1999) (Table 2). This property represents the strongest evidence to date for its role as an anchoring protein for both glycine receptors and GABAA receptors. It is unclear, however, whether tubulin binding is required to maintain gephyrin clusters in mature synapses, or whether it is only crucial in immature cells. Indeed, disruption of microtubules with colchicine strongly affects glycinergic transmission and gephyrin clustering in immature, but not in mature, spinal cord neurons in vitro (van Zundert et al., 2002). Likewise, disruption of microtubules in mature hippocampal neurons does not affect the clustering of GABAA receptor subunits and gephyrin (Allison et al., 1998). A second protein that is clustered selectively with GABAA receptors in a subset of synapses in the cerebral cortex, hippocampus, and cerebellum is dystrophin (Knuesel et al., 1999) (Table 2). While a direct or indirect interaction with GABAA receptor subunits has not been demonstrated, the absence of dystrophin in a mutant mouse (mdx) leads to a marked reduction in the apparent size and number of GABAA receptor clusters, but not gephyrin clusters, selectively in these regions (Knuesel et al., 1999). Dystrophin, therefore, might regulate the stability or size of GABAA receptor clusters, but does not influence the clustering of gephyrin. In cultured hippocampal neurons, several members of the dystrophin-associated protein complex (DPC), including dystrophin itself, a-dystroglycan, b-dystroglycan, and syntrophin, have also been shown to be clustered with GABAA receptors and gephyrin at GABAergic postsynaptic sites (Bru¨nig et al., 2002b; Levi et al., 2002). In view of the structural homology between GABAA and nicotinic acetylcholine receptors, the presence of the DPC in GABAergic synapses suggests that it regulates the function of both types of synapses via homologous mechanisms. The analysis of mutant mice lacking agrin, gephyrin, or dystrophin failed to influence the clustering of dystroglycan in cultured neurons (Levi et al., 2002). Furthermore, in dystroglycan-deficient neurons, formation of GABAergic synapses and clustering of GABAA receptors and gephyrin were not altered, although these synapses did not contain any dystrophin immunoreactivity (Levi et al., 2002). These results show that the DPC is not required for GABAergic synaptogenesis, confirming the analogy with the neuromuscular junction, where the DPC is also dispens-

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Table 2 GABAA receptor-associated proteins Protein (main references)

Gephyrin (Essrich et al., 1998; Kneussel et al., 1999; Sabatini et al., 1999; Kins et al., 2000; Fuhrmann et al., 2002)

Other binding partners

g2, g3

Tubulin, collybistin, GABARAP, profilin 1, dynein LC1 and LC2, RAFT1

No direct interaction demonstrated

Dystrophin-associated protein complex (dystroglycan, syntrophin, dystrobrevin) Gephyrin, P130, NSF, transferrin receptor

Postsynaptic density; co-localized with GABAA receptors and gephyrin in a subset of GABAergic synapses Immunoprecipitation (brain membranes); interaction site mapped

Dystrophin (Kneussel et al., 1999; Bru¨nig et al., 2002b; Levi et al., 2002)

GABARAP (Wang et al., 1999; Kneussel et al., 2000; Kittler et al., 2001; Kanematsu et al., 2002) AP2 (Kittler et al., 2000)

g2

In vitro interaction

GRIF-1 (Beck et al., 2002)

b1-, b3-, g2subunits (not a1 – 6) a- and b-subunits (not g2 or d) b2

GRAMP-1 (Keller et al., 2002)

g2

PKC (Brandon et al., 2000; Kumar et al., 2002b)

a1, a4, b1, b3

RACK-1

Pull-down assays, immunoprecipitation (brain membranes)

RACK1 (Brandon et al., 2002b)

b1, b3

PKC

Pull-down assays, immunoprecipitation (brain membranes)

GC1q-R (Schaerer et al., 2001)

bx (not a1 or g2) N.D.

PLIC-1 (Bedford et al., 2001)

GTAP34 (serine kinase) (Kannenberg et al., 1999a) D5 dopamine receptor (Liu et al., 2000)

Disruption of interaction with competing peptide Immunoprecipitation (brain membranes) Pull-down assays, immunoprecipitation (brain membranes) Pull-down assays

G2

N.D., not determined; NSF, N-ethylmaleimide-sensitive factor.

Immunoprecipitation (brain membranes) Phosphorylation of b3-subunit Pull-down assays, immunoprecipitation (brain membranes)

Subcellular localization

Co-localization with GABAA receptors In vitro

In vivo

Postsynaptic density of symmetric synapses

x

x

x

Intracellular (Golgi apparatus); no synaptic enrichment Clathrin-coated vesicles Inhibitory synapses and subsynaptic sites

Intracytoplasmic (Golgi apparatus)

x

x

x

x

x

x

Proposed functions

Anchoring GABAA receptor and glycine receptors at postsynaptic sites, interactions with the cytoskeleton, regulation of protein synthesis Stabilization of postsynaptic GABAA receptor clusters

Intracellular GABAA receptor trafficking, membrane targeting, receptor degradation Regulation of endocytosis

x

Regulation of GABAA receptor cell surface expression Unknown

x

Unknown

x

Regulation of GABAA receptor function and cell surface expression, target of ethanol regulation Potentiation of phosphorylation by PKC; regulation of GABAA receptor function by serotonin and acetylcholine Unknown

Intracellular

x

x

Modulation of receptor function Modulation of receptor function and/or cell surface expression

