Expression of connexin30.2 in interneurons of the central nervous system in the mouse

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 37 (2008) 119 – 134

Expression of connexin30.2 in interneurons of the central nervous system in the mouse Maria M. Kreuzberg,a Jim Deuchars,b Elisa Weiss,c Andreas Schober, d Stephan Sonntag,a Kerstin Wellershaus,a Andreas Draguhn,c and Klaus Willeckea,⁎ a

Institute of Genetics, Division of Molecular Genetics, University of Bonn, 53117 Bonn, Germany Institute of Membrane and Systems Biology, University of Leeds, Leeds, LS2 9JT, UK c Institute for Physiology and Pathophysiology, University of Heidelberg, 69120 Heidelberg, Germany d Department of Neuroanatomy, University of Heidelberg, 69120 Heidelberg, Germany b

Received 18 April 2007; revised 28 August 2007; accepted 5 September 2007 Available online 12 September 2007 Electrical synapses, particularly gap junctions composed of connexin (Cx) 36, have been suggested to synchronize neuronal network oscillations. Recently, we generated Cx30.2-deficient mice which express β-galactosidase under control of Cx30.2 gene regulatory elements. In the central nervous system β-galactosidase activity representing Cx30.2 expression was restricted to NeuN-positive cells, thus identifying Cx30.2 as new neuronal connexin. In the hippocampus, co-immunofluorescence analyses revealed β-galactosidase/Cx30.2 expression in GABAergic inhibitory interneurons such as parvalbuminand somatostatin-positive basket, axo-axonic, bistratified or oriens lacunosum-moleculare cells. ~94% of the Cx30.2 expressing parvalbumin-positive interneurons also expressed Cx36. Performing field potential recordings from hippocampal slices we found no differences in basal excitation and excitation–inhibition balance between Cx30.2+/+ and Cx30.2LacZ/LacZ mice. Furthermore, frequency and power of gap junction dependent γ and ripples oscillations were similar in these animals. This suggests that the lack of Cx30.2 in interneurons can be largely compensated by other connexins, most likely Cx36. © 2007 Elsevier Inc. All rights reserved. Keywords: Cx30.2; Electrical synapses; Gap junctions; Oscillations; Neurons

Introduction Electrical synapses provide bidirectional signal transfer and are important for the synchronization of subthreshold oscillations and spiking activity within clusters of neurons (Bennett, 1997; Perez Velazquez and Carlen, 2000; Connors and Long, 2004; Söhl et al., 2005). Gap junctional conduits directly connect the cytoplasm of

⁎ Corresponding author. Institut für Genetik, Abteilung Molekulargenetik, Universität Bonn, Römerstr. 164, 53117 Bonn, Germany. Fax: +49 228 734263. E-mail address: [email protected] (K. Willecke). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.09.003

adjacent cells to allow the diffusion of ions and metabolites (Kumar and Gilula, 1996). A gap junctional channel is composed of two hemichannels, socalled connexons, each provided by one adjacent cell. Connexons consist of six connexin (Cx) protein subunits. Several connexins, particularly Cx36, Cx45 and Cx57, have been described to be expressed in neurons of the central nervous system in mice (Söhl et al., 2005). Cx36 is the major neuronal connexin and widely expressed throughout the adult mouse brain, spinal cord and retina (Condorelli et al., 1998; Söhl et al., 1998; Feigenspan et al., 2001; Degen et al., 2004). Ablation of Cx36 leads to a reduction in power and synchronization of gamma frequency oscillations, whereas hippocampal theta oscillations are unaltered in vitro and in vivo (Hormuzdi et al., 2001; Buhl et al., 2003). In addition, Cx36deficient mice exhibit a loss of local synchrony in subthreshold oscillations within the thalamic reticular nucleus and in cortical interneuronal networks (Deans et al., 2001; Landisman et al., 2002; Blatow et al., 2003; Long et al., 2005). Furthermore, the deletion of Cx36 in mice causes alterations in the b-wave of the electroretinogram (ERG) (Güldenagel et al., 2001). During the last years, Cx57 and Cx45 proteins were also identified as neuronally expressed connexins. Cx57 protein is mainly restricted to horizontal cells of the adult mouse retina (Hombach et al., 2004), whereas Cx45 is widely expressed during embryonic development and is down-regulated postnatally, resulting in a distinct expression pattern in adult mice (Krüger et al., 2000; Maxeiner et al., 2003, 2005). Ablation of Cx57 in mice leads to a reduced receptive field size of retinal horizontal cells (Shelley et al., 2006). Conditional deletion of Cx45 coding DNA in Nestin-expressing cells by means of the Cre–loxP system causes a decrease of the b-wave in the electroretinogram, very similar to the findings in Cx36-deficient mice (Maxeiner et al., 2005). This phenotype is attributed to the lack of heterotypic Cx36/Cx45 gap junction channels between AII amacrine cells and OFF cone bipolar cells in the mouse retina (Maxeiner et al., 2005; Dedek et al., 2006). In addition,

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Fig. 1. Analyses of Cx30.2 expression by LacZ staining in different prenatal and postnatal developmental stages. (A) Whole mount LacZ staining of ED11.5– ED.14.5 Cx30.2+/LacZ embryos revealed the onset of the Cx30.2 expression on ED11.5 in central nervous tissue like spinal cord and brain stem as well as in other distinct regions of the Cx30.2+/LacZ embryo. The amount of LacZ staining increased during embryonic development. Scale bars: 1 mm. (B) β-Gal staining and NeuN immunohistochemical staining of coronal brain cryosections (20 μm) of neonatal and P7 Cx30.2/LacZ reporter mice. Cx30.2/LacZ is postnatally upregulated in different brain areas. While in P0 brain sections Cx30.2/LacZ staining in the hippocampal formation is primarily restricted to the fasciola cinereum (I), Cx30.2/LacZ at P7 is additionally expressed in the Ammon's horn of the hippocampus and can also be found in different thalamic nuclei (II). (III) Cx30.2/LacZ expression in different brainstem nuclei of P7 mice. Scale bars: 200 μm. Cb, cerebellum; Cx, cortex; Hc, hippocampus; Me, myelencephalon; Th, thalamus.

transient expression of Cx26 and Cx43 during neuronal development has been described (Bittman et al., 2002). We have recently characterized Cx30.2-deficient mice which express a NLS (nuclear localization signal)-LacZ reporter gene instead of the Cx30.2 coding DNA (Kreuzberg et al., 2006a). These mice are fertile and exhibit accelerated AV-nodal conduction in the heart. Here we show by further analyses of the LacZ reporter gene expression in Cx30.2-deficient mice that Cx30.2 is expressed in neurons of the central nervous system, thus identifying Cx30.2 as a new neuronal connexin. In contrast to Cx36-deficient brains, the constitutive deletion of Cx30.2, however, does not significantly alter excitability or fast network oscillations in the hippocampus, suggesting compensation by other connexins, potentially Cx36.

