Molecular and Cellular Neuroscience 41 (2009) 94–100

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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m c n e

Increased network excitability and impaired induction of long-term potentiation in the dentate gyrus of collybistin-deficient mice in vivo Peter Jedlicka a,b,⁎, Theofilos Papadopoulos b, Thomas Deller a, Heinrich Betz b, Stephan W. Schwarzacher a a b

Institute of Clinical Neuroanatomy, Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany Department of Neurochemistry, Max Planck Institute for Brain Research, 60528 Frankfurt, am Main, Germany

a r t i c l e

i n f o

Article history: Received 12 November 2008 Revised 1 February 2009 Accepted 6 February 2009 Available online 21 February 2009 Keywords: GABAA receptor Gephyrin GEF Synaptic plasticity Granule cell

a b s t r a c t Collybistin (Cb), a brain-specific guanine nucleotide exchange factor, has been shown to be essential for the gephyrin-dependent clustering of a specific set of GABAA receptors at inhibitory postsynaptic sites. Here, we examined whether the lack of Cb affects synaptic properties and neuronal activity in the intact hippocampus by monitoring network activity in the dentate gyrus of Cb-deficient mice after perforant-path stimulation in vivo. We found a decreased threshold for evoked population spikes of granule cells, indicating their increased excitability. Paired-pulse inhibition of the population spike, a measure for somatic GABAergic network inhibition, was enhanced. Mutant mice exhibited steeper slopes of field excitatory postsynaptic potentials, consistent with a reduced dendritic inhibition. In addition, the induction of long-term potentiation (LTP) was reduced. In line with these functional changes, the number of postsynaptic gephyrin and GABAA receptor clusters in the Cb-deficient dentate gyrus was significantly decreased. In conclusion, our data provide the first evidence that Cb-deficiency leads to significant changes of GABAergic inhibition, network excitability and synaptic plasticity in vivo. © 2009 Elsevier Inc. All rights reserved.

Introduction Balanced regulation of neuronal activity in the CNS depends on inhibitory neurotransmission. The majority of fast inhibition is mediated by GABAA receptor (GABAAR) proteins that are selectively concentrated at distinct postsynaptic sites. A better understanding of the complex mechanisms that regulate the expression and synaptic accumulation of these receptor proteins is crucial for unraveling the role of GABAergic inhibition in neuronal dynamics and its pathological changes in human disorders, such as epilepsy, anxiety, schizophrenia and autism (Gross and Hen, 2004; McDougle et al., 2005; Jacob et al., 2008). The clustering of major GABAAR subtypes at inhibitory synapses requires the scaffolding protein gephyrin (reviewed in Kneussel and Betz, 2000; Moss and Smart, 2001). Ablation of gephyrin expression prevents the postsynaptic clustering of α2- and γ2-subunits containing GABAARs in cultured neurons and in gephyrin knockout mice (Essrich et al., 1998; Kneussel et al., 1999; Lüscher and Keller, 2004). Gephyrin binds to collybistin (Cb), a brain-specific guanine nucleotide exchange factor (GEF) for small Rho-like GTPases (Kins et al., 2000; Grosskreutz et al., 2001). Transfection studies with mammalian cell lines and cultured hippocampal neurons have shown that the

⁎ Corresponding author. Institute of Clinical Neuroanatomy, Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Fax: +49 69 6301 6425. E-mail address: [email protected] (P. Jedlicka). 1044-7431/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2009.02.005

formation of plasma membrane-associated gephyrin scaffolds depends on Cb–gephyrin interactions (Kins et al., 2000; Harvey et al., 2004). Consistent with Cb regulating gephyrin deposition at developing inhibitory synapses, Cb-deficient mice display a loss of postsynaptic gephyrin and GABAAR clusters in specific regions of the central nervous system, including the amygdala and the hippocampus (Papadopoulos et al., 2007). This leads to reduced dendritic GABAergic transmission and altered synaptic plasticity of CA1 pyramidal cells in vitro. In agreement with these results, Cb-deficiency is accompanied by increased anxiety levels and impaired spatial learning (Papadopoulos et al., 2007). Notably, inactivation of the Cb gene at both embryonic and postnatal stages results in a loss of postsynaptic gephyrin and γ2-subunit containing GABAARs from the dendrites of CA1 pyramidal neurons (Papadopoulos et al., 2008). Thus, Cb is essential for both the initial localization as well as the subsequent maintenance of gephyrin and GABAARs at inhibitory postsynaptic sites in the hippocampus. Recent studies have implicated collybistin dysfunction in epilepsy, anxiety, insomnia, aggression, and mental retardation (Harvey et al., 2004; Marco et al., 2008; Kalscheuer et al., 2009). Together with the findings obtained from Cb-deficient mice the currently available data suggest that the loss of synaptic GABAARs resulting from impaired Cb function affects network activity and synaptic plasticity. However, this has not yet been directly shown in vivo. Here, we examined the consequences of Cb-deficiency on hippocampal network function using electrophysiological recordings in the dentate gyrus of anesthetized Cb KO mice. We report that the loss of Cb leads to significant

