Volume 24, Issue 5 pp. 1307-1315

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Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABA_A receptor α3 subunit-null mice

Remo Studer,

Remo Studer

R.S. and L.v.B. contributed equally to this work.

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Lotta Von Boehmer,

Lotta Von Boehmer

R.S. and L.v.B. contributed equally to this work.

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Tatjana Haenggi,

Tatjana Haenggi

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Claude Schweizer,

Claude Schweizer

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Dietmar Benke,

Dietmar Benke

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Uwe Rudolph,

Uwe Rudolph

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Jean-Marc Fritschy,

Jean-Marc Fritschy

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Remo Studer,

Remo Studer

R.S. and L.v.B. contributed equally to this work.

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Lotta Von Boehmer,

Lotta Von Boehmer

R.S. and L.v.B. contributed equally to this work.

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Tatjana Haenggi,

Tatjana Haenggi

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Claude Schweizer,

Claude Schweizer

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Dietmar Benke,

Dietmar Benke

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Uwe Rudolph,

Uwe Rudolph

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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Jean-Marc Fritschy,

Jean-Marc Fritschy

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH−8057 Zurich, Switzerland

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First published: 15 September 2006

https://doi.org/10.1111/j.1460-9568.2006.05006.x

Citations: 65

Professor Jean-Marc Fritschy, as above.
E-mail: [email protected]

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Abstract

Multiple GABA_A-receptor subtypes are assembled from α, β and γ subunit variants. GABA_A receptors containing the α3 subunit represent a minor population with a restricted distribution in the CNS. In addition, they predominate in monoaminergic neurons and in the nucleus reticularis thalami (nRT), suggesting a role in the regulation of cortical function and sleep. Mice with a targeted deletion of the α3 subunit gene (α3^0/0) are viable and exhibit a subtle behavioural phenotype possibly related to dopaminergic hyperfunction. Here, we investigated immunohistochemically the consequences of the loss of α3 subunit for maturation of GABA_A receptors and formation of GABAergic synapses in the nRT. Throughout postnatal development, the regional distribution of the α1, α2, or α5 subunit was unaltered in α3^0/0 mice and the prominent α3 subunit staining of nRT neurons in wildtype mice was not replaced. Subcellularly, as seen by double immunofluorescence, the α3 and γ2 subunit were clustered at postsynaptic sites in the nRT of adult wildtype mice along with the scaffolding protein gephyrin. In α3^0/0 mice, γ2 subunit clustering was disrupted and gephyrin formed large aggregates localized at the cell surface, but unrelated to postsynaptic sites, indicating that nRT neurons lack postsynaptic GABA_A receptors in mutant mice. Furthermore, GABAergic terminals were enlarged and reduced in number, suggesting a partial deficit of GABAergic synapses. Therefore, GABA_A receptors are required for gephyrin clustering and long-term synapse maintenance. The absence of GABA_A-mediated transmission in the nRT may have a significant impact on the function of the thalamo-cortical loop of α3^0/0 mice.

Introduction

GABA_A receptors mediate most of the fast phasic and tonic inhibition in the mammalian central nervous system (Moss & Smart, 2001; Farrant & Nusser, 2005). The existence of multiple GABA_A receptor subtypes, with distinct functional and pharmacological properties, is based on differential assembly from a large family of constituent subunit genes (Mohler et al., 2002; Sieghart & Sperk, 2002; Rudolph & Möhler, 2004). GABA_A receptors containing the α3 subunit represent approximately 10–15% of total GABA_A receptors in adult brain (McKernan & Whiting, 1996). While these receptors are expressed in multiple regions at all levels of the neuraxis, they also comprise the major GABA_A receptor subtype present in monoaminergic and basal forebrain cholinergic neurons, as well as in the nucleus reticularis thalami (nRT) (Gao et al., 1993; Fritschy & Mohler, 1995; Gao et al., 1995; Pirker et al., 2000). α3-GABA_A receptors are therefore likely to contribute to GABAergic control over a broad range of behavioural and cognitive states. To gain insight into the function of α3-GABA_A receptors, mice carrying a targeted deletion of the α3 subunit gene (α3^0/0 mice) have been generated (Yee et al., 2005). These mice are viable and fertile and show no gross morphological anomaly in the brain. However, they are hyperactive and exhibit a sensory-motor deficit most likely related to dopamine hyperfunction. The lack of a severe phenotype of α3^0/0 mice suggests that specific compensatory changes occur to substitute for the missing α3-GABA_A receptors.

In the present study, we have focused on potential changes in GABA_A receptor expression and in the organization of GABAergic synapses in α3^0/0 mice. Using immunohistochemistry, we investigated first potential changes in the regional distribution of major GABA_A receptor α subunit variants during postnatal development, because α3-GABA_A receptors are particularly abundant during ontogeny (Fritschy et al., 1994). Second, we investigated whether GABAergic synapses are altered in nRT neurons, which express a limited repertoire of GABA_A receptor subunits, consisting mainly of α3, β3, and γ2 subunits (Bentivoglio et al., 1991; Fritschy & Mohler, 1995; Browne et al., 2001). These receptors mediate phasic inhibition between nRT neurons and are essential for the modulation of rhythmic firing by thalamocortical neurons in the ventrobasal complex (Huntsman et al., 1999). At the subcellular level, α3-GABA_A receptors in the nRT are clustered at postsynaptic sites along with gephyrin (Baer et al., 2000; Sassoè-Pognetto et al., 2000). Therefore, changes in the subunit expression pattern and subcellular distribution of GABA_A receptors and gephyrin were sought as surrogates for postsynaptic alterations in nRT neurons of α3^0/0 mice. Presynaptic changes involving GABAergic terminals were monitored by staining for the vesicular GABA transporter (VGAT).