Results Analysis of LacZ reporter gene expression in the brain Analyzing β-galactosidase (β-Gal) staining of different organs in Cx30.2+/LacZ mice, we detected positive signals in the central nervous system (CNS) as well as in neural crest-derived tissue such as the adrenal gland. The Cx30.2/LacZ expression in spinal cord and brain stem was first observed after whole mount staining of ED11.5 embryos and up-regulated subsequently during development (Fig. 1). To characterize the Cx30.2-expressing cell type, we performed double immunofluorescence analyses with β-Galspecific antibodies and neuronal (NeuN), astrocytic (GFAP,

Fig. 2. Characterization of the Cx30.2/LacZ expression pattern in the CNS of Cx30.2+/LacZ mice. Brain sections (100 μm) are immunostained with antibodies to β-Gal (LacZ) and to cellular marker proteins. (A, B) NeuN-positive neurons of thalamus (A) and hippocampus (B) exhibited Cx30.2/LacZ expression. Neither GFAP- (C, D) nor S100β- (E, F) positive astrocytes nor CNPase-positive oligodendrocytes (G, H) expressed Cx30.2/LacZ. Or, stratum oriens; Py, stratum pyramidale; Ra, stratum radiatum; CNP, CNPase. Scale bars: 50 μm.

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Fig. 3. β-Gal staining and NeuN immunohistochemistry of coronal brain cryosections (20 μm). (A) β-Gal staining was found within different thalamic nuclei and within the hippocampus which showed prominent staining in the CA2 region and in particular in the fasciola cinereum. (B) Several brainstem nuclei revealed strong Cx30.2/LacZ expression. CA1–3, areas of the hippocampus; FC, fasciola cinereum; MD, mediodorsal thalamic nucleus; MVe, medial vestibular nucleus; Sol, solitary tract nucleus; Sp, spinal trigeminal nucleus; VPM, ventral posteromedial nucleus. Scale bars: 1 mm.

S100β) or oligodendrocytic (CNPase) markers on adult brain slices (Fig. 2). β-Gal and NeuN immunofluorescence signals were colocalized, indicating the expression of the NLS (nuclear localization signal)-LacZ reporter gene in neurons of the central nervous system (Figs. 2A, B). In contrast, β-Gal protein could never be detected in GFAP- or S100β-positive cells, excluding the expression of Cx30.2 in astrocytes (Figs. 2C–F). In addition, LacZ reporter gene expression was never found in CNPase-positive oligodendrocytes (Figs. 2G, H). In order to analyze Cx30.2/LacZ expression throughout the brain, we performed β-Gal staining and NeuN immunohistochemical analyses on serial brain cryosections of Cx30.2+/LacZ animals (Fig. 3). We found Cx30.2/LacZ expression in the anterior olfactory nucleus of the olfactory bulb, in different regions of the telencephalon, e.g. in cortex, hippocampus and several thalamic nuclei, in the interposed nucleus of the cerebellum, as well as in different nuclei of the myelencephalon (Fig. 3 and Table 1). In addition, Cx30.2/LacZ expression was found in the ganglion cell layer and the inner nuclear layer of the retina (results will be published elsewhere). Expression of Cx30.2 in the spinal cord In the spinal cord, labeling for Cx30.2/LacZ was most evident in the dorsal half and absent from motor neurons in the ventral horn (Fig. 4A). In the dorsal horn Cx30.2/LacZ expression was detected in laminae I, III and IV, but was more sparse in lamina II (Fig. 4B). Labeled cells were also apparent both dorsal and ventral to the central canal (Fig. 4C). Occasionally, cells were detected in the lateral horn, but staining was notably absent from sympathetic preganglionic neurons in the intermediolateral cell column.

Cx30.2 expression in inhibitory interneurons of the hippocampus The relatively sparse β-Gal staining in the hippocampus strongly indicated expression of the Cx30.2 gene in GABAergic inhibitory interneurons. To further determine the Cx30.2-expressing interneuronal subclasses, we performed co-immunofluorescence analyses for expression of the interneuronal markers parvalbumin, somatostatin and calbindin, in combination with β-Gal antibodies. Nearly 50% of the strongly parvalbumin-positive cells exhibited Cx30.2/LacZ expression, indicating an expression of Cx30.2 in parvalbumin-positive basket and axo-axonic cells (Figs. 5A–C). In addition, a subgroup of somatostatin-positive cells, especially in stratum oriens of the CA1 region, was β-Gal-positive (Figs. 5D–F). At least some of these cells were also positive for parvalbumin when tested by triple staining (data not shown) and thus might be oriens lacunosum-moleculare (O-LM) or bistratified cells. In contrast, calbindin-positive interneurons and pyramidal cells of the hippocampus in general did not express Cx30.2/LacZ (Figs. 5G–I). Furthermore, the Kv3.1 potassium channel protein, which is mainly expressed by fast-spiking parvalbumin-positive interneurons of the hippocampus, was shown to be colocalized with Cx30.2/LacZ expression (Figs. 5J–L), whereas calretinin-positive, interneuronspecific cells did not show Cx30.2/LacZ expression (data not shown).

Coexpression of Cx30.2 and Cx36 in the brain of Cx30.2+/LacZCx36+/CFP mice To analyze the extent of coexpression of the interneuronal Cx36 and Cx30.2 proteins, Cx30.2LacZ/LacZ animals were mated to Cx36CFP/CFP mice (K. Wellershaus, unpublished experiments).

