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• W2034100258 abstract "Article9 August 2007free access Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice Theofilos Papadopoulos Theofilos Papadopoulos Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Martin Korte Martin Korte Zoological Institute, Technical University Braunschweig, Braunschweig, Germany Search for more papers by this author Volker Eulenburg Volker Eulenburg Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Hisahiko Kubota Hisahiko Kubota Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Independent Hertie Research Group, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Marina Retiounskaia Marina Retiounskaia Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Robert J Harvey Robert J Harvey Department of Pharmacology, The School of Pharmacy, London, UK Search for more papers by this author Kirsten Harvey Kirsten Harvey Department of Pharmacology, The School of Pharmacy, London, UK Search for more papers by this author Gregory A O'Sullivan Gregory A O'Sullivan Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Bodo Laube Bodo Laube Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Swen Hülsmann Swen Hülsmann Department of Neuro- and Sensory Physiology, University of Göttingen, Göttingen, Germany Search for more papers by this author Jörg R P Geiger Jörg R P Geiger Independent Hertie Research Group, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Heinrich Betz Corresponding Author Heinrich Betz Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Theofilos Papadopoulos Theofilos Papadopoulos Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Martin Korte Martin Korte Zoological Institute, Technical University Braunschweig, Braunschweig, Germany Search for more papers by this author Volker Eulenburg Volker Eulenburg Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Hisahiko Kubota Hisahiko Kubota Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Independent Hertie Research Group, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Marina Retiounskaia Marina Retiounskaia Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Robert J Harvey Robert J Harvey Department of Pharmacology, The School of Pharmacy, London, UK Search for more papers by this author Kirsten Harvey Kirsten Harvey Department of Pharmacology, The School of Pharmacy, London, UK Search for more papers by this author Gregory A O'Sullivan Gregory A O'Sullivan Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Bodo Laube Bodo Laube Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Swen Hülsmann Swen Hülsmann Department of Neuro- and Sensory Physiology, University of Göttingen, Göttingen, Germany Search for more papers by this author Jörg R P Geiger Jörg R P Geiger Independent Hertie Research Group, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Heinrich Betz Corresponding Author Heinrich Betz Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Search for more papers by this author Author Information Theofilos Papadopoulos1, Martin Korte2, Volker Eulenburg1, Hisahiko Kubota1,3, Marina Retiounskaia1, Robert J Harvey4, Kirsten Harvey4, Gregory A O'Sullivan1, Bodo Laube1, Swen Hülsmann5, Jörg R P Geiger3 and Heinrich Betz 1 1Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany 2Zoological Institute, Technical University Braunschweig, Braunschweig, Germany 3Independent Hertie Research Group, Max-Planck-Institute for Brain Research, Frankfurt, Germany 4Department of Pharmacology, The School of Pharmacy, London, UK 5Department of Neuro- and Sensory Physiology, University of Göttingen, Göttingen, Germany *Corresponding author. Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrassee 46, 60528 Frankfurt am Main, Germany. Tel.: +49 69 96769 220; Fax: +49 69 96769 441; E-mail: [email protected] The EMBO Journal (2007)26:3888-3899https://doi.org/10.1038/sj.emboj.7601819 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor that has been implicated in plasma membrane targeting of the postsynaptic scaffolding protein gephyrin found at glycinergic and GABAergic synapses. Here we show that Cb-deficient mice display a region-specific loss of postsynaptic gephyrin and GABAA receptor clusters in the hippocampus and the basolateral amygdala. Cb deficiency is accompanied by significant changes in hippocampal synaptic plasticity, due to reduced dendritic GABAergic inhibition. Long-term potentiation is enhanced, and long-term depression reduced, in Cb-deficient hippocampal slices. Consistent with the anatomical and electrophysiological findings, the animals show increased levels of anxiety and impaired spatial learning. Together, our data indicate that Cb is essential for gephyrin-dependent clustering of a specific set of GABAA receptors, but not required for glycine receptor postsynaptic localization. Introduction Fast synaptic transmission in the nervous system is mediated by ligand-gated ion channels, which are highly concentrated at postsynaptic membrane specializations. At inhibitory synapses, the scaffolding protein gephyrin is required for the synaptic localization of glycine receptors (GlyRs) and major GABAA receptor (GABAAR) subtypes (Kneussel and Betz, 2000; Moss and Smart, 2001). Ablation of gephyrin expression by either antisense depletion in cultured neurons or gene knockout (KO) in mice prevents the clustering of GlyRs (Kirsch et al, 1993; Feng et al, 1998) and α2- and γ2-subunit containing GABAARs (Essrich et al, 1998; Kneussel et al, 1999; Lüscher and Keller, 2004) at developing postsynaptic sites. The lack of postsynaptic inhibitory receptors is believed to underlie the early postnatal (day of birth, P0) lethality of gephyrin-deficient mice (Feng et al, 1998). Gephyrin depends on both actin microfilaments and microtubules for synaptic targeting and submembraneous scaffold formation (Kirsch and Betz, 1995; Allison et al, 2000; Bausen et al, 2006; Maas et al, 2006) and binds to the cytoplasmic loop of the GlyR β-subunit (Meyer et al, 1995; Sola et al, 2004). In addition, the actin-binding proteins profilin (Mammoto et al, 1998) and Mena (mammalian enabled)/VASP (vasodilator stimulated phosphoprotein) (Giesemann et al, 2003; Bausen et al, 2006), as well as the dynein light chains 1 and 2 (Fuhrmann et al, 2002), interact with gephyrin. Gephyrin also binds to collybistin (Cb) (Kins et al, 2000), a guanine nucleotide exchange factor (GEF) for small Rho-like GTPases. Multiple Cb splice variants (I–III) have been identified (Kins et al, 2000; Harvey et al, 2004a), and the human Cb homologue, hPEM-2, has been demonstrated to function as a GEF specific for Cdc42 in fibroblasts (Reid et al, 1999). Cb I and II share the catalytic DH and membrane-binding PH domains, but differ by the presence of an src homology 3 (SH3) region and a coiled-coil structure in Cb I. Coexpression of gephyrin with Cb II in human embryonic kidney (HEK) 293 cells alters the subcellular distribution of gephyrin by relocating it from large intracellular aggregates to small clusters at the cellular cortex (Kins et al, 2000). Cb has therefore been proposed to participate in a membrane activation process that labels postsynaptic membrane domains for inhibitory synapse formation (Kneussel and Betz, 2000). Indeed, overexpression of a Cb II deletion mutant lacking the PH domain interfered with clustering of gephyrin at synaptic sites (Harvey et al, 2004a). Furthermore, in a human patient suffering from hyperekplexia and epilepsy a mutation in the SH3 domain of Cb has been identified. Overexpression of this Cb mutant in cultured neurons resulted in the loss of postsynaptic gephyrin clusters (Harvey et al, 2004a). Together, these data point to an important role of Cb in gephyrin localization at inhibitory synapses. To assess whether Cb is required for gephyrin-dependent clustering of inhibitory ionotropic receptors in vivo, we disrupted the Cb gene in mice. Herein, we show that in the absence of this neuronal GEF, gephyrin and gephyrin-dependent GABAAR subtypes, but not GlyRs, are lost from postsynaptic sites in specific brain regions. This results in reduced