The results demonstrate that the expression of other major GABA_A receptor subtypes remains unaffected throughout the brain during postnatal development and in adult α3^0/0 mice. As a consequence, α3-GABA_A receptors are not replaced in nRT neurons, disrupting clustering of postsynaptic proteins and leading to a reduction of presynaptic terminals. The loss of GABA_A receptors in nRT neurons may have a significant impact on the function of the thalamo-cortical loop.

Materials and methods

Animals

Wildtype and α3^0/0 mice (see Yee et al., 2005; for characterization) were maintained C57BL/6J background (B6.129 × 1-Gabra3tm2^Uru/Uru) and bred at the University of Zurich. As the α3 subunit gene is located on the X chromosome, mutant mice were either hemizygote male or homozygote female obtained from heterozygous/hemizygous breeding pairs or from α3^0/0 breeding pairs. Mice were genotyped by PCR analysis of tail biopsies. All experiments were approved by the veterinary office of the canton of Zurich and were performed in accordance with the European Community Council Directive (86/609/EEC).

Immunohistochemistry

The regional distribution of GABA_A receptor subunits was investigated by immunoperoxidase staining of transverse or parasagittal sections prepared from perfusion-fixed brain tissue, as described previously (Fritschy et al., 1998). Adult (n = 3 per genotype) and juvenile mice [postnatal day (P) 0, P4–5, P7, P10, P14, P21; n = 4–5 mice per time-point and genotype] were analysed. Briefly, mice were anaesthetized with pentobarbital (Nembutal; 50 mg/kg, i.p.) or with ice (P0, P4) and rapidly perfused through the ascending aorta with 4% paraformaldehyde in 0.15 m phosphate buffer (pH 7.4). The brains were removed, postfixed in the same solution, and incubated in sodium citrate buffer (pH 4.5). They were then irradiated in a microwave oven (650 W, 90 s), cryoprotected with 10% dimethylsulfoxide in PBS, and cut from frozen blocks with a sliding microtome. Sections were processed free-floating for immunohistochemistry. Guinea pig antibodies against the α1, α2, α3, or α5 subunit (see Fritschy & Mohler, 1995; Yee et al., 2005; for characterization) and rabbit antibodies against the α4 and α6 subunit (gift from Dr W. Sieghart, Vienna; see Sassoè-Pognetto et al., 2000; Kralic et al., 2006; for characterization) were used. Sections were incubated overnight at 4 °C in primary antibodies dissolved in Tris-saline buffer containing 2% normal goat serum and 0.2% Triton-X100. Biotinylated secondary antibodies (1 : 300; Jackson Immunoresearch, West Grove, PA, USA) were applied for 30 min, followed by the Vectastain Elite kit (Victor Laboratories, Burlingame, CA, USA) and incubation with diaminobenzidine as chromogen. Sections were mounted on gelatin-coated glass slides, air-dried, dehydrated and coverslipped.

The analysis of postsynaptic GABA_A receptor α2, α3, or γ2 subunit and gephyrin (mAb7a, Synaptic Systems, Göttingen, Germany) clusters was performed by double immunofluorescence staining of cryosections prepared from fresh-frozen tissue, as described (Fritschy et al., 1998). Seven adult mice per genotype were used. When required, presynaptic GABAergic terminals were detected using a rabbit antibody against the VGAT (Synaptic Systems). In some experiments, cell bodies in the nRT were labelled with parvalbumin (mouse monoclonal antibody, SWant, Bellinzona, Switzerland). Sections were incubated overnight at 4 °C in a mixture of primary antibodies derived from different species. Each antibody was then detected using the appropriate IgGs coupled to different fluorochromes (Alexa 488, Molecular Probes, Eugene, OR; Cy3, Cy5, Jackson Immunoresearch). Finally, sections were washed again, air-dried and coverslipped with buffered glycerol (pH 9.2).

Data analysis

Sections processed for immunoperoxidase staining were analysed by light microscopy (Axioskop Zeiss AG, Jena, Germany). For illustration, they were digitized with a high-resolution colour camera (Zeiss Axiocam). Sections from immunofluorescence staining were visualized by confocal laser scanning microscopy (Zeiss LSM510 Meta), using sequential acquisition of the different channels to avoid cross-talk between fluorochromes. Typically, stacks of 8–12 images (512 × 512 pixel, pixel size 70–150 nm) spaced by 0.3–0.5 µm were recorded, using a 100× objective with a numerical aperture 1.4. Images were processed with the software Imaris (Bitplane, Zurich, Switzerland). The numerical density of VGAT-positive terminals was quantified using three animals per genotype, using automatic 3D target recognition in a stack of ten confocal sections (Surpass module in Imaris version 4.2). The dimension of terminals was determined in single 2D confocal images using a threshold segmentation algorithm based on labelling intensity (min 90 on a 255 grey-level scale) and size (min 0.1 µm²) (MCID-M5 image analysis software; Imaging Research, St. Catherine, ON, Canada). Statistical analysis was performed on cumulative distribution plots (Kolmogorow–Smirnov) for size and with a nonparametric t-test (Kruskal–Wallis) for numerical density of terminals.