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changes of granule cell (GC) excitability, GABAergic inhibition and synaptic plasticity in the intact hippocampus. Results Excitatory synaptic transmission at perforant path–granule cell (PP–GC) synapses of Cb KO mice To assess whether the loss of Cb affects synaptic and neuronal physiology in the hippocampus in vivo, we recorded dentate gyrus network activity in urethane-anesthetized Cb KO mice and their WT littermates following perforant-path (PP) stimulation. First, we analyzed excitatory synaptic transmission at PP–GC synapses in the dentate gyrus of Cb mutants by recording evoked field excitatory postsynaptic potentials (fEPSP). Input–output relationship between the stimulus intensity and fEPSP slopes was significantly enhanced in Cb KO (n = 10, maximum slope 2.0 ± 0.2 mV/ms) relative to WT mice (n = 9; 1.4 ± 0.2 mV/ms; P = 0.04; Fig. 1A). This suggests a stronger postsynaptic response at excitatory synapses, presumably due to altered dendritic inhibition (see below). To examine presynaptic function at PP–GC synapses, we tested paired-pulse facilitation (PPF) of fEPSPs at intensities subthreshold for eliciting a population spike. PPF reflects a form of short-term plasticity of presynaptic origin (Zucker and Regehr, 2002) and is routinely measured as the ratio of fEPSP amplitudes in response to two successive stimuli at various inter-pulse intervals. No significant difference in PPF was found when comparing WT (n = 9) and Cb KO mice (n = 10, P N 0.3; Fig. 1B). This is consistent with the presynaptic function not being altered at excitatory synapses in the Cb-deficient dentate gyrus. GC excitability and network inhibition in the dentate gyrus of Cb KO mice The finding of Cb involvement in the regulation of synaptic inhibition in vitro (Papadopoulos et al., 2007) prompted us to investigate whether Cb plays an important role for network inhibition and excitability in vivo. To study excitability of GCs in Cb KO mice, we measured amplitudes of population spikes after PP stimulation. The population spike amplitude is proportional to the number of discharging cells at a given stimulus strength (Andersen et al., 1971; Chauvet and Berger, 2002). The analysis of the stimulus–response relationship revealed a significantly lower threshold for population spikes in Cb KO mice as compared to WT mice (Fig. 2A; stimulation threshold for a 1 mV spike in WT: 173 ± 23 μA; KO: 68 ± 8 μA; P b 0.001; see Experimental methods). In contrast, population spike amplitudes were not changed at maximal stimulation intensities. These results demonstrate an increased disposition of GCs to generate action potentials after stimulation of their inputs in Cb-deficient mice. To assess GABAergic network inhibition in the intact Cb KO dentate gyrus, we carried out paired-pulse stimulation at intensities above threshold for evoking a population spike. This paired-pulse protocol results in complete inhibition of the population spike at short inter-pulse intervals (paired-pulse inhibition of the population spike, PPI) and spike facilitation at longer intervals (paired-pulse disinhibition of the population spike, PPDI). PPI is a postsynaptic phenomenon that depends on the activity of GABAergic interneurons in the dentate network (Sloviter, 1991; DiScenna and Teyler, 1994; Bronzino et al., 1997). PPDI of the population spike is thought to be mediated by diminished summation of GABAAR currents at long inter-stimulus intervals and by GABAB autoreceptor mediated decrease of GABA release from presynaptic terminals (Lambert and Wilson, 1994; Brucato et al., 1995). PPI lasted longer in Cb KO mice (n = 11) leading to a rightward shift of the PPI/PPDI curve relative to the WT curve (n = 9, Fig. 2B). Quantification showed that the interval at which PPI changed to PPDI