M.M. Kreuzberg et al. / Mol. Cell. Neurosci. 37 (2008) 119–134 Table 1 Expression of Cx30.2/LacZ in neurons of the brain Labeled brain structure Olfactory bulb Anterior olfactory nucleus Telencephalon Cortex Hippocampal formation CA1 CA2 CA3 Dentate gyrus Subiculum Fasciola cinereum Indusium griseum Striatum Amygdala Thalamus Anteroventral thalamic nucleus Anteromedial thalamic nucleus Mediodorsal thalamic nucleus Ventral posteriomedial thalamic nucleus Dorsal lateral geniculate nucleus Mesencephalon Periaqueductal nuclei/gray Reticulotegmental pons nuclei/pontine nuclei Cerebellum Interposed nucleus Myelencephalon Medial vestibular nucleus Spinal trigeminal nucleus Solitary tract nucleus Area postrema Ventral and dorsal cochlear nucleus Cuneate nucleus Gracile nucleus Gigantocellular reticular nucleus Lateral reticular nucleus Nucleus of Roller Hypoglossal nucleus

Cx30.2/ Cx36/ Coexpression LacZ CFP +

−

−

+

++

✓

+ ++ + + ++ +++ +++ ++ +

+ + + − ++ + − ++ +

✓ ✓ ✓ − ✓ ✓ − ✓ ✓

++ ++ ++ ++

− − − −

− − − −

++

−

−

+ +

+ +

− ✓

++

++

−

++ ++ ++ ++ ++ ++ ++ + + ++ ++

− − + − ++ ++ n.d. − ++ − −

− − ✓ − − ✓ n.d. − ✓ − −

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In the hippocampal formation and cortex, triple immunofluorescence analyses likewise revealed a colocalization of LacZ, CFP and parvalbumin (Figs. 7B, C), suggesting coexpression of Cx30.2 and Cx36 in a subset of parvalbumin-positive basket or axo-axonic cells. We estimated the percentage of parvalbumin-positive interneurons expressing Cx30.2 and Cx36 in the Ammon's horn of the hippocampus and found ~48% of all parvalbumin-positive cells to be Cx30.2/LacZ-positive and ~93% to be positive for Cx36/CFP (number of cells counted n = 1719). Approximately 94% of all Cx30.2/LacZ-positive cells exhibited Cx36/CFP expression. Expression of Cx30.2 in the adrenal gland By analysis of β-Gal staining of adrenal gland cryosections, we recognized Cx30.2/LacZ expression in the medulla of the adrenal gland. Further characterization of the β-Gal-expressing cells in the adrenal gland using different cell type-specific markers revealed

+, ++, +++ indicate the strength of expression from faint to strong.

These mice express the cyan fluorescent protein under the control of the Cx36 gene regulatory elements. Heterozygous Cx30.2+/LacZ/ Cx36+/CFP mice were analyzed by double immunofluorescence analyses (Figs. 6 and 7). Thereby, β-Gal- and EGFP-specific antibodies (which also detect CFP) displayed colocalization of both reporter gene products in the striatum, amygdala, cortex, hippocampal formation and several brainstem nuclei (Figs. 6 and 7; Table 1). No coexpression of Cx30.2/LacZ and Cx36/CFP was present in neurons of cerebellum, olfactory bulb or thalamus (Fig. 6; Table 1). In the striatum, a high extent of Cx30.2 and Cx36 coexpression was detected (Fig. 7A). ~ 93% of all Cx30.2/ LacZ-positive cells expressed also the CFP reporter gene and 82% of the Cx36/CFP-expressing cells were found to be positive for Cx30.2/LacZ (number of cells counted n = 5110 and n = 5794, respectively). Triple immunofluorescence analyses with the β-Gal-, CFP- and parvalbumin-specific antibodies revealed that Cx30.2/Cx36 double positive cells were GABAergic inhibitory interneurons (Fig. 7A).

Fig. 4. (A) β-Gal staining of the thoracic spinal cord of a Cx30.2+/LacZ mouse. β-Gal-positive neurons were found predominantly in the dorsal horn (B) of the spinal cord, whereas staining was largely absent in the ventral horn (C). Panels B and C show insets of panel A in higher magnification. CC, central canal; DH, dorsal horn; VH, ventral horn; WM, white matter. Scale bars: 200 μm (A) or 100 μm (B, C).

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that β-Gal-positive cells were neither S100-positive Schwann-cells (data not shown) nor acetylcholinesterase (AChE)-positive preganglionic sympathetic nerve fibers (Fig. 8A, B). Co-immunofluorescence analyses with β-Gal- and tyrosine hydroxylase-specific antibodies demonstrated Cx30.2/LacZ expression in chromaffin cells (Fig. 8C-F). The low number of β-Gal-immunopositive cells points towards Cx30.2 expression in the noradrenaline producing population of chromaffin cells. Electrophysiological characterization of Cx30.2LacZ/LacZ mice In order to investigate possible changes in neuronal network activity due to lack of Cx30.2 expression in the hippocampus, extracellular recordings were performed on hippocampal slices. Schaffer collaterals were stimulated with increasing voltages and the maximal amplitude of extracellularly recorded population spikes in stratum pyramidale was assessed. Transgenic animals showed normal field EPSP-spike waveforms and had similar maximal population spike amplitudes as control animals (9.72 ± 1.37 mV [Cx30.2+/+] versus 8.19 ± 0.63 mV [Cx30.2LacZ/LacZ], n = 10 slices from 6 and 7 animals, respectively, P N 0.05). As a more sensitive measure of synaptic excitation–inhibition ratios and intrinsic excitability, we performed paired stimulations with varying time intervals at a stimulation strength yielding 70% of maximal population spike amplitude in the first spike. Again, the paired-pulse behavior did not differ between both groups of animals (Cx30.2+/+: n = 10 slices from 6 animals; Cx30.2LacZ/LacZ: n = 10 slices from 7 animals). These data are illustrated in Fig. 9. As interneuronal gap junctions have been reported to play an important role in the generation and synchronization of highfrequency network oscillations, we tested whether gamma network oscillations were altered in the absence of Cx30.2. This oscillation pattern was induced by bath-application of kainate (200 nM) and was measured as rhythmically oscillating field potential in stratum pyramidale of area CA3 (frequency range 28.8 to 40.5 Hz). Similar oscillations were concomitantly measured in the CA1 region (data not shown). Clearly discernible gamma oscillations could be induced in 19/93 slices tested (9/47 in control, 10/46 in ko). These field potential oscillations appeared similar in both groups of slices (see Fig. 10A). Statistical parameters for oscillation frequency, amplitude, frequency distribution and power were not different between Cx30.2+/+ and Cx30.2LacZ/LacZ animals (see Fig. 10B; n = 9 slices from 2 animals [Cx30.2+/+] and 10 slices from 4 animals [Cx30.2LacZ/LacZ]). Sharp wave-superimposed ripples (SPW-R) are another type of gap junction-dependent high-frequency oscillation at ~200 Hz. This pattern occurred spontaneously in almost all slices taken from the ventral portion of hippocampi from animals of both groups. SPW-R in Cx30.2LacZ/LacZ animals appeared similar to those recorded in control tissue. Again, quantitative analysis of sharp wave frequency, ripple frequency and ripple energy revealed no significant alterations in slices from Cx30.2-deficient mice in comparison to wild-type mice. Likewise, the increase in unit discharges during sharp waves was similar between both groups (15.8 ± 2.1 [Cx30.2+/+] versus 18.0 ± 3.0