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Interacting subunits

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able for synapse formation and initial clustering of acetylcholine receptors (Grady et al., 2000). Conversely, clustering of the DPC in GABAergic synapses does not require GABAA receptor or gephyrin clustering, as shown in cultures derived from g2-subunit-deficient mice, in which dystrophin and b-dystroglycan clusters were formed normally in the absence of GABAA receptor and gephyrin clustering (Bru¨nig et al., 2002b). With regard to the possible role(s) of the DPC in GABAergic synapses, two alternative views can be envisaged. Given the fact that dystrophin expression in the CNS is increased late during development (Knuesel et al., 2000), it is conceivable that the DPC stabilizes GABAergic postsynaptic densities after the stage of circuit formation and synaptic remodeling. According to this view, the DPC would maintain existing synaptic connections and prevent excessive remodeling, and might be important for long-term maintenance of GABAergic synapses. Conversely, since the DPC is expressed predominantly in brain regions displaying the highest levels of synaptic plasticity in adult animals, such as the hippocampus and neocortex, it is also possible that the DPC provides a scaffold, enabling changes in clustered GABAA receptor number without loosing the GABAergic postsynaptic apparatus. In view of the decisive role of the g2-subunit for postsynaptic clustering of GABAA receptors, proteins interacting with this subunit have been sought extensively (Table 2). A major candidate is GABAA receptor-associated protein (GABARAP), a microtubule-associated protein that binds to the intracellular loop of the g2-subunit (Wang et al., 1999; Nymann-Andersen et al., 2002). Disruption of this interaction using a membrane-permeant peptide inhibits cluster formation of recombinant GABAA receptors expressed in fibroblasts (Kittler et al., 2001). However, a role for GABARAP in GABAA receptor postsynaptic clustering in vivo is unlikely for several reasons: GABARAP is enriched in the Golgi apparatus and its subcellular distribution is unaffected in gephyrin-deficient mice (Kneussel et al., 2000); GABARAP interacts with N-ethylmaleimide-sensitive factor, a protein involved in intracellular membrane trafficking events (Kittler et al., 2001); and finally, GABARAP has several homologues, which have been implicated in axon elongation and vesicular transport (Okazaki et al., 2000). Current views, therefore, favor involvement of GABARAP in intracellular transport of GABAA receptors for membrane targeting and/or for degradation. The significance of the binding of GABARAP to the g2subunit has been highlighted in a recent publication describing a protein with a domain organization similar to phospholipase Cd, but catalytically inactive, which interacts with GABARAP and competitively inhibits binding of the g2subunit to GABARAP (Kanematsu et al., 2002) (Table 2). Inactivation of this protein, p130, by homologous recombination was related to deficits in pharmacological modulation of GABAA receptors by Zn2 + and diazepam, as well as altered sedative and anxiolytic effects of diazepam, suggesting a critical role for GABAA receptor function. It is not

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known, however, whether cell-surface targeting or clustering of GABAA receptors is affected in p130-deficient mice. Yeast-two-hybrid screens using the intracellular loop of a-subunit variants identified a ubiquitin-related protein, Plic-1, which is likely to regulate the number of GABAA receptors at the cell surface, either by facilitating their membrane insertion or by slowing their degradation following clathrin-dependent endocytosis (Bedford et al., 2001) (see Section 3.4). Additional proteins that might modulate intracellular trafficking of GABAA receptors are GABAA receptor interacting factor-1 (GRIF-1) (Beck et al., 2002) and GABA A receptor-associated membrane protein (GRAMP-1) (Keller et al., 2002), which bind to GABAA receptor subunits in in vitro assays (Table 2). Conclusive evidence for their function in vivo is still lacking. 3.3. Presynaptic regulation of GABAA receptor postsynaptic clustering In CNS neurons, which receive multiple inputs from different neurotransmitter systems, an appropriate match between the transmitter and its corresponding receptors and associated proteins is essential for appropriate synapse function. Since synapses using different neurotransmitters can be very close to each other in CNS neurons, highly efficient and precise sorting mechanisms are required to ensure appropriate matching. The role of presynaptic afferents and of neuronal activity for synapse formation and maintenance has been the object of intense scrutiny (for a review, see Craig & Boudin, 2001). The evidence available indicates that bi-directional interactions are required for appropriate formation and differentiation of synapses, but the nature of the signals involved remains largely not known. Presynaptic elements appear to initiate synapse formation, and this step requires homophilic interactions between pre- and postsynaptic SynCAM, a brain-specific immunoglobulin domain-containing protein (Biederer et al., 2002), and possibly heterophilic interactions between neurexin presynaptically and neuroligin postsynaptically (Song et al., 1999; Scheiffele et al., 2000). An elegant approach to determine whether the neurotransmitter itself plays a crucial role to specify the identity of the postsynaptic element was followed by Rao et al. (2000a), who cultured hippocampal neurons in isolation and correlated the distribution of preand postsynaptic markers of GABAergic and glutamatergic synapses with the neurotransmitter phenotype of the neuron. Under such conditions, where only autapses are permitted, a clear mismatch was observed, with clustering of GABAA receptors and gephyrin under presynaptic glutamatergic autapses in pyramidal cells and clustering of glutamate receptor subunits and PSD-95 in autapses of GABAergic neurons (Rao et al., 2000a). These results suggested the existence of a ‘‘general’’ synaptogenic factor that is sufficient to induce clustering of GABAA and ionotropic glutamate receptors and anchoring protins, irrespective of the nature of the neurotransmitter involved.

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Mismatched synapses were also observed in neurons receiving a dual glutamatergic and GABAergic innervation, selectively in dendrites receiving only few GABAergic contacts, in which GABAA receptors and gephyrin were clustered under glutamatergic terminals (Bru¨nig et al., 2002b; Christie et al., 2002) (Fig. 1c, d). Appropriately matched receptor clusters were seen apposed to GABAergic terminals, and no mismatched synapses were observed in neurons receiving extensive GABAergic innervation. The lack of mismatched synapses in the latter case suggests that the factor(s) specific for GABAergic synapses overrides the effect of the ‘‘general’’ factor for clustering of both GABAA receptors and gephyrin. Remarkably, clusters of dystrophin and the DPC were never observed in mismatched synapses (Bru¨nig et al., 2002b) (Fig. 1e), indicating that they are dependent on a factor specific for GABAergic synapses and are not responsive to the ‘‘general’’ synaptogenic factor. A more complex situation occurs in neurons co-expressing glycine receptors and GABAA receptors, which are both dependent on gephyrin for postsynaptic clustering. In cultures of purified motoneurons supplemented with either GABAergic neurons or with a mixture of GABAergic and glycinergic neurons, Levi et al. (1999) demonstrated that postsynaptic GABAA receptor clusters containing gephyrin occurred in motoneurons of both types of cultures, whereas glycine receptor clusters were seen only in the latter cultures. These results indicate that the presence of gephyrin clusters is not sufficient for clustering of glycine receptors, but that a presynaptic factor derived from glycinergic terminals is required in addition. A similar conclusion can be derived from a recent study on cultured neurons of the superior colliculus (Meier et al., 2002). In these cultures, there is a switch from glycinergic to GABAergic transmission during development, occurring both pre- and postsynaptically. At early stages in culture, glycine receptors are clustered (with gephyrin) postsynaptically to glycinergic afferents, whereas at later stages, presynaptic terminals express GAD65 and glycine receptors are replaced by GABAA receptors that are co-localized with gephyrin at postsynaptic sites. Intriguingly, glycine receptors are redistributed to extrasynaptic sites in mature cells, and chronic blockade of Type I metabotropic receptors prevents this redistribution, suggesting that synaptic clustering of glycine receptors is activity-dependent (Meier et al., 2002). Taken together, these results strongly support the existence of synapse-specific factors regulating clustering of GABAA and glycine receptors at appropriate synaptic sites. While a logical candidate as a synapse-specific factor would be the neurotransmitter itself, or a signaling cascade activated by the transmitter, the available evidence argues against this hypothesis, at least for GABAA receptors. Chronic blockade of action potential-mediated synaptic events with tetrodotoxin, or blockade of GABAA receptors with bicuculline, does not affect their postsynaptic clustering and co-localization with gephyrin (Craig et al., 1996; Rao et al., 2000b; Christie et al., 2002; Studler et al., 2002).