Results

Immunohistochemical staining of the six GABA_A receptor α subunit variants performed in sections from adult mice with subunit-specific antisera confirmed earlier studies in rat and mice (Fritschy & Mohler, 1995; Jones et al., 1997; Pirker et al., 2000; Peng et al., 2002; Kralic et al., 2006) that only the α3 subunit-immunoreactivity is present in the nRT (not shown). In α3^0/0 mice, as reported previously (Yee et al., 2005), staining for the α3 subunit was completely abolished, and no other α subunit variant, including the α4, α5, and α6 subunit, was detected in the nRT. These initial observations suggested that a deficit of postsynaptic GABA_A receptors might occur in adult nRT. For the rest of this study, we have therefore focused on the α subunit variants that contribute, at least in part, to postsynaptic GABA_A receptors (α1, α2, α3, α5) in brain.

Unaltered distribution of GABA_A receptor subunits during development of α3^0/0 mice

To establish whether a transient alteration in expression of postsynaptic GABA_A receptors occurs in immature α3^0/0 mice, the regional distribution of the α1, α2, and α5 subunit was analysed during postnatal development. As shown at P7, the pattern of expression of these subunits was highly similar in both genotypes (Fig. 1), indicating that it was not affected by the lack of α3 subunit expression in mutant mice. In particular, all regional and areal boundaries revealed by the selective distribution of these three subunits in wildtype mice were identical in α3^0/0 mice. In cerebral cortex, where each of these subunits exhibits an area-specific laminar distribution, no change was apparent in mutants. The lack of alteration was clearly evident in all regions strongly stained for the α3 subunit in wildtype, such as layers V–VI of primary visual cortex, the entorhinal cortex, the nRT, the superior colliculus, or the spinal trigeminal nucleus. The same results were obtained at the other stages of development examined (P0, P4–5, P10, P14, and P21; data not shown). Altogether, these results indicated that the α3 subunit is not replaced by another α subunit variant in α3^0/0 mice and its absence has no detectable influence on the distribution of other major GABA_A receptor subunits.

Details are in the caption following the image — **Figure 1**
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Normal distribution of the GABA_A receptor α1, α2, and α5 subunit in the brain of α3^0/0 mice compared to wildtype, as illustrated in parasagital sections of P7 mice processed for immunoperoxidase staining with subunit specific antisera. For comparison, the distribution of the α3 subunit is shown in wildtype. (A) On lateral sections, the area- and laminar-specific distribution of GABA_A receptor subtypes in the neocortex, forming sharp boundaries is clearly visible and is identical in both genotypes. Likewise, the subunit-specific distribution in basal ganglia and thalamus remains unchanged, including in all regions expressing the α3 subunit in wildtype. All sections from wildtype and mutant mice are from the same animals. (B) More medially, the olfactory bulb, tectum, cerebellum, and brainstem again show no change in subunit expression. The conserved subunit-specific pattern is particularly clear in the spinal trigeminal nucleus (Vs), for example. All sections are from the same wildtype and mutant mouse brains. CPu, striatum; Ent, entorhinal cortex; IC, inferior colliculus; S1, primary somatosensory cortex; SC, superior colliculus; V1, primary visual cortex.. Scale bars, 1 mm (A); 2 mm (B).

Maturation of GABA_A receptor α subunit-IR in the nRT

To understand better the formation of GABA_A receptors in the nRT, their distribution was monitored in detail during development in wildtype mice (Fig. 2). The α1 subunit was not detectable in the nRT at any age examined (not shown). As reported previously in rats, its expression increased gradually in relay nuclei, mainly during the third and fourth postnatal week. In contrast, α3 subunit immunoreactivity was prominent already at birth in the nRT, clearly outlining the boundaries of the nucleus (Fig. 2A). A weaker staining was evident in the ventrobasal complex (VB) and laterodorsal nucleus (LD). This distribution pattern did not change during development, except that α3 subunit staining intensity increased further in the nRT by P10 (Fig. 2C) and thereafter. In contrast, α3 subunit staining in VB and LD was decreased at P10 compared to P0 and P5. The α2 subunit, which is very prominent in VB during the first postnatal week (Fig. 2D–F), was virtually absent from the nRT at any age examined. Finally, the α5 subunit, which is not expressed in the thalamus of adult mice, was transiently detectable in the nRT at P5 (Fig. 2G–I). It subsided thereafter to become undetectable at P10. These results indicate that the α3 subunit represents the predominant GABA_A receptor subtype in the nRT at every stage of postnatal development, with a minor contribution of the α5 subunit around P5.