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[Cx30.2LacZ/LacZ]), indicating similar recruitment of pyramidal cells during these network bursts. Discussion In this study, we have identified Cx30.2 as a new neuronal connexin in the brain. By characterization of Cx30.2/LacZ reporter mice, the expression of Cx30.2/LacZ was demonstrated in mature neurons of the central nervous system. In contrast, astrocytes or oligodendrocytes did not exhibit Cx30.2/LacZ expression. In the brain, LacZ reporter gene expression was observed in different regions such as interneurons of the cortex and hippocampus, neurons of the striatum and diverse thalamic nuclei. In addition, moderate staining was apparent in the cerebellum, in contrast to strong labeling in different brainstem nuclei. In the spinal cord, Cx30.2 staining was mainly found in the dorsal horn. Cx30.2positive neurons of the hippocampus and cortex mostly displayed coexpression of the interneuronal markers parvalbumin and/or somatostatin. Thus, Cx30.2-positive cells in these areas presumably represent basket cells, axo-axonic cells, bistratified cells and O-LM cells. In neural-derived tissues, the expression pattern of Cx30.2 resembles that of Cx36. Both connexins are expressed in subtypes of chromaffin cells in the medulla of the adrenal gland, in distinct brain nuclei and in interneurons of cortex and hippocampus (Condorelli et al., 1998; Belluardo et al., 2000; Degen et al., 2004). In the adrenal gland, however, only a smaller portion of chromaffin cells, possibly the noradrenergic subfraction, expresses Cx30.2, whereas Cx36 is more widely expressed (Degen et al., 2004). In the brain, coexpression of both connexins within the same cell was found in neurons of different brainstem nuclei and striatum as well as in subpopulations of parvalbumin-positive basket or axo-axonic cells in hippocampus and cortex. The extent of coexpression was especially high in striatum constituting 93% and 82% of the Cx30.2/LacZ- and Cx36/CFP-positive cells, respectively. Interestingly, homotypic gap junctional channels composed of Cx30.2 or Cx36 exhibit similar electrophysiological properties: both have relatively small unitary conductance (9 pS and 14 pS, respectively) and exhibit a low sensitivity to changes in transjunctional voltage (Srinivas et al., 1999; Teubner et al., 2000; Kreuzberg et al., 2005). However, so far no investigations were performed to demonstrate functional Cx30.2/Cx36 heterotypic and heteromeric gap junctional channels, which might be formed in neurons of brain and spinal cord, within the retina or in the adrenal gland. Furthermore, similarities in the expression pattern of Cx30.2 and Cx45 can be expected. We previously described the coexpression of Cx30.2 and Cx45 within the same gap junctional plaques in the cardiac conduction system and proposed the formation of heterotypic and/or heteromeric channels (Kreuzberg et al., 2006b). In the adult central nervous system, strong expression of Cx45 was found in neurons of several thalamic nuclei (Maxeiner et al., 2003; Söhl et al., 2005). Some of them, such as the dorsolateral geniculate nucleus or the

Fig. 5. Co-immunofluorescence analyses with antibodies to β-Gal (LacZ) and different interneuronal markers in hippocampal slices (100 μm) of adult mice. (A–C) β-Gal- and parvalbumin-positive signals were often colocalized within the areas CA1 (A) and CA3 (C) of the hippocampus. (D, E, F) Some somatostatinpositive interneurons, especially in CA1, demonstrated Cx30.2/LacZ expression. (G–I) Calbindin-positive interneurons in general did not show Cx30.2/LacZ expression. (J–L) Kv3.1-positive interneurons of the hippocampus were found to be β-Gal-positive. Or, stratum oriens; Py, stratum pyramidale; Ra, stratum radiatum; PV, parvalbumin; SSN, somatostatin; CB, calbindin; Kv3.1, Kv3.1 potassium channel. Scale bars: 50 μm.

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ventrolateral posteromedial thalamic nucleus, also express Cx30.2. Thus, coexpression of both connexins in these regions is likely. Electrical coupling in hippocampus and cortex was described to be involved in the induction and maintenance of fast frequency rhythms such as gamma oscillations and sharp wave–ripple complexes (Buhl et al., 1998; Perez Velazquez and Carlen, 2000; Traub et al., 2001, 2003, 2004). GABAergic inhibitory interneurons, which are integrated into interneuronal networks via electrical synapses composed of Cx36 and possibly – as we describe here – of Cx30.2, were suggested to sharpen and strengthen such oscillatory patterns (Traub et al., 2001; Somogyi and Klausberger, 2005; Vida et al., 2006). In accordance, Cx36-deficient mice showed deficits in inducibility, synchrony and amplitude of gamma oscillations in vivo and in vitro (Hormuzdi et al., 2001; Buhl et al., 2003). However, residual gamma oscillations in these mice could be abolished by gap junctional blockers (Traub et al., 2003), indicating that Cx36 independent gap junctional coupling, for instance mediated by Cx30.2, exists in hippocampal neuronal networks. In the present study, we induced gamma oscillations in acute hippocampal slices of Cx30.2-deficient mice and found no significant differences in their frequency or amplitude. One explanation for these results can be the largely overlapping expression of Cx36, which is expressed in ~ 94% of all Cx30.2 expressing parvalbumin-positive GABAergic inhibitory interneurons of the hippocampus. Therefore, the lack of Cx30.2 in these cells might be compensated by Cx36 gap junctional channels. This hypothesis could be further elucidated when double mutated mice lacking Cx36 and Cx30.2 can be investigated. However, it should also be noted that the amplitude and power of gamma oscillations in Cx30.2LacZ/LacZ mice appeared somewhat reduced and that the frequency distribution was slightly broader, although these results were not statistically significant. This might hint towards incomplete compensation in electrical coupling by other gap junctionforming proteins such as Cx36 or pannexins (Vogt et al., 2005). In addition, we tested if sharp wave-related ripple oscillations (SPW-R) were altered in Cx30.2-deficient mice. Using gap junctional blockers, SPW-R could be demonstrated to depend on gap junctional coupling (Ylinen et al., 1995; Maier et al., 2003). However, the connexin or pannexin protein involved could not be clearly identified. In our recordings from Cx30.2-deficient mice, the oscillation pattern during spontaneously occurring sharp wave– ripple complexes was unchanged. Therefore, we can exclude that this new neuronal connexin is essential for the generation of high frequency oscillations in the 200-Hz domain. This is consistent with the assumption that field ripples are mainly due to the synchronized firing of pyramidal cells (Draguhn et al., 1998; Hormuzdi et al., 2001), which do not express Cx30.2 but high amounts of pannexin1 (Vogt et al., 2005) and probably low amounts of Cx45 (Maxeiner et al., 2003).