This is different, however, for glycine receptors, which do not form clusters upon chronic strychnine treatment in immature cells (Kirsch & Betz, 1998; Levi et al., 1998) and are rapidly internalized and degraded in mature neurons. Conflicting results were obtained with respect to gephyrin clustering, and the interpretation of these experiments is not straightforward. A recent study has shown, however, that functional glycine receptors can mature in dopaminergic neurons of the substantia nigra in the absence of glycinergic input or glycine receptor activation by another transmitter such as taurine (Mangin et al., 2002). Genetic approaches in Caenorhabditis elegans provide a more direct test for the role of neurotransmitter in receptor clustering. These results show that GABA is not required for differentiation of the neuromuscular junction and proper postsynaptic clustering of GABA receptors in these animals (Gally & Bessereau, 2002). If the transmitter is not essential for specifying the identity of the postsynaptic element, other candidates may include proteins, such as the cadherins and protocadherins, neuroligin, ephrins, and sidekicks, which have been involved in synaptogenesis and in the formation of topographic maps in the brain (Rao et al., 2000b; Wilkinson, 2001; Goda, 2002; Knoll & Drescher, 2002; Yamagata et al., 2002) and are present at synaptic sites. In the neuromuscular junction, anterograde signaling with agrin plays a preponderant role for synapse differentiation and acetylcholine receptor clustering. Although the contribution of the DPC for agrin signaling is debated (Ferns & Carbonetto, 2001; Sanes & Lichtman, 2001), it will be very interesting to determine whether a signaling pathway with a homologous function to the agrin/MusK pathway is present in central GABAergic synapses. It should be emphasized, however, that there is no reason to expect that neurotransmitter receptors and their associated proteins should be sorted by the same mechanism(s). The fact that dystrophin and other proteins of the DPC do not occur in mismatched synapses in vitro, whereas GABAA receptors and gephyrin can be found in both matched and mismatched synapses (Bru¨nig et al., 2002b), strongly suggests that the sorting mechanisms of these proteins are different. The identification of the ‘‘synapse-specific’’ clustering factors will be a major goal of future studies. The role of presynaptic terminals for initiating clustering of GABAA receptors and gephyrin and for long-term maintenance of the postsynaptic element following synapse maturation is also debated (Craig & Boudin, 2001). In cerebellar granule cells in vitro, which form relatively few GABAergic synapses compared with glutamatergic synapses, GABAergic afferents have been shown to influence the distribution of gephyrin clusters with a mechanism independent of GABAA receptor activity (Studler et al., 2002), confirming the existence of a synapse-specific signaling mechanism for induction of postsynaptic sites. However, in hippocampal neuron cultures, a substantial fraction of GABAA receptor clusters was reported not to be apposed

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to presynaptic terminals (Kannenberg et al., 1999b; Scotti & Reuter, 2001), suggesting that they might form at extrasynaptic sites. The latter studies did not examine whether these clusters contain gephyrin, and, therefore, it is difficult to evaluate the significance of these results in relation to the mechanism of synapse formation. In vivo, a recent study has evaluated the requirement of presynaptic afferents for long-term maintenance of the GABAA receptor and gephyrin clusters in two different mutant mouse lines undergoing postnatal degeneration of Purkinje cells (Lurcher mice and Purkinje-Cell-Degeneration mice) (Garin et al., 2002). While the initial formation of GABAergic synapses and gephyrin and GABAA receptor clusters was normal in neurons of the deep cerebellar nuclei in both mouse lines, the degeneration of Purkinje cells induced a loss of gephyrin clusters following the degeneration of GABAergic terminals. In contrast, GABAA receptor clusters were preserved, and even increased, in number and size (Garin et al., 2002). Therefore, the loss of gephyrin might be taken as evidence for the disappearance of the postsynaptic density following deafferentation, suggesting that presynaptic terminals are required for maintaining postsynaptic GABAergic specializations. The majority of the remaining GABAA receptor clusters was not facing one of the remaining GAD- or synapsin-positive terminals, suggesting their translocation to extrasynaptic sites. Taken together, the results discussed in this section emphasize the active role played by GABAergic presynaptic elements for formation and maintenance of postsynaptic sites containing appropriately clustered GABAA receptors, gephyrin, and other associated proteins such as the DPC. Communication across the synapse involves multiple signals, but activation of GABAA receptors seems to be dispensable for the building of GABAergic synapses. 3.4. Trafficking and internalization of GABAA receptors Regulation of the number of neurotransmitter receptors inserted in the postsynaptic membrane represents a powerful mechanism for rapid and transient changes in synaptic strength (Turrigiano, 2000; Kittler & Moss, 2001; Sheng & Lee, 2001). In principle, membrane receptor density at any given time point is the result of three major components: rate of membrane insertion, rate of endocytosis, and speed of lateral mobility. There is increasing evidence for the various members of the family of ligand-gated channels for dynamic regulation of synaptic and extrasynaptic receptor density, with endocytosis followed by recycling or degradation, representing, perhaps, the major factor for short-term regulation of neuronal function (Carroll et al., 1999; Kittler et al., 2000; Lin et al., 2000; Barnes, 2001). The analysis of the function of Plic-1 (see Section 3.2) also has emphasized the importance of the dynamic regulation of cell surface expression of GABAA receptors, and has suggested an alternative hypothesis to the concept of clus-