Selective loss of GABA_A receptor and gephyrin clusters in the nRT of α3^0/0 mice

An α subunit variant is necessary for assembly and cell-surface expression of GABA_A receptors (Fritschy et al., 1997; Jones et al., 1997; Sur et al., 2001). Postsynaptic GABA_A receptors are also required for postsynaptic clustering of gephyrin (Essrich et al., 1998; Schweizer et al., 2003; Kralic et al., 2006). In view of the predominance of α3-GABA_A receptors in the nRT, the formation of postsynaptic GABA_A receptor and gephyrin clusters might thus be affected in α3^0/0 mice. To verify this hypothesis, double immunofluorescence was performed in adult mice (Fig. 3). In wildtype mice, clusters of α3 and γ2 subunit immunofluorescence colocalized with gephyrin readily were detected in the nRT (Fig. 3A and B), as reported previously (Baer et al., 1999). In α3^0/0 mice, no clusters were seen and gephyrin formed large aggregates measuring up to 15 µm (Fig. 3C and D). Staining for the γ2 subunit was increased intracellularly, clearly revealing the soma of individual nRT neurons (Fig. 3D), suggesting a deficit in membrane targeting and intracellular retention of this subunit. As expected from the immunoperoxidase staining experiments, no staining was seen for the α1, α2 (Fig. 3E), and α5 subunit in the nRT of either wildtype or mutant mice. To determine whether the large gephyrin aggregates formed in mutant mice are localized postsynaptically, presynaptic terminals were visualized by staining for VGAT (Fig. 3F and G). In wildtype mice, an apposition between VGAT-positive boutons and gephyrin clusters was clearly evident (Fig. 3F). The number of such appositions was much reduced in α3^0/0 mice and most gephyrin aggregates were not in contact with a presynaptic terminal (Fig. 3G). As a control for the regional specificity of these synaptic changes, sections from α3^0/0 mice were examined in the dentate gyrus, where the α3 subunit is almost not expressed in wildtype mice. As expected, the distribution of γ2 subunit and gephyrin clusters was indistinguishable in the dentate gyrus among the two genotypes (Fig. 3H and I), confirming that the formation of gephyrin aggregates in nRT neurons is due to the absence of GABA_A receptors in α3^0/0 mice.

To understand better the alteration in subcellular distribution of gephyrin, the soma and dendrites of nRT neurons were visualized by immunofluorescence for the calcium-binding protein parvalbumin, which is abundantly expressed in the nRT. In wildtype mice, clusters of α3 subunit staining (Fig. 4A) and gephyrin (not shown) were seen on the soma and dendrites of individual nRT neurons. In α3^0/0 mice, gephyrin clusters were replaced by aggregates present mainly in the soma and in proximal dendrites and were located close to the cell surface (Fig. 4B). In contrast, the distribution of VGAT-positive terminals was less affected, although the size of individual terminals appeared larger and their number reduced (Fig. 4C and D). These observations were confirmed by a quantitative analysis in three mice per genotype, revealing a 25% decrease in areal density of VGAT-positive boutons in mutant mice compared to control (Fig. 4E; Kruskal–Wallis, P < 0.05), whereas their size was significantly increased in the nRT of α3^0/0 mice (Fig. 4F; Kolmogorov–Smirnov; P < 0.001).

In summary, the loss of the α3 subunit in the nRT prevents the formation of GABA_A receptors and alters postsynaptic clustering of gephyrin, resulting in a reduction of presynaptic boutons.

Discussion

The present results demonstrate that the absence of the α3 subunit is not compensated for by up-regulation or redistribution of GABA_A receptor subtypes containing the α1, α2, or α5 subunit in α3^0/0 mice. In nRT neurons, which selectively express the α3 subunit, none of the six α-subunit variants was detectable immunohistochemically in adult mutant mice. As a consequence, clustering of GABA_A receptor and gephyrin was disrupted in these cells, providing indirect evidence that these neurons do not express any other GABA_A receptor subtype interacting with gephyrin at postsynaptic sites. These observations confirm that GABA_A receptors are not assembled in the absence of an α subunit variant (Fritschy et al., 1997; Vicini et al., 2001; Fritschy et al., 2006) and that gephyrin depends on the presence of GABA_A receptors for postsynaptic clustering (Essrich et al., 1998; Schweizer et al., 2003; Alldred et al., 2005; Kralic et al., 2006). Furthermore, a partial reduction of VGAT-positive GABAergic terminals occurs in the absence of postsynaptic GABA_A receptors, accompanied by an enlargement of remaining terminals. Synaptic GABA_A receptor-mediated transmission therefore contributes to long-term maintenance of GABAergic terminals in adult nRT neurons.

As α3^0/0 mice exhibit no major deficit in brain morphology and have a subtle behavioural phenotype (Yee et al., 2005), compensatory mechanisms are likely activated in these mice during development to maintain proper function of neuronal circuits that are normally modulated by α3-GABA_A receptors. Most remarkably, these compensatory mechanisms do not include a detectable alteration of other GABA_A receptor subtypes, unlike that observed in mutant mice lacking more abundant GABA_A receptor subtypes, such as the α1 or δ subunit (Korpi et al., 2002; Peng et al., 2002; Kralic et al., 2006). In particular, the α4 subunit, which mediates tonic inhibition in thalamic relay nuclei, remains undetectable in the nRT of adult α3^0/0 mice. The missing α3 subunit is also not replaced transiently during postnatal development, when the relative abundance of α3-GABA_A receptors is higher than in adulthood. The absence of morphological abnormality in the brain of α3^0/0 mice suggests that α3-GABA_A receptors are not essential for mediating the proposed role of GABA as a paracrine regulator of brain development (Behar et al., 2000; Maric et al., 2001; Ben-Ari, 2002; Demarque et al., 2002). Furthermore, the expression of the α5 subunit remains transient around P5 in nRT neurons in α3^0/0 mice, indicating that the missing α3 subunit cannot be substituted for by the α5 subunit. This conclusion extends previous observations, notably in α1^0/0 mice, that a missing GABA_A receptor α subunit is not replaced by another α subunit variant expressed in the same cell (Kralic et al., 2006).