Fig. 1. Synaptic transmission at perforant path–granule cell (PP–GC) synapses in anesthetized Cb KO mice. (A) Analysis of fEPSPs in the dentate gyrus. Input–output curves show that the fEPSP slope size was significantly larger in Cb KO (n = 10) as compared to WT mice (n = 9). Top: Sample responses at maximal stimulation intensity. Inset: Note a significant increase of maximal slope value in Cb KO animals (⁎P b 0.05; two-tailed Student's t-test). (B) Paired-pulse facilitation of the fEPSP at various inter-stimulus intervals in the dentate gyrus of Cb KO (n = 10) and WT mice (n = 9). EPSP amplitude changes are presented as the ratio (in percent) of the second fEPSP amplitude to the first fEPSP amplitude. No significant difference was found between genotypes (P N 0.3; twotailed Student's t-test). Top: Sample recordings from WT and Cb KO mice show facilitation at 15 ms inter-pulse interval. Stimulus artefacts have been truncated. Calibrations: A, 2 mV, 2 ms; B, 0.5 mV, 5 ms.

was significantly prolonged in Cb-deficient mice (78 ± 10 ms) in comparison to WT littermates (43 ± 2 ms, P b 0.001). In addition, PPDI was significantly reduced. To exclude the possibility that the PPI/PPDI effect was present only at maximal stimulation intensity, we performed the paired-pulse test also at submaximal and minimal stimulation intensities (see Experimental methods). Likewise, we found strongly prolonged PPI in Cb KO animals in these experiments. Taken together, these findings suggest that the lack of Cb leads to alterations of GABAergic inhibition and dentate circuit excitability in vivo. Synaptic plasticity at PP–GC synapses in Cb KO mice To test whether Cb-deficiency influences synaptic plasticity at the PP–GC synapse in the dentate gyrus, we measured long-term potentiation (LTP) in anesthetized Cb KO mice and their WT littermates. As

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Layer-specific distribution of synaptic gephyrin and GABAAR clusters in the Cb KO dentate gyrus Our previous studies have revealed a region-specific loss of postsynaptic gephyrin and GABAAR clusters in the hippocampus and the basolateral amygdala of Cb KO mice (Papadopoulos et al., 2007). Here, we examined the effects of Cb-deficiency on the layer-specific distribution of gephyrin-dependent GABAAR clusters in the dentate gyrus in more detail by analyzing the postsynaptic localization of gephyrin and GABAAR subunits in the granule cell layer (GCL) and molecular layer (ML), respectively. In hippocampal sections derived from adult WT mice, punctate gephyrin immunoreactivity was present in both somatic (GCL) and dendritic (ML) layers of the dentate circuit (Figs. 4A, C, E and G). Double labeling with an antibody specific for vesicular inhibitory amino acid transporter (VIAAT), a marker protein of inhibitory presynaptic terminals, showed a clear apposition of gephyrin clusters to VIAAT immunoreactivity (Figs. 4A1, A2). In agreement with our previous observations (Papadopoulos et al. 2007), sections prepared from Cb KO mice showed reduced densities of gephyrin clusters in the dentate gyrus. The loss of punctate gephyrin immunoreactivity could be observed both in the GCL and ML (Figs. 4B, D, F, H). Quantification of the number of gephyrin-immunoreactive puncta per section area resulted in density values of 18.5 ± 1.6 versus 2.2 ± 0.5 puncta per 100 μm2 for the GCL, and of 41.2 ± 3.6 versus 7.2 ± 0.9 puncta per 100 μm2 for the ML of the dentate gyrus in WT and Cb KO sections, respectively (Fig. 4). Notably, the number of VIAAT-immunoreactive sites was not altered in both the GCL and ML of Cb KO mice (Figs. 4B1, B2). This confirms that the density of inhibitory nerve terminals is not altered upon Cb-deficiency (Papadopoulos et al., 2007).