GABAergic transmission, altered hippocampal synaptic plasticity, increased anxiety scores and impaired spatial learning. Our study discloses an essential role of Cb at selected GABAergic synapses. Results Generation of Cb KO mice To inactivate the mouse Cb gene (Arhgef9), located on the X-chromosome, in embryonic stem (ES) cells, we employed a targeting vector that allowed selective deletion of exon 5 by using the Cre-Lox system (Kuhn et al, 1995) (Figure 1A). Exon 5 encodes part of the catalytic RhoGEF domain, and its deletion is predicted to destroy exchange factor activity and to create a frameshift such that the entire C-terminus of Cb is lost. Using this strategy, we produced two independent properly targeted ‘knockout’ (KO) ES cell clones, which were injected into blastocysts to generate chimeric male mice. These animals were then crossed with C57BL/6 female mice to establish germline transmission of the mutation and to generate KO offspring. Mouse genotyping was carried out by Southern blot analysis of mouse tail DNA (Figure 1B). The absence of wild-type (WT) and truncated Cb transcripts and proteins in the Cb KO mice was confirmed by Northern blotting (Figure 1C) and Western blot analyses (Figure 1D), respectively. Figure 1.Targeted disruption of the mouse Cb gene. (A) Schematic representation of the targeting strategy showing the WT Cb gene, the targeting vector, the ‘floxed’ locus and the null allele. Exons are represented as black boxes. The neomycin resistance cassette (neo) and the herpes simplex virus thymidine kinase (tk) gene of the targeting vector are indicated as gray boxes, and loxP sites as triangles. P corresponds to the probe used for Southern analysis. SA, short arm; LA, long arm. Relevant restriction sites indicated are as follows: H, HindIII; N, NotI. (B) Southern blot analysis of HindIII-digested mouse tail DNA from WT, Cb KO and heterozygous (±) offspring. The 4.2 kb and the 3.1 kb bands represent the WT and the KO alleles, respectively. (m), male; (f), female. (C) Upper panel: Northern blot of brain total RNA with a 430-bp cDNA probe encoding exons 1–3 of the mouse Cb gene reveals 5.5 and 6.0 kb Cb transcripts in WT but not in Cb KO mice. Lower panels: control hybridizations with random-primed probes derived from a 714-bp fragment of the mouse β-actin (β-act) cDNA or a 490-bp fragment of the mouse GAPDH cDNA demonstrate that comparable amounts of total RNA were loaded. (D) Western blot analysis of brain homogenates (50 μg protein/lane) using either a polyclonal Cb antibody (upper panel) or a monoclonal antibody specific for the N-terminally located SH3 domain of the Cb protein (lower panel). Note the absence of Cb protein (indicated by arrows) in all KO samples. Asterisks indicate unspecific bands stained under the experimental conditions used. A β-tubulin (β-tub)-specific antibody was used to confirm that roughly equal amounts of protein were loaded. (E) Western blot analysis of crude membrane fractions prepared from different CNS regions of WT and Cb KO mice. Equal amounts of protein (50 μg protein/lane) were loaded and probed with the indicated antibodies. Note that the expression levels of all proteins tested were not significantly different between genotypes; densitometric scanning of the band intensities determined in three independent experiments did not reveal significant differences for any of the immunoreactive bands examined (data not shown). Geph, gephyrin; γ2, GABAAR γ2-subunit; GlyR, glycine receptor; VIAAT, vesicular inhibitory amino acid transporter; NL2, neuroligin 2; Syn, synaptophysin. Download figure Download PowerPoint Initial characterization of Cb KO mice Female mice heterozygous for the Cb mutant allele (X+X−) appeared phenotypically normal and showed unimpaired development and fertility. Crossing of the heterozygous female mice with C57BL/6 males, or intercrossing them with Cb KO (X−Y) males, generated KO mice at Mendelian ratios. These animals also proved to be viable and fertile. For further analysis of the consequences of Cb deficiency, we