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tering (Bedford et al., 2001). Indeed, assuming that GABAA receptors are shuffled continuously between the postsynaptic membrane and a subsynaptic compartment, shifting this equilibrium in one or the other direction is likely to have a major influence on the number of receptors available for synaptic transmission at a given time-point. Furthermore, the importance of other mechanisms should not be underestimated, as underscored by the recent demonstration that glycine receptors and AMPA receptors are highly mobile in the plasma membrane and can reversibly enter or leave zones of confinement most likely corresponding to postsynaptic sites, in which they stay immobile only for short periods of time (Meier et al., 2001; Borgdorff & Choquet, 2002). The ‘‘release’’ of receptors from zones of confinement stresses the importance of considering receptor clustering as a dynamic process, and not as a permanent anchoring and immobilization in the postsynaptic density. Clathrin-dependent endocytosis is a major mechanism for recycling and degradation of membrane proteins, and it plays an essential role in desensitization of G-proteincoupled receptors (for reviews, see Ferguson, 2001; Tsao & von Zastrow, 2001). For GABAA receptors, it has been suggested to occur constitutively in A293 cells expressing recombinant a1b3g2 receptors, in cultured hippocampal neurons, and in rat cerebral cortex (Connolly et al., 1999a; Kittler et al., 2000; Kittler & Moss, 2001; Kumar et al., 2002a). In addition, clathrin-independent endocytosis has also been reported (Cinar & Barnes, 2001). A putative interaction between the clathrin adaptor protein AP2 and the intracellular domains of the b1-, b3-, and g2-subunits, but not of any of the a1-a6-subunit, was identified using pull-down assays (Table 2). Most importantly, blockade of clathrin-dependent endocytosis with a peptide interfering with the association between amphiphysin and dynamin resulted in a large increase in the amplitude of miniature inhibitory postsynaptic currents (IPSCs) in cultured neurons (Kittler et al., 2000). These results support the conclusion that the number of GABAA receptors at the cell surface depends on a dynamic equilibrium between insertion and removal. Protein phosphorylation mechanisms, notably mediated by protein kinase A, protein kinase C (PKC), as well as several serine and tyrosine kinases, have been shown to modulate GABAA receptor function (Brandon et al., 2001, 2002a; Balduzzi et al., 2002). In addition to direct effects on channel-gating properties, phosphorylation of GABAA receptor subunits have multiple effects related to cycling between synaptic sites and intracellular compartments. In recombinant expression systems, for example, activation of PKC increases the rate of GABAA receptor internalization (Connolly et al., 1999a, 1999b; Filippova et al., 2000). It should be emphasized, however, that a direct phosphorylation of GABAA receptor subunits does not appear to regulate cell surface stability. Intermediate substrates, possibly including GABAA receptor-interacting proteins, could be involved (Brandon et al., 2000). In addition, it is not

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known whether PKC activation stimulates the rate of endocytosis or the rate of degradation of internalized receptors in vivo (Brandon et al., 2000). Recent evidence indicates that PKC and its anchoring protein, receptor for activated C kinase (RACK) 1, directly bind to specific sites on the GABAA receptor b-subunits and that RACK1 potentiates GABAA receptor phosphorylation by PKC (Brandon et al., 2002b) (Table 2). RACK1 binding is also important for modulation of GABAA receptor function upon activation of metabotropic serotonin and acetylcholine receptors that are positively coupled to phospholipase C and PKC (Feng et al., 2001; Brandon et al., 2002b). Suppression of GABAA receptor signaling by PKC phosphorylation, therefore, might represent an important, novel mechanism for serotonergic and cholinergic modulation of neuronal activity in vivo. Finally, evidence that PKC might regulate cell surface expression of GABAA receptors in cerebral cortex in vivo has been provided recently by Kumar et al. (2002b), who demonstrated a direct association between PKC-g and GABAA receptors containing the a1- or a4subunit. This association was shown to be increased for the a4-subunit and decreased for the a1-subunit upon chronic ethanol treatment. Since ethanol also increases a1-subunitimmunoreactivity in clathrin-coated vesicles (Kumar et al., 2002a), these ethanol-induced changes in association of PKC with GABAA receptors could influence surface expression or functional properties of GABAA receptors. Activation of tyrosine kinase receptors, in particular, ligand-activated receptors, has also been shown to regulate the function (Tanaka et al., 1997; Henneberger et al., 2002) and cell-surface expression of GABAA receptors (Bru¨nig et al., 2001). The regulation is bi-directional, with insulin treatment leading to a rapid recruitment of cell-surface GABAA receptors (Wan et al., 1997), and brain-derived neurotrophic factor (BDNF) application having the opposite effect (Bru¨nig et al., 2001). The effects of BDNF are mediated postsynaptically, since application of this neurotrophin leads within minutes to a decrease in amplitude, but not in frequency or kinetic, of miniature IPSCs in cultured hippocampal neurons (Bru¨nig et al., 2001; Jovanovic et al., 2001). Furthermore, this effect is paralleled by a decreased cell surface immunoreactivity of GABAA receptor subunits that can be prevented by blockade of tyrosine kinase signaling. It has also been shown that BDNF application leads to a rapid and transient phosphorylation of the b3-subunit, but it is not established which kinase is involved, nor whether this effect relates to changes in cell surface expression (Jovanovic et al., 2001). However, Tanaka et al. (1997) showed that the reduction of GABAA receptor function by BDNF is prevented by postsynaptic Ca2 + chelation or of phospholipase C inhibition, suggesting a possible involvement of PKC. There is evidence from early studies that agonist-induced activation of GABAA receptors increases clathrin-dependent endocytosis (Barnes & Calkin, 1994). Likewise, chronic benzodiazepine treatment in vivo and in recombinant receptors increases GABAA receptor internalization (Tehrani &

Barnes, 1991). This mechanism has even been proposed to explain allosteric uncoupling between GABA and benzodiazepine site agonists observed following chronic treatment (Ali & Olsen, 2001). Since benzodiazepines are membranepermeant, binding to internalized receptors remains possible, whereas agonist activation is reduced; hence, the uncoupling observed in pharmacological assays. It should be stressed, however, that this hypothesis is based on a heterologous expression system in which GABAA receptors are not clustered in specialized domains such as the postsynaptic density, and, most importantly, in which they lack many of their interacting proteins such as gephyrin. While the in vivo relevance of this observation remains to be established, the concept is appealing because it would explain how pharmacodynamic tolerance to chronic benzodiazepine treatment could occur without major changes in GABAA receptor gene expression or protein levels (Tietz et al., 1999).