Fig. 2. GC excitability and network inhibition are changed in the dentate gyrus of Cb KO mice. (A) Input–output curve for population spikes recorded in Cb KO (n = 10) and WT mice (n = 9). The population spike amplitude reflects the number of granule cells firing at a given stimulus intensity. Top: Sample responses at 100 μA stimulation intensity. Inset: Data were fitted using a Boltzmann equation from which a stimulation threshold (in μA) for a population spike of 1 mV was determined. Note a significantly lower threshold for the population spike in Cb KO mice, consistent with an increased GC excitability (⁎⁎⁎P b 0.001; two-tailed Mann–Whitney U-test). (B) Paired-pulse inhibition and disinhibition of the population spike (PPI/PPDI) in the dentate gyrus of Cb KO (n = 9) and WT mice (n = 10). PPI reflects GABAergic network inhibition. Top: Sample traces show paired-pulse responses at 50 ms inter-stimulus interval. Note a significant rightward shift in the PPI/PPDI curve of Cb KO mice. Inset: mean inter-pulse interval (in ms) at which equal amplitudes of the first and second population spike were observed (⁎⁎⁎P b 0.001; two-tailed Mann–Whitney U-test). Calibration bars: A, 1 mV, 2 ms; B, 2 mV, 10 ms.

shown in Fig. 3, following theta-burst stimulation (TBS), LTP was significantly impaired in Cb KO mice relative to WT animals. Already the initial increase in the fEPSP slope (0 –10 min) was significantly lower in mutant (n = 7, 112 ± 4%) than WT mice (n = 9, 153 ± 5%; P b 0.001; two-tailed Student's t-test; Fig. 3A). One hour after the LTP-inducing TBS, the potentiation of the fEPSP slope remained significantly decreased in Cb KO (50–60 min: 102 ± 2%) as compared to WT animals (50–60 min: 133 ± 5%; P b 0.001; twotailed Mann–Whitney U-test). Similarly, immediately after the TBS, Cb KO mice showed a weaker potentiation of the population spike amplitude (Fig. 3B; 0–10 min: 142 ± 16%; 50–60 min: 122 ± 14%) in comparison to their WT littermates (0–10 min: 198 ± 16%; P = 0.03; 50–60 min: 189 ± 19%; P = 0.02; two-tailed Student's t-test). These data demonstrate that Cb-deficiency affects the induction of longterm synaptic plasticity in the intact dentate gyrus.

Fig. 3. Cb KO mice show impaired synaptic plasticity at PP–GC synapses. Induction of long-term potentiation (LTP) was impaired in the dentate gyrus of anesthetized Cb KO mice as compared to WT littermates. Group data (WT n = 9, Cb KO n = 7) for fEPSP recordings before and after theta-burst stimulation (TBS; 6 series of 6 trains of 6 pulses at 400 Hz, 200 ms between trains, 20 s between series). Mean normalized fEPSP slope (A) and population spike amplitude (B) are plotted as a function of time. The potentiation is expressed as a percentage change relative to the mean response during the 10 min prior to the TBS (arrow).Top in A: Sample potentials collected before (grey) and immediately after induction of LTP (black). Calibration bars: 1 mV, 2 ms.

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Fig. 4. Layer-specific reduction of synaptic gephyrin staining and reduced clustering of the GABAAR γ2-, α2-, and α1-subunits in the Cb KO dentate gyrus. Sections from adult Cb KO mice and their WT littermates were stained with antibodies specific for gephyrin, VIAAT and the GABAAR, γ2-, α2-, and α1-subunits and processed for confocal microscopy (A–H). I: Quantification of immunoreactivities. For both genotypes, each bar corresponds to counts performed on 2 sections from 3–4 individual brains (⁎⁎P b 0.01; ⁎⁎⁎P b 0.001; Student's t-test). The punctate staining of sections from KO animals for gephyrin and the γ2-, α2-, and α1-subunits was strongly reduced in the dentate gyrus as compared to WT sections (C–I). In contrast, VIAAT immunoreactivity was unaffected by Cb-deficiency (A, B). ML, molecular layer of the dentate gyrus; GCL, granule cell layer. Scale bar, 16 μm.