focused on Cb KO males and their WT littermates. A histological analysis of cresyl-violet-stained sections from WT and KO brains failed to disclose differences in both brain size and morphology between genotypes (data not shown). Western blot analysis performed on hippocampal, cerebellar, brainstem and spinal cord membranes prepared from adult WT and Cb KO mice revealed that the immunoreactivities of the postsynaptic proteins gephyrin, the GABAAR γ2-subunit, the GlyR α-subunits and neuroligin 2, and of the presynaptic proteins VIAAT (vesicular inhibitory amino-acid transporter) and synaptophysin, all were similar in both genotypes (Figure 1E). Thus, inactivation of the Cb gene did not affect the expression of proteins found at inhibitory synapses. Cb KO mice show normal locomotor behavior but enhanced anxiety The motor performance of the Cb KO mice was normal at all postnatal stages examined. The mutant mice showed no signs of ‘hind feet clasping’, increased tremor or altered gait/righting responses, that is, symptoms indicative of impaired glycinergic inhibition (Gomeza et al, 2003). When locomotor behavior was tested in the open field, also no significant differences were detected between genotypes. Figure 2A and B indicate representative paths traveled by a WT (A) and a Cb KO (B) animal during an observation period of 10 min; the total path length covered by Cb KO mice was about 96% of that measured for the WT group (Figure 2C). Notably however, Cb KO mice spent significantly less time in the center than their WT littermates (Figure 2D), suggesting behavioral differences in anxiety-related responses between mutants and controls. Furthermore, in the elevated plus-maze, the KO mice spent significantly less time on the open arms than their WT littermates (Figure 2E). In addition, KO animals dipped their heads into the open arms less often than WT animals (Figure 2F). Thus, inactivation of the Cb gene resulted in a reduced exploratory behavior and increased anxiety scores without affecting motor performance. Figure 2.Behavior of Cb KO mice and WT littermates in the open field and elevated plus-maze tests. Male Cb KO mice and their WT littermates were tested between 8 and 10 weeks of age (open field, n=10 per group; elevated plus-maze, n=7 per group). All values represent means±s.e.m. (A–D) Open field. (A, B) indicate representative paths traveled by a WT (A) and a Cb KO (B) animal during a period of 10 min. (C) No significant differences between genotypes were observed in the total distance traveled in the open field. (D) Cb KO mice showed a strong reduction in the time spent in the center of the open field and stayed significantly longer in the periphery as compared to WT (**P<0.01; Student's t-test). (E, F) Elevated plus-maze. (E) Cb KO mice spent less time on the open arms as compared with WT controls (*P<0.05; Student's t-test). (F) The number of head dips into open arms was strongly reduced with Cb KO animals as compared with WT littermates (***P<0.001; Student's t-test). Download figure Download PowerPoint Region-specific reduction of synaptic gephyrin and GABAAR clusters in the Cb KO CNS As the expression levels of different synaptic proteins in the CNS of the Cb KO mice were unaltered (Figure 1E), we hypothesized that the observed behavioral deficits may be related to redistribution rather than loss of inhibitory postsynaptic proteins. Therefore, the postsynaptic localization of gephyrin was examined in brain sections prepared from adult WT and Cb KO mice. In WT sections, punctate gephyrin immunoreactivity was found throughout all CNS regions examined (Figure 3A, C and E). These gephyrin clusters were synaptically localized, as revealed by their apposition to presynaptic VIAAT immunoreactivity (Figure 3G, I and K). In contrast, brain sections derived from Cb KO mice showed region-specific reductions in the density of gephyrin clusters. The loss of punctate gephyrin immunoreactivity was most pronounced in hippocampal structures including the CA1–CA3 of stratum radiatum, stratum oriens (Figure 3B and J), dentate gyrus (hilus, molecular layer), lateral and ventral parts of the thalamus (data not shown), the amygdala (not shown) and the cerebellum (Purkinje cells, molecular layer; Figure 3D). In the granule cell layer of the olfactory bulb (not shown), the stratum pyramidale of the hippocampus (Figure 3B and H) and the granule cell and molecular layers of the cerebellum (Figure 3D), gephyrin was found in larger punctate structures that did not colocalize with VIAAT and corresponded to cytoplasmic gephyrin aggregates (data not shown). In contrast, when neocortex, striatum, medial thalamic areas, brainstem (caudal medulla) or spinal cord were analyzed, punctate gephyrin immunoreactivities were indistinguishable between KO and WT (Figure 3E, F, K and L, and data not shown). A summary of the anatomical results obtained is given in Supplementary Table I. Figure 3.Region-specific reduction of synaptic gephyrin staining in the Cb KO CNS. (A–L) Sections from adult Cb KO mice and their WT littermates were stained with gephyrin and VIAAT antibodies. (A, B) The punctate staining of sections from KO animals for gephyrin was strongly reduced in the CA1 region of SR and SO as compared with WT sections. Note the significant increase of gephyrin deposits (arrows in B) in the SP of KO sections (A). (C, D) A strong reduction of gephyrin immunoreactivity was also observed in cerebellar regions (GCL, PCL, ML) of Cb KO animals. (E, F) BS CM sections in contrast showed similar densities of gephyrin puncta in both WT and KO animals. (G–L) Double immunostainings of gephyrin and VIAAT puncta in the SP (G, H) and SR (I, J) of the hippocampus, and in brainstem (K, L) of WT and Cb KO mice. Note that, in contrast to the loss of gephyrin puncta seen in (H, J), VIAAT immunoreactivity was unaffected by Cb deficiency (H, I). Scale bars, 32 μm (A–F), 16 μm (G–L). (M, N) Quantification of gephyrin and VIAAT immunoreactivities. For both genotypes, each bar corresponds to mean values (±s.e.m.) obtained with sections from 3–4 individual brains (***P<0.001; Student's t-test). SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; GCL, granule cell layer; PCL, Purkinje cell layer; Cer ML, molecular layer of cerebellum; BS CM, caudal medulla of the brainstem; DG HL, hilus of the dentate gyrus; BLA, basolateral amygdala. Download figure Download PowerPoint Quantification of the number of gephyrin-immunoreactive puncta per 1000 μm2 section area resulted in density values of 329±27 versus 7±0.6 for the stratum radiatum of the hippocampal CA1 region, 204±4 versus 43±5 for the hilus of the dentate gyrus, 240±5 versus 90±19.5 for the basolateral amygdala, 219±28 versus 43±5 for the granule cell layer of the cerebellum, 247±43 versus 9±0.5 for the molecular layer of the cerebellum, and 262±35 versus 225±31 for the brainstem (caudal medulla) in WT and Cb KO sections, respectively (Figure 3M). The number of VIAAT-immunoreactive sites was unaffected by the loss of Cb even in areas showing a marked reduction of gephyrin clusters (Figure 3N). This indicates that the density of inhibitory nerve terminals was not changed in the KO mice. Similar results were obtained when primary cultures of hippocampal neurons derived from WT and Cb KO littermate embryos were compared for dendritic gephyrin and VIAAT immunoreactivities (Supplementary Figure 1). Since gephyrin is essential for synaptic clustering of both GlyRs (Feng et al, 1998; Harvey et al, 2004b) and α2- and γ2-subunit containing GABAARs (Essrich et al, 1998; Kneussel et al, 1999, 2001a), we examined the effects of Cb deficiency on GlyR and GABAAR localization. The distribution of GlyR clusters revealed by mAb4a staining was unchanged in three different CNS areas expressing GlyRs, the caudal medulla of the brainstem, the basolateral amygdala and the hilus of the dentate gyrus (Figure 4A–C). GlyR cluster numbers per 1000 μm2 area were 327.8±22.7 versus 319.8±29.5 for brainstem, 29±2.8 versus 28.8±2.5 for the basolateral amygdala and 30.8±4.6 versus 35.5±3.5 for the hilus in