The disruption of gephyrin clustering and the formation of intracellular aggregates was noted also in thalamic and cerebellar neurons of α1^0/0 mice (Kralic et al., 2006), indicating that postsynaptic targeting and clustering of gephyrin requires the presence of GABA_A receptors (or probably glycine receptors). These gephyrin aggregates might therefore represent a morphological marker of neurons lacking postsynaptic GABA_A receptors. Here, we extend these conclusions by showing that gephyrin aggregates are located in the soma and dendrites of nRT neurons that have otherwise a normal morphology. The shape of these aggregates is strongly reminiscent of those formed by gephyrin in recombinant expression systems that lack interaction partners, such as collybistin, for cell-surface targeting of gephyrin (Kins et al., 2000; Harvey et al., 2004). This observation is intriguing, because it means either that gephyrin interaction with GABA_A receptors is required for cell-surface targeting, or that gephyrin is not stably anchored at synaptic sites in neurons lacking GABA_A receptors and forms such aggregates prior to degradation. It is of note, however, that no gephyrin aggregates were reported in the brain of γ2^0/0 mice, which lack postsynaptic GABA_A receptor and gephyrin clusters throughout the brain, but die within a few days after birth (Essrich et al., 1998; Baer et al., 1999). It is therefore possible that aggregates seen in α1^0/0 and α3^0/0 mice are formed slowly and become apparent only in adult animals.

The presence of numerous GABAergic terminals in the nRT of α3^0/0 mice shows that GABAergic synaptogenesis occurs normally even in the absence of GABA_A receptors. However, it cannot be excluded that the presence of the α5 subunit during a short time-window possibly contributes to GABAergic synaptogenesis in mutant mice. The maintenance of GABAergic terminals in the absence of GABA_A receptor-mediated transmission also occurs in the cerebellum of α1^0/0 mice (Fritschy et al., 2006). Ultrastructural analysis will be required to demonstrate directly the presence of GABAergic synapses on nRT neurons in mutant mice. It is unclear whether the enlarged size of these terminals represents a potential compensatory mechanism to increase GABA release as no immunohistochemical evidence for extrasynaptic GABA_A receptors was obtained in this study. However, a functional analysis remains to be carried out to settle this issue.

Functional implications

The nRT is an important gate regulating the function of the thalamo-cortical loop and it likely contributes to bottom-up and top-down processing in multiple sensory and motor modalities (reviewed in Pinault, 2004). It is reciprocally connected to first and higher order relay nuclei and receives collaterals from layer VI corticothalamic axons (Jones, 2002). The crucial role of GABA_A receptors in the nRT for regulating rhythmic firing and synchronization of thalamocortical neurons was first demonstrated in β3^0/0 mice, which, like α3^0/0 mice, lack GABA_A receptors in the nRT (Huntsman et al., 1999). However, these mice have a severe phenotype and a curtailed survival, and exhibit major changes in GABA_A receptor expression in other brain areas (Ramadan et al., 2003), which limits their use for studying the role of the nRT in thalamocortical function.

The nRT has been proposed to serve as pacemaker for generating oscillations in the spindle frequency range and is critically involved in inhibition of relay neurons during absence seizures (reviewed in Fuentealba & Steriade, 2005; Steriade, 2005). It also plays a general modulating or resetting role of thalamocortical oscillations that underlie discrete conscious events and regulate arousal states. The functional consequences of loss of GABAergic inhibition in the nRT of α3^0/0 mice are difficult to predict, in particular as GABAergic control of layer VI corticothalamic neurons, which mainly express α3-GABA_A receptors, is also likely impaired. Furthermore, tight-junction coupling between nRT neurons (Landisman et al., 2002) might contribute to stabilize the activity of the entire population of nRT neurons in the absence intranuclear inhibition. Finally, compensatory adaptations involving other key mechanisms of synaptic inhibition in nRT neurons, notably expression of GABA_B receptor and/or low threshold (T-type) Ca²⁺ channels might occur.

However, pharmacological blockade of intrareticular inhibition facilitates spike-wave discharges from thalamocortical cells (Sohal & Huguenard, 2003). Increased propensity or pharmacological sensitivity of α3^0/0 mice to exhibit absence seizures may therefore be expected. Furthermore, thalamic dysrhythmia, a pathological condition underlying negative and positive symptoms in neurological and psychiatric disorders (reviewed in Llinas et al., 2005) can emerge as a consequence of excessive inhibition of thalamocortical neurons. The subtle phenotype described so far in α3^0/0 mice (Yee et al., 2005) might reflect the fact that the thalamocortical system might operate normally under baseline conditions. The effects of excessive and uncoordinated inhibition of thalamocortical cells hypothesized above might become evident only after a specific challenge or under pathophysiological conditions, suggesting that α3^0/0 mice represent a potential animal model of thalamocortical dysrhythmia.