4. Regulation of GABAA receptor expression by neurosteroids: physiological and pathophysiological implications A major group of endogenous modulators of GABAA receptors that is gaining considerable interest are the neurosteroids, and in particular, the metabolites of progesterone and of deoxycorticosterone, which appear to regulate important physiological functions and pathophysiological states through a direct, non-genomic interaction with specific GABAA receptor subtypes. Indeed, neuroactive steroids, such as allopregnanolone (3a-hydroxy-5a-pregnan-20-one) or allotetrahydrodeoxycorticosterone (3a,2ldihydroxy-5a-pregnan-20-one), are positive allosteric modulators of GABAA receptors (for a review, see Lambert et al., 2001), with highest potency for those containing the dsubunit (Mihalek et al., 1999; Adkins et al., 2001; Wohlfarth et al., 2002). The physiological significance of the action of neurosteroids of GABAA receptors in vivo has been investigated during development (Cooper et al., 1999; Grobin & Morrow, 2001), in relation to stress (Zinder & Dar, 1999; Barbaccia et al., 2001), and in relation to the fluctuation of circulating progesterone and metabolites during pregnancy and parturition or during the estrous cycle (Concas et al., 1998; Brussaard & Herbison, 2000; Smith, 2002). Finally, a novel avenue of research has been opened to establish potential interactions between ethanol and neurosteroids, which could lead to the development of novel therapeutic strategies for the treatment of ethanol dependence (Morrow et al., 2001). 4.1. Interactions between GABAA receptors and neurosteroids in the stress response Acute stress increases the concentration of allopregnanolone and deoxycorticosterone in the brain and plasma, and this effect has been suggested to counteract the stress-

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related decrease in GABAA receptor function (Barbaccia et al., 2001). The interaction appears to be bi-directional, since pharmacological reduction of GABAergic synthesis, or application of negative allosteric GABAA receptor modulators, increases neurosteroid expression. By acting on GABAA receptors, neurosteroids, therefore, might represent a homeostatic mechanism to control neuronal overexcitation elicited by acute stress. For example, a direct link between stress-induced enhancement of allotetrahydrodeoxycorticosterone synthesis and increased seizure threshold has been established for several models of seizures in mice (Reddy & Rogawski, 2002). In the hippocampus, prolonged exposure to corticosterone induces complex changes in GABAA receptor pharmacology in discrete hippocampal subfields, as shown by autoradiography with [35S]tert-butylbicyclophosphorothionate and [3H]flunitrazepam (Orchinik et al., 2001). It is not clear, however, how these changes relate to the expression or subcellular distribution of GABAA receptor subtypes in hippocampal neurons. In contrast to acute stress, prolonged stress, which can be modeled by social isolation in rodents, decreases the concentration of several neuroactive steroids in the brain and plasma, and leads to reduced GABAA receptor function, as tested by Cl flux and radioligand-binding assays (Serra et al., 2000). These changes were accompanied with an anxiety-like behavioral profile, confirming a role for both neurosteroids and GABA A receptors in the modulation of emotional behavior and mood. 4.2. Changes in GABAA receptor function in relation to fluctuating progesterone levels A remarkable example of plasticity of GABAA receptor function in the adult brain related to physiological fluctuation of circulating progesterone and its metabolites is provided by the profound and rapid alterations in the firing properties of oxytocin neurons in the supraoptic nucleus, which occur at the end of gestation to enable parturition and lactation (Brussaard & Herbison, 2000). These alterations are triggered off by the sudden drop in circulating progesterone metabolites, and correlate with multiple changes of GABAA receptor synaptic currents around parturition. During late gestation, GABAergic IPSCs are characterized by a relatively fast decay that can be markedly prolonged by allopregnanolone. After parturition, these currents have slower decay kinetics, but are insensitive to neurosteroid modulation. Since these changes are accompanied by a switch in the relative expression of the a1- and a2-subunits, it was first hypothesized that the corresponding GABAA receptor subtypes are differentially sensitive to neurosteroids and have different deactivation kinetics (Brussaard et al., 1997). Further studies have suggested, however, that steroid sensitivity is linked to PKC-dependent phosphorylation mechanisms, with activation of PKC being required for subsequent neurosteroid modulation of decay kinetics of GABAergic IPSCs (Brussaard et al., 2000). Therefore,

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multiple mechanisms are likely to contribute to the change in GABAA receptor function in oxytocin neurons around parturition. Profound alterations of GABAA receptor function related to changes in circulating neurosteroid levels have also been reported in a rodent model of premenstrual syndrome (Smith et al., 1998). These studies revealed that expression of the a4- and d-subunit mRNAs is markedly increased in rats following withdrawal from chronic progesterone treatment. These changes in expression are paralleled by a loss in benzodiazepine responsiveness and by increased ethanolinduced potentiation of GABA-induced currents in dentate gyrus granule cells (Sundstrom-Poromaa et al., 2002). Progesterone withdrawal also increases the expression of the a4-subunit in cultured cerebellar granule cells and cortical neurons (Follesa et al., 2001), indicating that the effect is not limited to a single cell type. These changes in GABAA receptor expression and function are believed to contribute to the physiological alterations and behavioral effects induced by progesterone withdrawal and to underlie the symptoms of premenstrual syndrome, including dysphoria and increased anxiety. 4.3. Interactions between neurosteroids and ethanol: a common mechanism of action at GABAA receptors? Although ethanol can activate GABAA receptors, many in vivo electrophysiological effects of ethanol cannot be reproduced in vitro, suggesting the involvement of endogenous modulators such as the neurosteroids (Criswell et al., 1999). Experimentally, several lines of evidence support the existence of a link between ethanol and neurosteroids. For example, it has been shown that neurosteroid sensitivity of GABAA receptors is increased following ethanol withdrawal (Devaud et al., 1996). Ethanol administration strongly increases the expression of allopregnanolone and allotetrahydrodeoxycorticosterone in alcoholpreferring rats, suggesting that they mediate the anxiolytic and rewarding effects of ethanol in these animals (Barbaccia et al., 1999). Investigations of d-subunit-deficient mice, which exhibit a greatly reduced sensitivity to neurosteroids, revealed altered behavioral responses to ethanol, suggesting a causal relationship (Mihalek et al., 1999, 2001). The link between the d-subunit and ethanol is particularly interesting in view of the finding that a4bdsubunit-containing receptors are selectively modulated by low doses of ethanol (Sundstrom-Poromaa et al., 2002). Since these subunits are up-regulated in the dentate gyrus upon progesterone withdrawal (see Section 4.2), the effect of ethanol on GABAA receptor function was tested in this model and was found to increase in parallel with upregulation of these receptors. These findings provide a molecular and cellular substrate for explaining enhanced ethanol sensitivity following progesterone withdrawal and in premenstrual syndrome (Sundstrom-Poromaa et al., 2002).