WT and Cb KO sections, respectively (Figure 4D). Double labeling with mAb4a and the VIAAT antibody showed that only 28.8±3.4 and 27.9±2.7% of the GlyR clusters in the hilus of WT and Cb KO preparations, respectively, were apposed to VIAAT (data not shown). This low percentage of synaptic GlyR clusters in the hippocampus is in agreement with a previously published study (Danglot et al, 2004). Figure 4.Reduced clustering of the GABAAR γ2-subunit in the hippocampus and basolateral amygdala of Cb KO mice. Sections from adult Cb KO mice and their WT littermates were stained with the GlyR specific mAb4a (A–C) or an antibody specific for the γ2-subunit of GABAARs (E–I) and processed for confocal microscopy. (A–C) The punctate staining of GlyRs was comparable in BS CM, BLA and DG HL of both Cb KO and WT mice. (D) Quantification of GlyR and GABAAR γ2-subunit immunoreactivities. For both genotypes, each bar corresponds to counts performed on sections from 3–4 individual brains. Data represent means±s.e.m. (**P<0.01; ***P<0.001; Student's t-test). (E, H) In BS CM and Cer ML, γ2-staining was unaffected by the loss of Cb, as compared to controls. (F, G, I) In contrast, the γ2-subunit punctate staining was selectively reduced in the BLA, the hilus of the DG and the CA1 region of SR in Cb KO sections, as compared with WT. Scale bar, 16 μm. BS CM, caudal medulla of the brainstem; BLA, basolateral amygdala; DG HL, hilus of the dentate gyrus; Cer ML, molecular layer of cerebellum; SR, stratum radiatum. Download figure Download PowerPoint For examining the distribution of GABAARs in the brain of Cb KO mice, we used an γ2-subunit-specific antibody. No significant difference was observed between WT and Cb KO sections in the number of GABAAR γ2-immunoreactive puncta in brainstem (caudal medulla) and cerebellum (Figure 4E and H), as well as in spinal cord, neocortex, thalamus and striatum (Supplementary Table I, and data not shown). In contrast, we consistently found a strong reduction of GABAAR γ2-subunit-positive puncta in the basolateral amygdala and the hippocampus of the Cb KO mice as compared with their WT littermates (Figure 4F, G and I; Supplementary Table I). Quantification of the number of γ2-positive clusters per 1000 μm2 section area revealed density values of 188±13.5 versus 24±1 for the basolateral amygdala, 321±16.5 versus 51±2 for the hilus of the dentate gyrus, and 327±71 versus 46.4±5.3 for the stratum radiatum, in WT and Cb KO sections, respectively (Figure 4D). A similar reduction of gephyrin-dependent dendritic GABAAR clustering was observed when primary cultures of hippocampal neurons derived from WT and Cb KO littermate embryos were stained with a GABAAR α2-subunit-specific antibody (Supplementary Figure 1). In conclusion, Cb deficiency leads to a region-specific reduction of gephyrin-dependent GABAAR clusters. Reduced dendritic GABAergic transmission in the hippocampus of Cb KO mice To determine the physiological consequences of the reduced GABAAR γ2-subunit clustering seen in the hippocampus, we recorded GABAergic miniature inhibitory postsynaptic currents (mIPSCs) from CA1 pyramidal neurons in slices prepared from KO mice and littermate WT controls (Figure 5A and B). The most prominent difference found was a strong reduction (43.3%) in mean mIPSC frequency in KO neurons as compared with controls (WT: 3.3±0.32 ms versus Cb KO: 1.87±0.19 ms; P<0.001; Figure 5C). In addition, a significant decrease in the mean amplitude of mIPSCs (WT: 57.08±2.74 pA versus Cb KO: 48.3±2.53 pA; P<0.05; Figure 5B and D) and a slower rise time (WT: 1.31±0.046 ms versus Cb KO: 1.57±0.068 ms; P<0.01; Figure 5B and E) of mIPSCs was found in Cb KO mice, whereas mean mIPSC decay times were similar in both genotypes (WT: 24.59±1.45 ms versus Cb KO: 22.67±1.68 ms; P=0.39; Figure 5F). These results indicate postsynaptic changes and show that the loss of Cb results in a substantial reduction of action potential-independent GABAergic inhibition of CA1 pyramidal cells. Figure 5.Altered GABAergic mIPSCs in CA1 pyramidal