Acknowledgements

This study was supported by the Swiss National Science Foundation (grant No. 3100A0–108260 to JMF and 3100A0–102113 to UR). We would like to thank Dr Werner Sieghart (University of Vienna) for a generous gift of antisera against the GABA_A receptor α4 and α6 subunit, Corinne Sidler and Franziska Parpan for excellent technical assistance, and Ruth Keist for her essential contribution to the generation and breeding of α3^0/0 mice.

Abbreviations

nRT: nucleus reticularis thalami
P: postnatal day
VB: ventrobasal complex
VGAT: vesicular GABA transporter

References

Alldred, M.J., Mulder-Rosi, J., Lingenfelter, S.E., Chen, G. & Lüscher, B. (2005) Distinct γ2 subunit domains mediate clustering and synaptic function of postsynaptic GABA_A receptors and gephyrin. J. Neurosci., 19, 594–603.
10.1523/JNEUROSCI.4011-04.2005
CAS Google Scholar
Baer, K., Essrich, C., Balsiger, S., Wick, M.J., Harris, R.A., Fritschy, J.M. & Luscher, B. (2000) Rescue of γ2 subunit-deficient mice by transgenic overexpression of the GABA_A receptor γ2S or γ2L subunit isoforms. Eur. J. Neurosci., 12, 2639–2643.
10.1046/j.1460-9568.2000.00159.x
CAS PubMed Web of Science® Google Scholar
Baer, K., Essrich, C., Benson, J.A., Benke, D., Bluethmann, H., Fritschy, J.M. & Luscher, B. (1999) Postsynaptic clustering of γ-aminobutyric acid type A receptors by the γ3 subunit in vivo. Proc. Natl Acad. Sci. USA, 96, 12860–12865.
10.1073/pnas.96.22.12860
CAS PubMed Web of Science® Google Scholar
Behar, T.N., Schaffner, A.E., Scott, C.A., Greene, C.L. & Barker, J.L. (2000) GABA_A receptor antagonists modulate postmitotic cell migration in slice cultures of embryonic rat cortex. Cereb. Cortex, 10, 899–909.
10.1093/cercor/10.9.899
CAS PubMed Web of Science® Google Scholar
Ben-Ari, Y. (2002) Excitatory actions of GABA during development: The nature of the nurture. Nature Rev. Neurosci., 3, 728–739.
10.1038/nrn920
CAS PubMed Web of Science® Google Scholar
Bentivoglio, M., Spreafico, R., Alvarez-Bolado, G., Sanchez, M.P. & Fairen, A. (1991) Differential expression of the GABA_A receptor complex in the dorsal thalamus and reticular nucleus: an immunohistochemical study in the adult and developing rat. Eur. J. Neurosci., 3, 118–125.
10.1111/j.1460-9568.1991.tb00072.x
PubMed Web of Science® Google Scholar
Browne, S.H., Kang, J., Akk, G., Chiang, L.W., Schulman, H., Huguenard, J.R. & Prince, D.A. (2001) Kinetic and pharmacological properties of GABA_A receptors in single thalamic neurons and GABA_A subunit expression. J. Neurophysiol., 86, 2312–2322.
10.1152/jn.2001.86.5.2312
CAS PubMed Web of Science® Google Scholar
Demarque, M., Represa, A., Becq, H., Khalilov, I., Ben-Ari, Y. & Aniksztejn, L. (2002) Paracrine intercellular communication by a Ca²⁺– and SNARE-independent release of GABA and glutamate prior to synapse formation. Neuron, 36, 1051–1061.
10.1016/S0896-6273(02)01053-X
CAS PubMed Web of Science® Google Scholar
Essrich, C., Lorez, M., Benson, J.A., Fritschy, J.M. & Luscher, B. (1998) Postsynaptic clustering of major GABA_A receptor subtypes requires the γ2 subunit and gephyrin. Nature Neurosci., 1, 563–571.
10.1038/2798
CAS PubMed Web of Science® Google Scholar
Farrant, M. & Nusser, Z. (2005) Variations on an inhibitory theme: Phasic and tonic activation of GABA_A receptors. Nature Rev. Neurosci., 6, 215–229.
10.1038/nrn1625
CAS PubMed Web of Science® Google Scholar
Fritschy, J.M., Benke, D., Johnson, D.K., Mohler, H. & Rudolph, U. (1997) GABA_A-receptor α subunit is an essential prerequisite for receptor formation in vivo. Neuroscience, 81, 1043–1053.
10.1016/S0306-4522(97)00244-3
CAS PubMed Web of Science® Google Scholar
Fritschy, J.M. & Mohler, H. (1995) GABA_A-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J. Comp. Neurol., 359, 154–194.
10.1002/cne.903590111
CAS PubMed Web of Science® Google Scholar
Fritschy, J.M., Panzanelli, P., Kralic, J.E., Vogt, K.E. & Sassoè-Pognetto, M. (2006) Differential dependence of axo-dendritic and axo-somatic GABAergic synapses on GABA_A receptors containing the α1 subunit in Purkinje cells. J. Neurosci., 26, 3245–3255.
10.1523/JNEUROSCI.5118-05.2006
CAS PubMed Web of Science® Google Scholar