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The interactions between ethanol and neurosteroids might explain how ethanol increases GABAergic neurotransmission in vivo. Furthermore, neurosteroids may protect against ethanol dependence, both by alleviating the symptoms of withdrawal and by decreasing drinking behavior. Indeed, it has been shown that, while allopregnanolone treatment increases ethanol intake in non-dependent rats, it leads to a marked reduction in ethanol self-administration following chronic ethanol consumption in alcohol-preferring rats (for a review, see Morrow et al., 2001). GABAA receptor plasticity induced by neurosteroids, therefore, may represent a new mechanism of ethanol action, and may lead to novel therapeutic strategies for alcoholism (Morrow et al., 2001).

5. Alterations of GABAA receptor expression and function in neurological and psychiatric disorders The investigation of the cellular mechanisms regulating GABAA receptor expression and function (Section 3) revealed three principal findings: (1) that the neuron- and synapse-specific expression of GABAA receptor subtypes is largely cell-autonomous (Bru¨nig et al., 2002a); (2) that clustering of GABAA receptors at postsynaptic sites and the maintenance of GABAA receptor clusters depends on distinct mechanisms (Levi et al., 1999, 2002; Bru¨nig et al., 2002b; Garin et al., 2002); and (3) that the number of GABAA receptors in the cell membrane is dynamically regulated on a short-term basis by multiple trafficking mechanisms (Bru¨nig et al., 2001; Kittler & Moss, 2001; Kneussel, 2002). Understanding alterations of GABAA receptor function in disease states will require in-depth analysis of GABAA receptor regulation in the adult brain. In this section, we discuss the results of recent studies providing evidence for long-term regulation of GABAA receptor expression and localization in various disease states. This overview includes experimental studies, as well as clinical investigations, to underscore the relevance of these findings for understanding the pathophysiology of neurological and psychiatric disorders and for designing novel therapeutic strategies. 5.1. Regulation of GABAA receptor expression following deafferentation: implications for neurodegenerative diseases Degeneration of GABAergic neurons is one of the hallmarks of Huntington’s disease. In the caudate nucleus and putamen, it is accompanied by a profound reduction in benzodiazepine-binding sites and GABAA receptor subunit immunoreactivity, corresponding to the loss of neurons (Faull et al., 1993; Kunig et al., 2000). However, in the globus pallidus, a major target of striatal neurons, GABAA receptors are increased, suggesting compensatory up-regulation in the remaining synapses. Experimentally, following

quinolinic acid-induced lesions of the striatum to mimic the pattern of neuronal degeneration of Huntington’s disease, increased GABAA receptor b2/3-subunit immunoreactivity has been demonstrated in the substantia nigra, pars reticulata (Brickell et al., 1999). A detailed electron microscopic analysis, using post-embedding techniques, revealed a selective increase of GABAA receptor labeling in symmetric synapses, but not of AMPA receptors in asymmetric synapses, in lesioned animals (Fujiyama et al., 2002). Most strikingly, the increased expression of GABAA receptors is long-lasting (>15 months), but it is induced very rapidly, being detectable by autoradiography within 2 hr following intrastriatal quinolinic acid injection (Brickell et al., 1999). The signals involved in this rapid induction have not been investigated. While increasing the number of postsynaptic GABAA receptors might represent a compensatory response to the loss of GABAergic afferent, such a response can also occur in response to a loss of glutamatergic terminals. Thus, the number of postsynaptic GABAA receptor clusters associated with gephyrin increases in the molecular layer of the dentate gyrus following entorhinal cortex lesions (Simburger et al., 2001). Deafferentation of excitatory input to the dentate gyrus, therefore, appears to induce a profound synaptic remodeling on dendrites of granule cells, possibly affecting GABAergic circuits. Interestingly, the effect was cell-specific, since GABAA receptor clusters on interneurons, distinguished by the presence of the a1-subunit, were not affected in this experimental paradigm (Simburger et al., 2001). To understand the significance of this remodeling, it will be necessary to determine whether novel GABAergic synapses are formed and which neurons respond to the lesion by reactive sprouting. 5.2. GABAA receptor regulation after focal ischemia Focal ischemic brain infarcts induce long-lasting changes of neuronal excitability in widespread structurally intact brain regions (Luhmann, 1996; Redecker et al., 1998; Hagemann et al., 2000). It is unclear whether these alterations contribute to functional deficits or whether they are part of plastic restorative mechanisms. Analysis of GABAA receptor subunit expression following transient middle cerebral artery occlusion revealed widespread and long-lasting subunit-specific changes in the ipsi-and contralateral hemisphere (Redecker et al., 2002). While a bihemispheric reduction in a1-, a2-, a5-, and g2-subunit immunoreactivity occurred within 30 days following the infarct, the a3-subunit was increased contralaterally and changed its laminar distribution, suggesting recapitulation of a juvenile expression pattern. Changes in GABAA receptor staining were not restricted to the neocortex, but were also seen in the hippocampus and thalamus, indicating that focal infarcts cause changes in network activity that are paralleled by a long-lasting reduction in