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In summary, several electrophysiological properties of the hippocampal network (including neuronal synchronization) are unchanged in the absence of Cx30.2. Thus, our analyses so far could not provide unequivocal evidence that Cx30.2 forms gap junctional channels that mediate electrical coupling in the hippocampus. However, Cx30.2 could fulfill other functions not directly related to electrical coupling and the formation of synchronized networks between connexins in neurons. In recent years, several groups described the opening of hemichannels, which results in non-exocytotic release of ATP, ions and glucose, and their physiological relevance in the central nervous system (Pearson et al., 2005; Thompson et al., 2006, Romanov et al., 2007). We recently demonstrated the general ability of Cx30.2 to form functional hemichannels in HeLa-Cx30.2 transfectants (Bukauskas et al., 2006). Cx30.2 hemichannels, are relatively insensitive to differences in applied voltages of either polarity and their open probability only modestly depends on the extracellular Ca2+ level (Bukauskas et al., 2006). Therefore, Cx30.2 hemichannels if present in the brain might be opened under physiological conditions. We hypothesize that due to their very low unitary conductance of ~ 20 pS such hemichannels might favor the passage of small ions rather than somewhat larger molecules such as cAMP or ATP (Bedner et al., 2006; Bukauskas et al., 2006). In addition, Elias et al. (2007) most recently described the involvement of gap junctions in cell adhesion during neuronal migration. Future experiments such as dye coupling and dye uptake experiments might help to estimate to what extent Cx30.2 gap junctional channels or hemichannels exist in the central nervous system. In addition, paired cell recordings, could help to further elucidate the coupling properties in certain brain areas. Experimental methods Animals Mice were raised and maintained according to governmental and institutional care instructions under 12:12-h light/dark cycle. For the generation of Cx30.2+/LacZCx36+/CFP animals, Cx30.2LacZ/LacZ mice expressing nuclear β-galactosidase (β-Gal) protein under control of Cx30.2 gene regulatory elements (Kreuzberg et al., 2006a) were bred to Cx36CFP/CFP mice, which express the CFP reporter protein under control of Cx36 gene regulatory elements (Kerstin Wellershaus, unpublished experiments). β-Gal staining and NeuN immunohistochemistry Adult mouse brains were dissected from cervical dislocated mice, frozen on dry ice and cryosectioned to 20 μm sections. Staining for β-Gal activity was performed according to Krüger et al. (2000) using 2% paraformaldehyde (PFA)/0.2% glutaraldehyde/PBS− (phosphate-buffered saline) for fixation of the cryosections. β-Gal stained slices were postfixed with 4% PFA for immunohistochemical analyses. NeuN immunohistochemical investigations were

Fig. 6. Double immunofluorescence analyses of β-Gal (LacZ, red) and CFP (green) expression in slices (100 μm) of adult brain. Coexpression of both reporter genes was found in the cuneate nucleus (F), lateral reticular nucleus (J) and solitary tract nucleus (K) of the brainstem as well as in the CA1 area of the hippocampus (B) and the subiculum (C). No coexpression was detectable in the olfactory bulb (A), in the thalamus (D, E) or other brainstem nuclei (G–L). Scale bars: 50 μM. GrO, granule layer of the olfactory bulb; OA, anterior olfactory nucleus; FC, fasciola cinereum; CA1, CA1 region of the hippocampus; S, subiculum; AM, anteromedial thalamic nucleus; DLG, dorsal lateral geniculate nucleus, Cu, cuneate nucleus; DC, dorsal cochlear nucleus; Hyp, hypoglossal nuclei; GI, gigantocellular nucleus; IO, inferior olive; LRt, lateral reticular nucleus; Sol, solitary tract nucleus; Sp, spinal trigeminal nucleus.

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performed with the MOM detection kit following the manufacturer's instructions using a 1:100 dilution of NeuN (Chemicon, Temecula, USA) antibodies. For β-Gal staining of the spinal cord, anesthetized adult animals were perfused with 4% glutaraldehyde/PBS−. The spinal cord was dissected and sectioned into 30 μm slices with a Leica VT1000S vibratome. β-Gal staining and NeuN immunohistochemistry were documented using a Zeiss Axiophot photomicroscope.

anti-β-galactosidase primary antibodies (1:15,000, Chemicon) for 48 h at 4 °C. Afterwards, cryosections were subsequently incubated with the biotin-conjugated anti sheep IgG (1:200) for 1 h, Cy2-conjugated streptavidin (1:2000, Jackson) for 1 h and Cy3-conjugated anti-mouse IgG (1:500, Jackson) for 2 h at room temperature.

Whole mount β-Gal staining of embryos

Experiments were performed in hippocampal slices from wild-type (Cx30.2+/+) and Cx30.2-deficient mice (Cx30.2LacZ/LacZ) of both sexes aged 2 to 3 months. Procedures were approved by the state government of Baden–Württemberg. Mice were anaesthetized with ether, decapitated and the brain was removed. Brains were kept in cooled (~1–4 °C) artificial cerebrospinal fluid (ACSF), containing (mM): NaCl 124, KCl 3, MgSO4 1.8, CaCl2 1.6, glucose 10, NaH2PO4 1.25, NaHCO3 26, gassed with 95% O2/5 % CO2 (pH 7.4 at 37 °C). After removal of the cerebellum, we cut horizontal slices of 450 μm on a vibratome (Leica, VT 1000 S, Germany). Recordings were performed at 35 ± 0.5 °C in a modified Haas-type interface chamber after 2 h of equilibration. Extracellular glass microelectrodes (tip diameter ~5 μm) were filled with ACSF before use. In order to test for alterations in excitability, we evoked population spikes in the CA1 pyramidal cell layer by supramaximal stimulation of Schaffer collaterals. In addition, we tested responses to paired pulses with varying intervals (5 to 1000 ms) at a stimulation strength yielding 70% of the maximal amplitude of population spikes. For gamma frequency network oscillations, kainate (200 nM, Tocris, Northpoint, UK) was added to the bath for 1 h and recordings were performed in stratum pyramidale of area CA3. Sharp wave– ripple complexes (SPW-R) occurred spontaneously in normal ACSF and were recorded in stratum pyramidale of CA1 (Maier et al., 2002).