Since gephyrin and Cb are known to be important for synaptic clustering of GABAAR-subunits (Essrich et al., 1998; Kneussel et al., 1999, 2001; Papadopoulos et al., 2007, 2008), we examined the effects of Cb-deficiency on GABAAR localization in the dentate gyrus by using γ2-, α2-, and α1-subunit-specific antibodies. This revealed a strong reduction in the number of GABAAR γ2- and α2immunopositive puncta in both the ML and GCL of the Cb KO mice (Figs. 4D, F) as compared to their WT littermates (Figs. 4C, E). Quantification of the number of immunoreactive clusters per 100 μm2 section area revealed density values of 39.8 ± 1.6 (γ2), 38.5 ± 2.6 (α2) versus 10.6 ± 1.0 (γ2), 2.7 ± 0.3 (α2) for the ML, and 22.2 ± 2.5 (γ2), 17.3 ± 1.3 (α2) versus 8.5 ± 0.8 (γ2), 9.6 ± 0.9 (α2) for the GCL, in WT and Cb KO sections, respectively (Fig. 4I). Interestingly, the loss of GABAAR α2-subunit-positive puncta was stronger in the dendritic ML than in the somatic GCL of the Cbdeficient dentate gyrus. Furthermore, whereas a reduction of GABAAR α1-immunoreactive puncta could be found in the ML of Cb KO dentate gyrus (KO: 4.7 ± 0.8 clusters/100 μm2; WT: 14.2 ± 2.0 clusters/100 μm2), no significant difference was observed in the dentate GCL between WT (5.2 ± 1.4 clusters/100 μm2) and Cb KO sections (5.0 ± 2.0 clusters/100 μm2; Figs. 4G, H). In conclusion, the lack of Cb leads to a layer-specific reduction of gephyrin-dependent GABAAR clusters in the dentate gyrus network. Discussion In this study, we show that Cb is required for normal GC excitability, GABAergic network inhibition and synaptic plasticity in the dentate gyrus in vivo. Moreover, we find that the functional deficits in the dentate network of Cb KO mice correlate with a significant reduction of synaptic gephyrin and GABAAR clusters. Thus, our work demonstrates that Cb is an important determinant of

gephyrin-dependent GABAergic mechanisms that regulate network excitability in the intact hippocampus. Postsynaptic excitatory responses are enhanced, and presynaptic function is normal at PP–GC synapses of Cb KO mice To assess excitatory transmission in the dentate gyrus of Cb KO mice, we recorded evoked field potentials in the GC layer after electric stimulation of the PP. Cb KO mice displayed significantly higher fEPSP slopes than WT littermates. An increased slope of the fEPSP indicates a higher efficacy of excitatory synapses in Cb-deficient hippocampus. It is known that dendritic inhibitory terminals regulate the efficacy of afferent excitatory inputs, probably by shunting dendritic EPSPs (Miles et al., 1996; Maglóczky and Freund, 2005). Therefore, the differences in the fEPSP slope may be explained by decreased GABAergic control of dendritic EPSPs due to reduced inhibition of GC dendrites. Previous experiments in acute hippocampal slices in the CA1 region yielded similar results, revealing a trend to higher fEPSP slope values (Papadopoulos et al., 2007). This was consistent with both measurements of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and with pharmacological analysis of dendritic fEPSPs, which demonstrated reduced dendritic inhibition of CA1 pyramidal neurons (Papadopoulos et al., 2007). An additional cause of increased fEPSP slopes might be a pre-potentiation of PP–GC synapses in Cb KO mice as a result of their disturbed dendritic inhibition (see below). To examine presynaptic function, we studied PPF, a form of short-term plasticity that is inversely related to the probability of glutamate release and is predominantly mediated by presynaptic mechanisms (Thomson, 2000). In line with unaltered PPF at the Schaffer collateral-CA1 synapse in vitro (Papadopoulos et al., 2007), we found no PPF deficits in Cb-deficient mice in vivo,