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GABAA receptor expression. Unexpectedly, these alterations could be prevented by a single injection of the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 during lesion induction, pointing to the involvement of N-methyl-D-aspartate-dependent excitatory processes at an early stage of circuit remodeling (Redecker et al., 2002). Experimental cortical malformations such as dysplasias induced by a freeze-lesion also induce widespread and long-lasting hyperexcitability in structurally intact adjacent cortical regions (Redecker et al., 2000). Interestingly, although these lesions are produced in neonatal rats, they caused changes in GABAA receptor expression persisting in adult animals, and extending up to several millimeters away from the dysplasia, as well as to the ipsilateral hippocampus (Redecker et al., 2000). The most parsimonious explanation for these long-lasting changes, seen after a lesion in either the developing or adult brain, is that they reflect profound changes in network properties and, perhaps, in synaptic organization distributed in multiple brain areas. 5.3. GABAA receptors in epilepsy Genetic evidence that GABAA receptors are involved in human idiopathic epilepsy has been provided for three distinct mutations in the g2-subunit gene and one mutation in the a1-subunit gene. The g2K289M point mutation in the extracellular loop between TM2 and TM3 of the g2-subunit was reported in a family with generalized epilepsy with febrile seizures (Baulac et al., 2001). In recombinant a1b2g2 receptors expressed in Xenopus oocytes, this mutation reduced the amplitude of GABA-induced currents. However, potentiation by diazepam was not affected. Two other mutations, g2R43Q (Wallace et al., 2001) and a single nucleotide exchange at the splice donor site of intron 6 (Kananura et al., 2002), were reported in two families with childhood epilepsy and febrile seizures. The g2R43Q mutation, when expressed in Xenopus oocytes, did not affect GABA-gating of recombinant a1b2g2 receptors, but suppressed diazepam potentiation. The splice-donor site mutation most likely results in a nonfunctional allele. Finally, a loss-of-function mutation of a1-GABA A receptors (a1A322D) was detected in a family with an autosomal dominant form of juvenile myoclonic epilepsy (Cossette et al., 2002). The effects of the g2K289M and g2R43Q point mutations reported in the original publications could not be reproduced in a different expression system (Bianchi et al., 2002). However, this study uncovered distinct alterations in the functional properties of the mutated receptors, which might be due to defective receptor assembly or membrane expression. How these alterations contribute to seizure disorders remains to be elucidated. A possible contribution of GABAA receptors to other forms of epilepsy, notably to temporal lobe epilepsy, is suggested by the profound changes in expression that have

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been reported in patients and in various rodent models of temporal lobe epilepsy with hippocampal sclerosis (for reviews, see Duncan, 1999; Olsen et al., 1999; Coulter, 2001; Treimann, 2001). In patients, the extensive neuronal loss in CA1, which is one of the characteristic features of hippocampal sclerosis, is accompanied by a marked decrease in benzodiazepine-binding sites (Savic et al., 1988; Debets et al., 1997). However, a detailed examination at the cellular and subcellular level, using immunohistochemistry with subunit-specific antibodies, revealed a complex pattern of changes, characterized above all by an increased staining intensity on surviving neurons, and by subtype-specific changes in subcellular distribution of GABAA receptors in epileptic tissue (Loup et al., 2000). Among the most pronounced and consistent changes was the increased a1- and a2-subunit immunoreactivity in the soma and apical dendrites of the dentate gyrus granule cells and an apparent translocation of the a3-subunit immunoreactivity from the somatic region to the distal dendrites in CA2 pyramidal cells (Loup et al., 2000). In experimental temporal lobe epilepsy, changes in GABAA receptor subunit expression have been analyzed in several animal models, with largely convergent results. The main observation was increased expression of GABAA receptors in the dentate gyrus granule cells, with changes in pharmacological properties, suggesting aberrant expression of GABAA receptors in these cells. Notably, an increase in a3-, a4-, and d-subunit expression has been reported in rats experiencing chronic recurrent seizures following i.p. injection of the muscarinic agonist pilocarpine or the glutamate receptor agonist kainic acid (Schwarzer et al., 1997; BrooksKayal et al., 1998; Fritschy et al., 1999). However, chronic recurrent seizures induced by i.p. injection of kainic acid or pilocarpine do not mimic the complex partial seizures experienced by most patients with temporal lobe epilepsy. Also the pattern of neuronal loss is significantly different from that reported in neuropathological studies of hippocampal sclerosis. These limitations are partially overcome in a mouse model of temporal lobe epilepsy, in which spontaneous recurrent partial seizures are induced following unilateral injection of kainic acid into the dorsal hippocampus (Bouilleret et al., 1999; Riban et al., 2002). In these mice, the dentate gyrus granule cells become hypertrophic and undergo a prominent dispersion. Although the a4- and d-subunits were not analyzed, a marked increase in a1-, a2-, a5-, and g2-subunit immunoreactivity was observed in the epileptic dentate gyrus (Bouilleret et al., 2000), which corresponded to an increase in the size and density of postsynaptic clusters co-localized with gephyrin and dystrophin (Knuesel et al., 2001). These findings are strongly suggestive of the formation of novel GABAergic synapses, possibly reflecting sprouting of GABAergic axons in the epileptic dentate gyrus. Furthermore, they show that GABAA receptor-associated proteins increase in parallel with GABAA receptors at postsynaptic sites. In addition to the well-known recurrent mossy fiber sprouting in the

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dentate gyrus and CA3 area, the formation of aberrant GABAergic connections might thus also be considered as a contributing factor of temporal lobe epilepsy. A direct demonstration for a prominent increase in the density of GABAergic axons in the dentate gyrus has been provided in the pilocarpine model, using dual labeling for GAD and GABA transporter Type 1 (GAT-1) (Andre´ et al., 2001). These findings suggest that reactive sprouting of GABAergic axons in response to lesion might be a common response in the dentate gyrus. In addition, they indicate that increased, rather than decreased, GABAergic inhibition might be a key feature of epileptognesis and seizure expression in the dentate gyrus.

In summary, direct evidence for morphologic alterations in GABAergic circuits and distribution of GABAA receptors is available for major neurological and psychiatric disorders. While the analysis of causal mechanisms is impossible in human studies, some of these changes can be reproduced experimentally in animal models and their molecular and cellular basis analyzed in vitro. A fruitful interaction, therefore, can be expected between in vitro studies of GABAA receptor trafficking and clustering and in vivo analyses of animal models to identify the contribution of GABAergic plasticity to these disorders.

6. Conclusions and outlook 5.4. Synapse-specific alterations of GABAA receptors in schizophrenia Alterations in several biochemical and anatomical markers of GABAergic transmission have been reported in schizophrenic patients, including changes in GAD expression, muscimol binding, and number of interneurons (for reviews, see Lewis, 2000; Benes & Berretta, 2001; Nutt & Malizia, 2001; Blum & Mann, 2002). The regions affected include the hippocampus, anterior cingulate cortex, and medial prefrontal cortex. In most cases, the specificity of these alterations with regard to the disease type, medication, and brain region affected has not been established. One study has reported, however, a selective reduction in the number of GABAergic axon terminals formed by chandelier neurons onto the axon initial segment of pyramidal cells in areas 9 and 46 of the prefrontal cortex, labeled with antibodies to GAT-1 (Woo et al., 1998). This decrease was not seen in age-matched, non-schizophrenic psychiatric patients, and was independent of antipsychotic medication at the time of death. Since the number and size of parvalbumin-positive neurons, which include chandelier neurons, was not affected, these results were taken as evidence for an alteration in GAT-1 expression and not for a decrease in the number of axon terminals. A recent study by the same group demonstrates compensatory upregulation of a2-GABAA receptor immunoreactivity in the axon initial segment, again occurring selectively in schizophrenic patients independently of antipsychotic medication (Volk et al., 2002). These findings, therefore, support the hypothesis of a disturbed GABAergic transmission in the prefrontal cortex of schizophrenic subjects due to a selective alteration of GABAergic function in the synapses formed by chandelier cells. Additional interesting information provided by these studies is the considerable variability in the density of immunopositive terminals or receptor clusters in axon initial segments of control subjects, ranging from 5 to 70/mm2 in 40-mm thick sections in area 46. These differences were not due to technical variations, such as post-mortem delay or tissue quality, but are likely to reflect the variability of GABAergic circuits between individuals (Volk et al., 2002).