After dissection, ED11.5–ED14.5 Cx30.2+/LacZ embryos were fixed for 30 min to 1 h in 4% PFA/PBS− at room temperature. LacZ staining was performed as previously described (Krüger et al., 2000) with prolonged washing steps after fixation. After LacZ staining, embryos were dehydrated using an ethanol series (60%→100%) and afterwards cleared with benzylalcohol/benzylbenzoate (1:2) overnight at 4 °C (Eng et al., 2001). LacZ staining was documented using a binocular equipped with a digital camera. Immunofluorescence analysis of brain thick slices Animals were anesthetized with Rompun (0.2%) and Ketavet (0.5%) (10 ml/kg) and transcardially perfused with 10 ml 0.9% NaCl and 40 ml 4% PFA/PBS−. Brains were postfixed at 4 °C overnight with 4% PFA/PBS− and sectioned with a Leica VT1000S vibratome in 100 μm slices. For βgalactosidase, NeuN, GFAP (glial fibrillary acidic protein), S100β, parvalbumin, somatostatin and calbindin immunofluorescence analyses, floating slices were washed three times for 10 min in PBS−/0.1% Triton X100, blocked for 2 h at room temperature in washing solution containing 2% normal goat serum and incubated overnight at 4 °C with a mixture of primary antibodies diluted in blocking solution (Table 2). After three washing steps, slices were incubated for 2 h at room temperature with appropriate secondary antibodies (Dianova, Hamburg, Germany or Molecular probes, Eugene, USA; Table 2) diluted in blocking solution and washed again. Cover slips were mounted with Permafluor aqueous mounting solution. During EGFP immunofluorescence analysis slices were washed with PBS−, permeabilized with PBS−/0.25% Triton X-100 and blocked with PBS−/0.25% Triton X-100/4% BSA. Kv3.1 immunofluorescence analyses were carried out as previously described (Deuchars et al., 2001). Immunofluorescence signals were visualized using a confocal Zeiss LSM510 microscope. Histochemical and immunochemical staining in the adrenal gland Cx30.2LacZ/LacZ animals were anesthetized and transcardially perfused with 2% PFA/PBS−. After post-fixation in 2% PFA for 1 h at room temperature, adrenal glands were cryoprotected overnight (30% sucrose in 0.1 M PB), cryosectioned and mounted on chromalaun pre-coated slides. In order to combine β-Gal staining with AChE (acetylcholinesterase) enzyme histochemical analyses, cryosections were β-Gal stained as described above without further fixation. These pre-stained cryosections were incubated for 15 min in 0.05 M Tris–maleate buffer (pH 5.0) and AChE staining was performed according to the protocol of Andrä and Lojda (1986). Sections were incubated for 1 h at 37 °C in the following solution: 45 ml 0.05 M Tris–maleate (pH 5.0), 6 ml 0.4 M sodium citrate, 6 ml 0.12 M copper sulfate, 3 ml 0.16 M potassium hexacyanoferrate (III), 0.6 ml 0.001 M isoOMPA and 0.1% Triton X-100. For immunofluorescence analyses, cryosections were washed three times with PB, blocked for 1 h at room temperature in PB/0.1% Triton X100/5% NGS and incubated with anti-tyrosine hydroxylase (1:400) and

Electrophysiology

Data processing and analysis Extracellular field potentials were measured with an EXT 10-C amplifier (npi electronics, Tamm, Germany), digitized at 5000 to 10,000 Hz with an ADC interface (CED 1401) and sampled on hard disk using Signal 3.0 and Spike-2 software (CED, Cambridge, UK). Before digitization, data were low-pass filtered at 3000 Hz. Analysis of network oscillations was performed off-line with custom-made programs in Signal 3.0 and MatLab. For gamma oscillations, we calculated the power spectrum from 180 s of raw data and assessed the parameters frequency, width of frequency distribution and power. The amplitude of gamma oscillations was computed as the mean of the amplitude between positive and negative peaks of raw data. Sharp waves were detected after low-pass filtering of original data at 50 Hz using a peakdetection algorithm. Sharp wave frequency was calculated as the number of sharp waves per time interval. A sharp wave-superimposed ripple was defined by at least three consecutive high-frequency oscillations at N140 Hz. In order to analyze ripple frequency and energy, we performed continuous wavelet transform (complex Morlet wavelet) starting 33 ms before and ending 67 ms after the peak of the detected sharp wave. Extracellularly recorded single-action potentials were detected after high-pass filtering at 500 Hz and by setting a negative threshold at four times standard deviation of event-free baseline. Sharp wave-correlated increase in unit activity was calculated as unit activity during sharp waves divided by unit activity in eventfree episodes at 1 s to 400 ms before sharp waves. Quantitative results are given as means ± S.E.M. Data were compared using the Student's t-test after testing for normality. The Welch-correction was used in cases of different standard deviations between groups (InStat,

Fig. 7. Triple immunofluorescence analyses of β-Gal (LacZ, red) (I), CFP (green) (II) and parvalbumin (III) expression in slices (100 μm) of adult brain. (A) CFP (I) and β-Gal (II) immunofluorescence signals strongly colocalized in the striatum (IV). Cx30.2/Cx36 double positive cells exhibited parvalbumin expression (III, IV) demonstrating that these cells are GABAergic inhibitory interneurons. (B, C) In the cortex (B) and hippocampus (C) a subset of parvalbumin-positive GABAergic inhibitory interneurons colocalized with LacZ and CFP immunofluorescence signals suggesting coexpression of Cx36 and Cx30.2 in basket cells and/or axo-axonic cells. Or, stratum oriens; Py, stratum pyramidale; Ra, stratum radiatum. Scale bars: 50 μm.

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Fig. 8. Characterization of Cx30.2-expressing cells located in the adrenal medulla of Cx30.2+/LacZ mice. (A, B) Combined β-Gal and acetylcholinesterase (AChE) staining. No colocalization between β-Gal (blue) and AChE (brown) activity could be observed in medullary nerve fibers, indicating the absence of Cx30.2 from preganglionic sympathetic nerve fibers. (C–F) Double immunofluorescence analyses with β-Gal- (red) and tyrosine hydroxylase- (TH, green) specific antibodies. Nuclear β-Gal (D, E) and cytoplasmic TH immunofluorescence (F) signals were colocalized in a subset of adrenal medullary chromaffin cells (F). AC, adrenal cortex; AM, adrenal medulla. Nuclei were stained with DAPI. Scale bars: 100 μm (A, C) or 50 μm (B, D–F).