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indicating normal presynaptic functioning of their glutamatergic PP–GC synapses. GC excitability is increased, and network inhibition is altered in the dentate gyrus of Cb KO mice To test the effects of Cb-deficiency on the excitability of principal neurons in the dentate circuit in vivo, we determined the relationship between stimulus intensities and population spike amplitudes, which reflect GC firing. A highly significant decrease of the threshold for evoked population spikes was detected in Cb KO animals in comparison to WT mice. This enhancement of GC excitability is consistent with our immunohistochemical data demonstrating the loss of markers of GABAergic inhibitory synapses in the dentate gyrus of Cb KO mice. The most straightforward interpretation of these data is that Cb KO mice have reduced dendritic inhibition (Papadopoulos et al., 2007), which results in an enhanced ability of GCs to fire action potentials in response to PP stimulation. In contrast to the increase of GC excitability seen after singlepulse stimulation, our PPI experiments revealed an augmented suppression of GC population spikes after the second pulse in Cb KO mice. Since PPI of spiking activity in the population of GCs depends on the activity of GABAergic interneurons in the dentate network, it is a measure of GABAAR-mediated, predominantly somatic, inhibition of GCs (Sloviter, 1991). As a reduction of gephyrin and GABAAR α2 and γ2 clusters was found in the GCL of the Cb-deficient dentate gyrus, the enhancement of PPI disclosed here is unexpected. However, our immunohistochemical results and previous electrophysiological data (Papadopoulos et al., 2007) may provide an explanation. Perisomatic inhibition is predominantly mediated by soma-targeting synapses of parvalbumin-positive basket cells that activate α1-subunit containing GABAARs (Freund, 2003; Freund and Katona, 2007; but see also Prenosil et al., 2006). GABAAR α1-subunits were not reduced in the GCL of Cb-deficient mice. Thus, perisomatic inhibition should be less affected than dendritic inhibition (see below the discussion of immunohistological data). Indeed, PPI as an indicator of somatic inhibition was not reduced but enhanced in Cb KO mice, suggesting that paired-pulse stimulation recruited more inhibitory basket cells in Cb mutants than in WT littermates. This might be a consequence of a nonlinear network effect (Kapfer et al., 2007): reduced dendritic inhibition makes GCs more excitable, and hence GCs may recruit GABAergic feedback inhibition, mediated by soma-targeting interneurons, more effectively (Miles et al., 1996; Fig. 5). Additional factors might contribute to the compartment-specific alteration of GABAergic inhibition, e.g. a loss of Cb- and gephyrin-dependent GABAARs from soma-targeting interneurons (Simbürger et al., 2001), leading to their disinhibition (Fig. 5). In agreement with perisomatic inhibition being less impaired than dendritic inhibition, the analysis of evoked IPSCs (eIPSCs) had not disclosed a general reduction of CA1 pyramidal cell inhibition in Cb-deficient hippocampal slices (Papadopoulos et al., 2007). Recordings of eIPSCs mainly assess somatic inhibition (Miles et al., 1996). Thus, strong perisomatic inhibition might mask diminished dendritic inhibition. Cb-deficiency leads to changes in synaptic plasticity in the dentate gyrus Because the induction of synaptic potentiation depends on the level of postsynaptic depolarization during high frequency stimulation (Bliss and Collingridge, 1993), changes in GABAergic transmission may influence synaptic plasticity (Wigström and Gustafsson, 1983; Nosten-Bertrand et al., 1996; Casasola et al., 2004; Kleschevnikov et al., 2004; Nikonenko et al., 2006). Here, we observed an impairment of LTP induction at PP–GC synapses in the dentate gyrus of Cb KO mice. This contrasts with our previous in vitro data for the hippocampal CA1 region, in which the threshold for LTP induction was found to be reduced, resulting in enhanced LTP and reduced long-term depression

Fig. 5. Dentate gyrus network schematic. Basic dentate gyrus circuitry: PP: perforant path, GC: granule cells, IN: GABAergic interneurons. A: PP-stimulation initiates feedforward excitation of GCs (PP → GC) along with feedforward (PP → IN → GC) and feedback inhibition (PP → GC → IN → GC) responsible for the paired-pulse inhibition of GC spikes. B: Electrophysiological data from Cb KO mice indicate that the reduction of dendritic GABAergic inhibition leads to enhanced ability of GCs to fire evoked action potentials (indicated by the larger blue arrow; see Fig. 2A). The altered paired-pulse inhibition (see Fig. 2B) might be a network consequence of dendritic inhibition changes which lead to increase of GC excitability and enhanced recruitment of somatic GABAergic feedback inhibition (indicated by the larger red arrow).