Fritschy, J.M., Paysan, J., Enna, A. & Mohler, H. (1994) Switch in the expression of rat GABA_A-receptor subtypes during postnatal development: an immunohistochemical study. J. Neurosci., 14, 5302–5324.
10.1523/JNEUROSCI.14-09-05302.1994
CAS PubMed Web of Science® Google Scholar
Fritschy, J.M., Weinmann, O., Wenzel, A. & Benke, D. (1998) Synapse-specific localization of NMDA- and GABA_A-receptor subunits revealed by antigen-retrieval immunohistochemistry. J. Comp. Neurol., 390, 194–210.
10.1002/(SICI)1096-9861(19980112)390:2<194::AID-CNE3>3.0.CO;2-X
CAS PubMed Web of Science® Google Scholar
Fuentealba, P. & Steriade, M. (2005) The reticular nucleus revisited: intrinsic and network properties of a thalamic pacemaker. Prog. Neurobiol., 75, 125–141.
10.1016/j.pneurobio.2005.01.002
CAS PubMed Web of Science® Google Scholar
Gao, B., Fritschy, J.M., Benke, D. & Mohler, H. (1993) Neuron-specific expression of GABA_A-receptor subtypes: differential associations of the α1- and α3-subunits with serotonergic and GABAergic neurons. Neuroscience, 54, 881–892.
10.1016/0306-4522(93)90582-Z
CAS PubMed Web of Science® Google Scholar
Gao, B., Hornung, J.P. & Fritschy, J.M. (1995) Identification of distinct GABA_A-receptor subtypes in cholinergic and parvalbumin-positive neurons of the rat and marmoset medial-septum-diagonal band complex. Neuroscience, 65, 101–117.
10.1016/0306-4522(94)00480-S
CAS PubMed Web of Science® Google Scholar
Harvey, K., Duguid, I.C., Alldred, M.J., Beatty, S.E., Ward, H., Keep, N.H., Lingenfelter, S.E., Pearce, B.R., Lundgren, J., Owen, M.J., Smart, T.G., Luscher, B., Rees, M.I. & Harvey, R.J. (2004) The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering. J. Neurosci., 24, 5816–5826.
10.1523/JNEUROSCI.1184-04.2004
CAS PubMed Web of Science® Google Scholar
Huntsman, M.M., Porcello, D.M., Homanics, G.E., DeLorey, T.M. & Huguenard, J.R. (1999) Reciprocal inhibitory connections and network synchrony in the mammalian thalamus. Science, 283, 541–543.
10.1126/science.283.5401.541
CAS PubMed Web of Science® Google Scholar
Jones, E.G. (2002) Thalamic circuitry and thalamocortical synchrony. Phil. Trans. R. Soc. Lond. B, 357, 1659–1673.
10.1098/rstb.2002.1168
PubMed Web of Science® Google Scholar
Jones, A., Korpi, E.R., McKernan, R.M., Pelz, R., Nusser, Z., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H. & Wisden, W. (1997) Ligand-gated ion channel subunit partnerships: GABA_A receptor α6 subunit gene inactivation inhibits delta subunit expression. J. Neurosci., 17, 1350–1362.
10.1523/JNEUROSCI.17-04-01350.1997
CAS PubMed Web of Science® Google Scholar
Kins, S., Betz, H. & Kirsch, J. (2000) Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin. Nature Neurosci., 3, 22–29.
10.1038/71096
CAS PubMed Web of Science® Google Scholar
Korpi, E.R., Mihalek, R.M., Sinkkonen, S.T., Hauer, B., Hevers, W., Homanics, G.E., Sieghart, W. & Luddens, H. (2002) Altered receptor subtypes in the forebrain of GABA_A receptor δ subunit-deficient mice: recruitment of γ2 subunits. Neuroscience, 109, 733–743.
10.1016/S0306-4522(01)00527-9
CAS PubMed Web of Science® Google Scholar
Kralic, J.E., Sidler, C., Parpan, F., Homanics, G., Morrow, A.L. & Fritschy, J.M. (2006) Compensatory alteration of inhibitory synaptic circuits in thalamus and cerebellum of GABA_A receptor α1 subunit knockout mice. J. Comp. Neurol., 495, 408–421.
10.1002/cne.20866
CAS PubMed Web of Science® Google Scholar
Landisman, C.E., Long, M.A., Beierlein, M., Deans, M.R., Paul, D.L. & Connors, B.W. (2002) Electrical synapses in the thalamic reticular nucleus. J. Neurosci., 22, 1002–1009.
10.1523/JNEUROSCI.22-03-01002.2002
CAS PubMed Web of Science® Google Scholar
Llinas, R., Urbano, F.J., Leznik, E., Ramirez, R.R. & Van Marle, H.J.F. (2005) Rhythmic and dysrhythmic thalamocortical dynamics: GABA systems and the edge effect. TINS, 28, 325–333.
10.1016/j.tins.2005.04.006
CAS PubMed Web of Science® Google Scholar
Maric, D., Liu, Q.Y., Maric, I., Chaudry, S., Chang, Y.H., Smith, S.V., Sieghart, W., Fritschy, J.M. & Barker, J.L. (2001) GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA_A autoreceptor/Cl^– channels. J. Neurosci., 21, 2343–2360.
CAS PubMed Web of Science® Google Scholar