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Fig. 9. Field potential responses to stimulation of the Schaffer collateral, recorded in CA1 pyramidal layer. (A) Population spike responses to stimuli of increasing strength. Representative traces show a field EPSP, population spike response to weak stimulation, and maximal population spike, respectively. (B) Mean amplitude of maximal population spike, Cx30.2+/+ versus Cx30.2LacZ/LacZ. (C, D) Paired pulse protocol at a stimulation strength yielding 70% of maximal population spike. (C) Representative frames showing paired population spike at different time intervals. (D) Comparison of the paired-pulse ratio (amplitude of second population spike/amplitude of first population spike) for all tested interstimulus intervals. GraphPad Software Inc., San Diego, USA). P b 0.05 was regarded as significant.

Acknowledgments We thank Gerda Hertig, Richard Hertel and Nicole Karch for technical assistance and Larysa Voytenko for help with Cx30.2/ Cx36 coexpression studies. Elisa Weiß is a fellow of the MD/PhDProgram at the University of Heidelberg. This work was supported by the German Research Association (Wi 270/22–5,6 to K.W., Dr 326/1–4 to A.D. and SFB 636 TP A5 to A.S.). References Andrä, J., Lojda, Z., 1986. A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes. Histochemistry 84, 575–579. Bedner, P., Niessen, H., Odermatt, B., Kretz, M., Willecke, K., Harz, H., 2006. Selective permeability of different connexin channels to the second messenger cyclic AMP. J. Biol. Chem. 281, 6673–6681. Belluardo, N., Mudo, G., Trovato-Salinaro, A., Le Gurun, S., Charollais, A., Serre-Beinier, V., Amato, G., Haefliger, J.A., Meda, P., Condorelli, D.F.,

2000. Expression of connexin36 in the adult and developing rat brain. Brain Res. 865, 121–138. Bennett, M.V., 1997. Gap junctions as electrical synapses. J. Neurocytol. 26, 349–366. Bittman, K., Becker, D.L., Cicirata, F., Parnavelas, J.G., 2002. Connexin expression in homotypic and heterotypic cell coupling in the developing cerebral cortex. J. Comp. Neurol. 443, 201–212. Blatow, M., Rozov, A., Katona, I., Hormuzdi, S.G., Meyer, A.H., Whittington, M.A., Caputi, A., Monyer, H., 2003. A novel network of multipolar bursting interneurons generates theta frequency oscillations in neocortex. Neuron 38, 805–817. Buhl, E.H., Tamas, G., Fisahn, A., 1998. Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro. J. Physiol. 513, 117–126. Buhl, D.L., Harris, K.D., Hormuzdi, S.G., Monyer, H., Buzsaki, G., 2003. Selective impairment of hippocampal gamma oscillations in connexin36 knock-out mouse in vivo. J. Neurosci. 23, 1013–1018. Bukauskas, F.F., Kreuzberg, M.M., Rackauskas, M., Bukauskiene, A., Bennett, M.V., Verselis, V.K., Willecke, K., 2006. Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels: implications for atrioventricular conduction in the heart. Proc. Natl. Acad. Sci. U. S. A. 103, 9726–9731. Condorelli, D.F., Parenti, R., Spinella, F., Trovato, S.A., Belluardo, N., Cardile, V., Cicirata, F., 1998. Cloning of a new gap junction gene ODT

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Fig. 10. High-frequency network oscillation patterns. (A) Gamma oscillations in vitro induced by bath-application of kainic acid (200 nM) in slices from a wildtype (top) and Cx30.2-deficient mouse (bottom). (B) Comparison of the means for the following parameters: amplitude of the oscillation, frequency, power and full-width of half-maximum of the frequency distribution (FWHM). (C) Sharp wave–ripple complexes in slices from wild-type (top) and Cx30.2-deficient (bottom) mice. Voltage traces in the right panels show the marked events (⁎) at higher temporal resolution. Original traces reveal no significant changes in waveform or any disruption of the pattern. (D) Comparison of the means of sharp wave frequency, ripple frequency and ripple energy.

M.M. Kreuzberg et al. / Mol. Cell. Neurosci. 37 (2008) 119–134 Table 2 Antibody

Company

Species/Order No.

Dilution

Anti-NeuN Anti-GFAP Anti-S100 (β-subunit) Anti-CNPase Anti-parvalbumin Anti-somatostatin Anti-calbindin Anti-Kv3.1

Chemicon Sigma Sigma

Mouse/MAB377 Mouse/G-3893 Mouse/S-2532

1:1000 1:1000 1:1000

Sigma Sigma Chemicon Swant Alomone Laboratories Sigma

Mouse/C 5922 Mouse/P3088 Rat/MAB354 Rabbit/CB-38 Rabbit/APC-002

1:1000 1:1000 1:1000 1:1000 1:1000

Mouse/G-8021

1:1000

ICN/Cappel

Rabbit/55976

1:3000

Chemicon

Rabbit/ AB1211-5MG Chicken/ab13970 Goat/A-11029

1:15,000 1:2500 1:1000

Goat/A-11008

1:1000

Goat/103-165-155 Donkey/715-165-151 Donkey/711-165-152 Goat/112-165-102 Goat/115-175-166

1:500 1:1000 1:1000 1:1000 1:1000

Antiβ-galactosidase Antiβ-galactosidase Antiβ-galactosidase Anti-EGFP Anti-mouseAlexa488 Anti-rabbitAlexa488 Anti-chicken-Cy3 Anti-mouse-Cy3 Anti-rabbit-Cy3 Anti-rat-Cy3 Anti-mouse-Cy5