The remarkable progress in our knowledge of GABAergic synaptic transmission opens novel horizons and challenges classical views about the formation, maintenance, and function of GABAergic synapses in the healthy and diseased brain. These findings underscore the necessity of considering GABAA receptor-mediated neurotransmission as a dynamic and highly flexible process controlled by multiple mechanisms operating at the molecular, cellular, and systemic level. In our opinion, four major novel concepts have emerged from the work reviewed here. (1) The number of GABAA receptors available for synaptic transmission is regulated on both a short-and a long-term basis, and their functional properties can be adjusted very rapidly in response to numerous stimuli. (2) There are multiple modes of GABAA receptor-mediated neurotransmission, including tonic inhibition mediated by functionally and pharmacologically specialized extrasynaptic receptors, and distinct forms of phasic inhibition produced by distinct GABAA receptor subtypes, localized in defined neuronal circuits operating in parallel within a given brain structure, as shown for hippocampal pyramidal cells. (3) Neurosteroids most likely represent the major endogenous modulator of GABAA receptor function and represent a powerful homeostatic mechanism to enhance GABAergic function in response to increased excitability. (4) Long-term dysregulation of GABAA receptor expression and localization is a key aspect of multiple neurological and psychiatric disorders, most likely reflecting changes in GABAergic circuits, including axonal sprouting and formation of novel synapses. From a pharmacological point of view, such conclusions are encouraging because they show the feasibility to target specific GABAA receptor subtypes to produce highly specific therapeutic action with minimal side effects. We would like to underscore, however, that major issues remain to be answered for a better understanding of the biology of GABAA receptors and for designing appropriate therapeutic strateies: . GABAA receptor subtypes: In addition to the elucidation of GABAA receptor stoichiometry, which will be essential to understand the mechanism of channel gating and allosteric modulation, a major open question related to

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GABAA receptor subtypes is the role of their subunit composition for determining their functional properties in vivo. This question will need to be resolved both at the level of single-channel properties and at the level of synapse physiology. . GABAA receptor postsynaptic clustering: Four major questions await answers in this field. First, it remains unknown how the interaction of GABAA receptors with proteins of the postsynaptic specialization and their regulation by post-translational modification determines GABAergic synaptic function and pharmacology. In other words, how different is the function of GABAA receptors in a heterologous expression system compared with a synaptic context? Second, the nature of the interaction between GABAA receptors and gephyrin needs to be elucidated. Is it mediated by an additional protein(s) or are both GABAA receptors and gephyrin stabilized and clustered by the cytoskeleton? Third, the identification of the ‘‘general’’ and ‘‘synapse-specific’’ clustering signals will be a major advance for elucidating how receptors and signaling proteins are sorted to the appropriate synapses. Finally, the role of the DPC in GABAergic synapses remains hypothetical. In addition to possible functions in relation to synaptic stabilization, a role in signal transduction also represents an attractive possibility, especially in analogy with the neuromuscular junction. . Regulation of cell-surface expression: The concept that GABAA receptors might shuffle between the cell surface and an intracellular, subsynaptic compartment is new and is largely derived from corresponding findings in the field of glutamate receptor biology. The role of GABAA receptor internalization, its regulation by physiological and pharmacological means, and the fate of internalized receptors along the degradation/recycling pathways will require considerable attention to understand how these mechanisms determine the number of receptors available for synaptic transmission at any given time point in a neuron. . Allosteric modulation by neurosteroids: One of the major issues in this field is the site of synthesis of neuroactive steroids in the brain, along with the regulation of their synthesis. Are neurosteroids produced by astrocytes and released in the vicinity of synapses, and are locally synthesized neurosteroids playing an important role in the modulation of GABAA receptors under physiological conditions? In addition, the function and the subcellular localization of the steroid-sensitive GABAA receptor subtypes, in particular those induced by fluctuating progesterone concentrations, remain largely enigmatic. . GABAergic synaptic plasticity in disease states: The rapid, but long-lasting, alterations in the distribution of GABA A receptors and other markers of GABAergic synapses reviewed in Section 5 raise fundamental issues with regard to their functional significance. In particular, it remains unresolved whether sprouting of GABAergic axons and synaptogenesis occur under pathophysiological situations, for instance in Huntington’s disease or in epilepsy, or

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whether the apparent increase in the number of GABAA receptor clusters and GABAergic axons merely reflects an up-regulation of these proteins in existing structures. In the field of epilepsy, a key issue will be to clarify the role of the mutations identified in the a1- and g2-subunits for producing the associated syndrome. More generally, the mechanisms of epileptogenesis and seizure expression in relation to the reorganization of GABAergic circuits and GABAA receptor up-regulation are to be understood. Finally, in stroke and ischemia, the functional significance of the long-lasting reduction of GABAA receptor expression needs to be clarified. Are there homeostatic mechanisms to compensate for this alteration of inhibitory function? The ultimate goals of these studies are to further our understanding of brain function, but also to design novel therapeutic strategies based on selective targets, for enhancing efficacy while minimizing side effects. Providing effective treatments, or relief of symptoms, for neurological and psychiatric disorders remains a major challenge for biomedical science. Given the clinical importance of GABAA receptor pharmacology, the elucidation of the role of GABAA receptor subtypes in specific neuronal circuits will represent a major advance toward this goal.

Acknowledgments Our work was supported by the Swiss National Science Foundation (Grants Nr. 31-52869.97 and 31-63901.00 and NCCR Neural Plasticity and Repair). We are grateful to Corinne Sidler for excellent technical support and Verena Du¨nki for secretarial assistance.

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