McKernan, R.M. & Whiting, P.J. (1996) Which GABA_A-receptor subtypes really occur in the brain? TINS, 19, 139–143.
10.1016/S0166-2236(96)80023-3
PubMed Web of Science® Google Scholar
Mohler, H., Fritschy, J.M. & Rudolph, U. (2002) A new benzodiazepine pharmacology. J. Pharm. Exp. Ther., 300, 2–8.
10.1124/jpet.300.1.2
CAS PubMed Web of Science® Google Scholar
Moss, S. & Smart, T. (2001) Constructing inhibitory synapses. Nature Rev. Neurosci., 2, 240–250.
10.1038/35067500
CAS PubMed Web of Science® Google Scholar
Peng, Z., Hauer, B., Mihalek, R.M., Homanics, G.E., Sieghart, W., Olsen, R.W. & Houser, C.R. (2002) GABA_A receptor changes in δ subunit-deficient mice: Altered expression of α4 and γ2 subunits in the forebrain. J. Comp. Neurol., 446, 179–197.
10.1002/cne.10210
CAS PubMed Web of Science® Google Scholar
Pinault, D. (2004) The thalamic reticular nucleus: structure, function and concept. Brain Res. Rev., 46, 1–31.
10.1016/j.brainresrev.2004.04.008
PubMed Web of Science® Google Scholar
Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W. & Sperk, G. (2000) GABA_A receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience, 101, 815–850.
10.1016/S0306-4522(00)00442-5
CAS PubMed Web of Science® Google Scholar
Ramadan, E., Fu, Z., Losi, G., Homanics, G.E., Neale, J.H. & Vicini, S. (2003) GABA_A receptor β3 subunit deletion decrases α2/3 subunits and IPSC duration. J. Neurophysiol., 89, 128–134.
10.1152/jn.00700.2002
CAS PubMed Web of Science® Google Scholar
Rudolph, U. & Möhler, H. (2004) Analysis of GABA_A receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annu. Rev. Pharmacol. Toxicol., 44, 475–498.
10.1146/annurev.pharmtox.44.101802.121429
CAS PubMed Web of Science® Google Scholar
Sassoè-Pognetto, M., Panzanelli, P., Sieghart, W. & Fritschy, J.M. (2000) Co-localization of multiple GABA_A receptor subtypes with gephyrin at postsynaptic sites. J. Comp. Neurol., 420, 481–498.
10.1002/(SICI)1096-9861(20000515)420:4<481::AID-CNE6>3.0.CO;2-5
CAS PubMed Web of Science® Google Scholar
Schweizer, C., Balsiger, S., Bluethmann, H., Mansuy, I.M., Fritschy, J.M., Mohler, H. & Lüscher, B. (2003) The γ2 subunit of GABA_A receptors is required for maintenance of receptors at mature synapses. Mol. Cell. Neurosci., 24, 442–450.
10.1016/S1044-7431(03)00202-1
CAS PubMed Web of Science® Google Scholar
Sieghart, W. & Sperk, G. (2002) Subunit composition, distribution and function of GABA_A receptor subtypes. Cur. Topics Med. Chem., 2, 795–816.
10.2174/1568026023393507
CAS PubMed Google Scholar
Sohal, V.S. & Huguenard, J.R. (2003) Inhibitory interconnections control burst pattern and emergent network synchrony in reticular thalamus. J. Neurosci., 23, 8978–8988.
10.1523/JNEUROSCI.23-26-08978.2003
CAS PubMed Web of Science® Google Scholar
Steriade, M. (2005) Sleep, epilepsy and thalamic reticular inhibitory neurons. TINS, 28, 317–324.
10.1016/j.tins.2005.03.007
CAS PubMed Web of Science® Google Scholar
Sur, C., Wafford, K.A., Reynolds, D.S., Hadingham, K.L., Bromidge, F., Macaulay, A., Collinson, N., O'Meara, G., Howell, O., Newman, R., Myers, J., Atack, J.R., Dawson, G.R., McKernan, R.M., Whiting, P.J. & Rosahl, T.W. (2001) Loss of the major GABA_A receptor subtype in the brain is not lethal in mice. J. Neurosci., 21, 3409–3418.
10.1523/JNEUROSCI.21-10-03409.2001
CAS PubMed Web of Science® Google Scholar
Vicini, S., Ferguson, C., Prybylowski, K., Kralic, J., Morrow, A.L. & Homanics, G.E. (2001) GABA_A receptor α1 subunit deletion prevents developmental changes of inhibitory synaptic currents in cerebellar neurons. J. Neurosci., 21, 3009–3016.
10.1523/JNEUROSCI.21-09-03009.2001
CAS PubMed Web of Science® Google Scholar
Yee, B.K., Keist, R., Von Boehmer, L., Studer, R., Benke, D., Hagenbuch, B., Dong, Y., Malenka, R.C., Fritschy, J.M., Bluethmann, H., Feldon, J., Mohler, H. & Rudolph, U. (2005) A schizophrenia-related sensorimotor deficit links α3-containing GABA_A receptors to a dopamine hyperfunction. Proc. Natl Acad. Sci. USA, 102, 17154–17159.
10.1073/pnas.0508752102
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume24, Issue5

September 2006

Pages 1307-1315

Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABA_A receptor α3 subunit-null mice

Abstract

Introduction