(LTD) (Papadopoulos et al., 2007). It is known that GABAergic inputs on the dendrites of principal cells can limit synaptic plasticity (Miles et al., 1996). As a result of compromised dendritic inhibition, excitatory inputs to dendrites of neurons may undergo excessive potentiation (Maglóczky and Freund, 2005). In light of the results obtained with hippocampal slices, we interpret our in vivo finding of reduced LTP as a consequence of excessive potentiation of many synapses within the hippocampal network of living Cb KO animals. Accordingly, reduced dendritic inhibition in the Cb-deficient dentate gyrus would lead to a pre-potentiation of synaptic transmission (see Fig. 1A), and thereby saturate LTP and prevent further potentiation (see e.g. Saghatelyan et al., 2001). Consistent with this view, reduced LTP in the dentate gyrus in vivo correlates with the impairment of spatial learning observed in Cb KO mice (Papadopoulos et al., 2007). Alternatively, reduced LTP seen here in vivo and enhanced LTP found in slice preparations previously (Papadopoulos et al., 2007) could reflect different signaling pathways which mediate LTP induction in Cb-deficient CA1 pyramidal cells and GCs, respectively (Cooke et al., 2006; see also Nosten-Bertrand et al., 1996). Future studies are needed to address this possibility. Taken together, we conclude that Cb contributes to the regulation of excitation/inhibition balance, which represents an important modulator of hippocampal synaptic plasticity. Layer-specific reduction of synaptic gephyrin and GABAAR clusters in the Cb KO dentate gyrus We consistently found a strong reduction of gephyrin, α2- and γ2subunit containing GABAARs in the ML and GCL of the Cb-deficient

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dentate gyrus. In contrast, α1-subunit containing GABAARs were only reduced in the ML but not the GCL. These results are consistent with previous studies showing that, in the hippocampus, GABAARs containing α2- and γ2-subunits depend on gephyrin (Essrich et al., 1998; Kneussel et al., 1999; Levi et al., 2004; Yu et al., 2007; Tretter et al., 2008; see also Schweizer et al., 2003) and Cb (Papadopoulos et al., 2007) for postsynaptic localization. Perisomatic inhibition provided by parvalbumin-containing basket cells is largely mediated by α1 containing GABAARs (Freund and Katona, 2007). Accordingly, our immunohistological data support the conclusion that Cbdeficiency in the hippocampus mainly affects gephyrin-dependent dendritic inhibition (Papadopoulos et al., 2007; see also Viltono et al., 2008; and this study). Consistent with this proposal, the intensity of immunolabeling for gephyrin and the α2-subunit increases gradually in the direction of distal dendritic regions of GCs (Simbürger et al., 2001), suggesting their importance for dendritic inhibition. Electron microscopic studies have revealed that 75% of all GABAergic synapses in the dentate gyrus are located on GC dendrites and 25% on GC somata (Halasy and Somogyi, 1993). Thus, although gephyrin and GABAAR α2- and γ2-subunit immunoreactivities were reduced both in the dentate GCL and ML of Cb KO mice, the clustering deficits seem to preferentially affect dendritic GABAergic synapses. In conclusion, our combined immunocytochemical and electrophysiological analysis indicates that the impairment of GABAAR subtype-specific clustering in Cb KO mice has important functional consequences in the dentate gyrus in vivo, including significant changes of GABAergic inhibition and synaptic plasticity. Cb has been implicated in human diseases like epilepsy, anxiety and learning deficits (Harvey et al., 2004; Marco et al., 2008; Kalscheuer et al., 2009). Thus, our data may help to understand the pathophysiological consequences of Cb-deficiency for in vivo neuronal network activity and behaviour. Experimental methods Cb KO mice The generation of Cb KO mice has been described in detail previously (Papadopoulos et al., 2007). Adult male Cb KO mice and their WT littermates (derived from heterozygous breeding pairs) were used for electrophysiology and immunohistology. All experiments were performed in accordance with German laws governing the use of laboratory animals. Animal surgery and electrophysiology Electrophysiological characterization of Cb KO and WT mice was carried out as described before (Kienzler et al., 2006; Jedlicka et al., 2009; Winkels et al., 2009). Briefly, Cb KO mice and their WT littermates (2–3.5 months old) were anesthetized with urethane (Sigma, 1.2 g/kg i.p.; supplemental doses of 0.3–0.6 g/kg s.c. as needed). All recordings were made with the investigator blind to the genotype. The body temperature of the mice was monitored continuously and kept at 37 °C. Recordings and stimulation were made in the granule cell layer of the dentate gyrus (1.7 mm posterior and 1 mm lateral to bregma; approximate depth from the brain surface: 1.7 mm; Tungsten recording electrode) and in the medial perforant path (3.8 mm posterior to bregma and 2.1 mm lateral to lambda; approximate depth from the brain surface: 1.6 mm; bipolar stimulation electrode: tip separation 0.5 mm), respectively. Stimulus–response relationships were determined using a range of stimulation intensities from 30–800 μA. To determine the stimulation threshold for a population spike of 1 mV, data for each mouse were fitted using a Boltzmann equation. To measure paired-pulse facilitation (PPF) of the fEPSP amplitude, a double-pulse stimulation at intensities subthreshold to a population spike was applied, with