neurons of Cb KO slice preparations. (A) Representative traces of mIPSCs recorded in the absence or presence of the GABAA receptor antagonist bicuculline (10 μM) from WT and Cb KO neurons. (B) Superimposed averaged traces calculated from 3316 (WT) and 1316 (Cb KO) events over a recording period of 15 min. Dotted lines indicate peak positions of mIPSCs in each trace. (C–F) Cumulative distributions of mIPSC frequencies (C), amplitudes (D), rise times (E) and decay times (F) recorded from 14 neurons per genotype. In total, 40 351 (WT) and 23 370 (KO) events were analyzed. Each dot represents the mean value for an individual cell. Lines in each cumulative distribution indicate the averaged mean values of WT (open dots) and Cb KO (closed dots). P-values for each cumulative distribution are indicated (two-tailed Mann−Whitney U-test). All experiments were performed in the presence of 1 μM TTX, 10 μM CNQX and 1 μM D-AP5 at a VH of −65 mV. Download figure Download PowerPoint To test for action potential-dependent GABAergic inhibition, we performed experiments in brain slices and recorded evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and GABAAR-mediated IPSCs from the somata of CA1 pyramidal cells (Supplementary data). However, the ratio of the peak amplitudes of GABAergic IPSCs and glutamatergic EPSCs was not significantly different between WT and Cb KO mice (P>0.2; data not shown). This might be explained by the fact that strong perisomatic inhibition (Freund, 2003), largely mediated by gephyrin-independent α1 containing GABAARs (Kneussel et al, 2001a), masks differences in dendritic inhibition suggested by our immunohistochemical results (Figure 4; Supplementary Figure 1). To disclose selective alterations in evoked dendritic inhibition, we performed field potential recordings on acute hippocampal slices by stimulating the Schaffer collateral-CA1 synapse and compared the local dendritic field potential response to extracellular stimulation both in the absence and presence of the GABAAR antagonist picrotoxin (100 μM). Application of picrotoxin enhanced the slope of field excitatory postsynaptic potentials (fEPSPs) to a lesser extent in slices derived from Cb KO mice as compared with controls (WT: 130.1±4.1% versus Cb KO: 115.3±7.5%; P=0.04; Figure 6A), indicating a significant difference in GABAergic inhibition between genotypes. In conclusion, our analysis of mIPSCs and dendritic fEPSPs revealed a reduction of dendritic GABAAR-mediated transmission in Cb KO mice. Figure 6.Analysis of fEPSPs in the hippocampal CA1 region. (A) Left: fEPSP slope size was significantly increased by bath-application of 100 μM picrotoxin. This enhancement was significantly larger in WT as compared with Cb KO mice (*P<0.05; two-tailed Student's t-test). Right: sweeps from individual experiments before (aCSF) and after application of picrotoxin (aCSF+Pic), averaged over three trials. (B) Left: fEPSP slope size at various stimulus intensities (FV: fiber volley). Right: series of original traces recorded from WT and Cb KO slices. (C) Left: paired-pulse facilitation of the fEPSP at various interstimulus intervals (ISI) from Cb KO and WT slices. Right: single sweeps for WT and Cb KO mice recorded at 10–160 ISI. (D) Left: NMDA receptor and AMPA receptor contributions to the fEPSP recorded in the presence of the indicated antagonists under low (0.5 mM) Mg2+ conditions. Right: original traces from individual experiments. In panels A−C, there were no significant differences between KO and WT slices (P>0.1; Student's t-test). All values represent means±s.e.m. Download figure Download PowerPoint Consistent with the unaltered punctate GlyR staining seen in brainstem of Cb KO mice, we found no differences in glycinergic neurotransmission between WT and KO animals. Recordings from hypoglossal motoneurons (Gomeza et al, 2003) di" @default.
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• W2034100258 title "Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice" @default.
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