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inter-pulse intervals of 15–100 ms. To study paired-pulse inhibition and disinhibition (PPI/PPDI) of the population spike, maximal (800 μA), medium (250 μA) and minimal (evoking population spikes of 1 mV) stimulation intensities were used (inter-pulse intervals of 15–1000 ms; data shown only for maximal stimulation intensity). PPI/PPDI curves were fitted using a Boltzmann equation to obtain the mean inter-pulse interval, at which equal amplitudes of the first and second population spike were observed. LTP was induced by thetaburst stimulation (TBS): six series of six trains of six stimuli at 400 Hz, with 200 ms between trains and 20 s between series (Jones et al., 2001). Pulse width and stimulus intensity was doubled during TBS in comparison to baseline recordings. Baseline fEPSP slopes were calculated from the average of responses over the 10 min prior to TBS. Baseline stimulus intensity was set to evoke a population spike of approximately 1 mV before LTP induction. The potentiation of the fEPSP slope was expressed as a percentage change relative to the baseline. Immunofluorescence labeling Immunostainings were performed similarly for all sections prepared from brains of Cb KO mice and their WT littermates. For double immunostaining with antibodies against the following subunits of GABAA receptors: α1 (rabbit polyclonal; upstate, NY; 1:1000), γ2 (guinea pig; 1:4000) or α2 (guinea pig; 1:2000; the γ2- and α2specific antibodies were kindly provided by Dr. Jean-Marc Fritschy, University of Zurich, Switzerland), and the gephyrin specific antibody mAb7a (mouse monoclonal; 1:400), mice were deeply anesthetized and decapitated. The brains were immediately removed and frozen on dry ice. Coronal hippocampal cryostat sections (14 μm) were fixed with 4% (w/v) paraformaldehyde, 4% (w/v) sucrose in phosphatebuffered saline (PBS) for 10 min at 4 °C, washed twice for 2 min in PBS and once with SC buffer (10 mM sodium citrate, 0.05% (v/v) Tween20, pH 8.0). Sections were immersed in a pre-heated staining dish containing SC buffer and incubated for 30 min at 95 °C. After allowing the slides to cool at room temperature for 20 min, sections were rinsed twice for 2 min in PBS, permeabilized with 0.3% (w/v) Triton X-100, 4% (v/v) goat serum in PBS, blocked for 3 h with 10% goat serum in PBS and incubated overnight at 4 °C with primary antibodies at appropriate dilutions in PBS/10% goat serum. For double-immunostaining with the mAb7a and an antibody specific for the Vesicular Inhibitory Amino Acid Transporter (VIAAT, affinity purified polyclonal rabbit; Synaptic Systems GmbH, Goettingen, Germany; 1:500), mice were deeply anesthetized and decapitated. The brains were immediately removed and incubated overnight at 4 °C in 50 ml fixative containing 4% (w/v) paraformaldehyde and 0.1% (w/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The tissue was then cryoprotected overnight at 4 °C with 30% (w/v) sucrose in phosphate-buffered saline (PBS). Frontal 20 μm sections were prepared from frozen tissue and collected free-floating in PBS. Sections were washed once with PBS, mounted on SuperFrost Plus slides (Menzel GmbH, Braunschweig, Germany) and air-dried. Sections were then postfixed for 10 min with 4% (w/v) paraformaldehyde in PBS at 4 °C, washed twice for 2 min in PBS and once with SC buffer. Sections were immersed in a pre-heated staining dish containing SC buffer and incubated for 30 min at 95 °C. After allowing the slides to cool at room temperature for 20 min, sections were rinsed twice for 2 min in PBS, permeabilized with 0.3% (w/v) Triton X-100, 4% (v/v) goat serum in PBS, blocked for 3 h with 10% (v/v) goat serum in PBS and incubated overnight at 4 °C with the primary antibodies at appropriate dilutions in PBS/10% goat serum, and for 1 h with secondary antibodies (Alexa488 and Alexa 546; Invitrogen, Karlsruhe, Germany; 1:1000). Serial confocal images of immunostained slices were captured at a total magnification of either 400× or 630× on a Leica TCS-SP confocal laser-scanning microscope (Leica Microsystems, Bensheim, Germany).

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