Abcam Molecular Probes Molecular Probes Dianova Dianova Dianova Dianova Dianova

(Cx36) highly expressed in mammalian brain neurons. Eur. J. Neurosci. 10, 1202–1208. Connors, B.W., Long, M.A., 2004. Electrical synapses in the mammalian brain. Annu. Rev. Neurosci. 27, 393–418. Deans, M.R., Gibson, J.R., Sellitto, C., Connors, B.W., Paul, D.L., 2001. Synchronous activity of inhibitory networks in neocortex requires electrical synapses containing connexin36. Neuron 31, 477–485. Dedek, K., Schultz, K., Pieper, M., Dirks, P., Maxeiner, S., Willecke, K., Weiler, R., Janssen-Bienhold, U., 2006. Localization of heterotypic gap junctions composed of connexin45 and connexin36 in the rod pathway of the mouse retina. Eur. J. Neurosci. 24, 1675–1686. Degen, J., Meier, C., Van Der Giessen, R.S., Söhl, G., Petrasch-Parwez, E., Urschel, S., Dermietzel, R., Schilling, K., De Zeeuw, C.I., Willecke, K., 2004. Expression pattern of lacZ reporter gene representing connexin36 in transgenic mice. J. Comp. Neurol. 473, 511–525. Deuchars, S.A., Brooke, R.E., Frater, B., Deuchars, J., 2001. Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit Kv3.1. Neuroscience 106, 433–446. Draguhn, A., Traub, R.D., Schmitz, D., Jefferys, J.G., 1998. Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro. Nature 394, 189–192. Elias, L.A., Wang, D.D., Kriegstein, A.R., 2007. Gap junction adhesion is necessary for radial migration in the neocortex. Nature 448, 901–907. Eng, S., Gratwick, K., Rhee, J.M., Fedtsova, N., Gan, L., Turner, E.E., 2001. Defects in sensory axon growth precede neuronal death in Brn3adeficient mice. J. Neurosci. 21, 541–549. Feigenspan, A., Teubner, B., Willecke, K., Weiler, R., 2001. Expression of neuronal connexin36 in AII amacrine cells of the mammalian retina. J. Neurosci. 21, 230–239. Güldenagel, M., Ammermüller, J., Feigenspan, A., Teubner, B., Degen, J., Söhl, G., Willecke, K., Weiler, R., 2001. Visual transmission deficits in mice with targeted disruption of the gap junction gene connexin36. J. Neurosci. 21, 6036–6044. Hombach, S., Janssen-Bienhold, U., Söhl, G., Schubert, T., Bussow, H., Ott, T., Weiler, R., Willecke, K., 2004. Functional expression of connexin57 in horizontal cells of the mouse retina. Eur. J. Neurosci. 19, 2633–2640. Hormuzdi, S.G., Pais, I., LeBeau, F.E., Towers, S.K., Rozov, A., Buhl, E.H.,

133

Whittington, M.A., Monyer, H., 2001. Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice. Neuron 31, 487–495. Kreuzberg, M.M., Söhl, G., Kim, J.S., Verselis, V.K., Willecke, K., Bukauskas, F.F., 2005. Functional properties of mouse connexin30.2 expressed in the conduction system of the heart. Circ. Res. 96, 1169–1177. Kreuzberg, M.M., Schrickel, J.W., Ghanem, A., Kim, J.S., Degen, J., Janssen-Bienhold, U., Lewalter, T., Tiemann, K., Willecke, K., 2006a. Connexin30.2 containing gap junction channels decelerate impulse propagation through the atrioventricular node. Proc. Natl. Acad. Sci. U. S. A. 103, 5959–5964. Kreuzberg, M.M., Willecke, K., Bukauskas, F.F., 2006b. Connexin-mediated cardiac impulse propagation: connexin 30.2 slows atrioventricular conduction in mouse heart. Trends Cardiovasc. Med. 16, 266–272. Krüger, O., Plum, A., Kim, J.S., Winterhager, E., Maxeiner, S., Hallas, G., Kirchhoff, S., Traub, O., Lamers, W.H., Willecke, K., 2000. Defective vascular development in connexin 45-deficient mice. Development 127, 4179–4193. Kumar, N.M., Gilula, N.B., 1996. The gap junction communication channel. Cell 84, 381–388. 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. Long, M.A., Jutras, M.J., Connors, B.W., Burwell, R.D., 2005. Electrical synapses coordinate activity in the suprachiasmatic nucleus. Nat. Neurosci. 8, 61–66. Maier, N., Güldenagel, M., Söhl, G., Siegmund, H., Willecke, K., Draguhn, A., 2002. Reduction of high-frequency network oscillations (ripples) and pathological network discharges in hippocampal slices from connexin 36-deficient mice. J. Physiol. 541, 521–528. Maier, N., Nimmrich, V., Draguhn, A., 2003. Cellular and network mechanisms underlying spontaneous sharp wave–ripple complexes in mouse hippocampal slices. J. Physiol. 550, 873–887. Maxeiner, S., Krüger, O., Schilling, K., Traub, O., Urschel, S., Willecke, K., 2003. Spatiotemporal transcription of connexin45 during brain development results in neuronal expression in adult mice. Neuroscience 119, 689–700. Maxeiner, S., Dedek, K., Janssen-Bienhold, U., Ammermüller, J., Brune, H., Kirsch, T., Pieper, M., Degen, J., Krüger, O., Willecke, K., Weiler, R., 2005. Deletion of connexin45 in mouse retinal neurons disrupts the rod/ cone signaling pathway between AII amacrine and ON cone bipolar cells and leads to impaired visual transmission. J. Neurosci. 25, 566–576. Pearson, R.A., Dale, N., Llaudet, E., Mobbs, P., 2005. ATP released via gap junction hemichannels from the pigment epithelium regulates neural retinal progenitor proliferation. Neuron 46, 731–744. Perez Velazquez, J.L., Carlen, P.L., 2000. Gap junctions, synchrony and seizures. Trends Neurosci. 23, 68–74. Romanov, R.A., Rogachevskaja, O.A., Bystrova, M.F., Jiang, P., Margolskee, R.F., Kolesnikov, S.S., 2007. Afferent neurotransmission mediated by hemichannels in mammalian taste cells. EMBO J. 26, 657–667. Shelley, J., Dedek, K., Schubert, T., Feigenspan, A., Schultz, K., Hombach, S., Willecke, K., Weiler, R., 2006. Horizontal cell receptive fields are reduced in connexin57-deficient mice. Eur. J. Neurosci. 23, 3176–3186. Söhl, G., Degen, J., Teubner, B., Willecke, K., 1998. The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development. FEBS Lett. 428, 27–31. Söhl, G., Maxeiner, S., Willecke, K., 2005. Expression and functions of neuronal gap junctions. Nat. Rev., Neurosci. 6, 191–200. Somogyi, P., Klausberger, T., 2005. Defined types of cortical interneurone structure space and spike timing in the hippocampus. J. Physiol. 562, 9–26. Srinivas, M., Rozental, R., Kojima, T., Dermietzel, R., Mehler, M., Condorelli, D.F., Kessler, J.A., Spray, D.C., 1999. Functional properties of channels formed by the neuronal gap junction protein connexin36. J. Neurosci. 19, 9848–9855. Teubner, B., Degen, J., Söhl, G., Güldenagel, M., Bukauskas, F.F., Trexler, E.B., Verselis, V.K., De Zeeuw, C.I., Lee, C.G., Kozak, C.A., PetraschParwez, E., Dermietzel, R., Willecke, K., 2000. Functional expression of

134

M.M. Kreuzberg et al. / Mol. Cell. Neurosci. 37 (2008) 119–134

the murine connexin 36 gene coding for a neuron-specific gap junctional protein. J. Membr. Biol. 176, 249–262. Thompson, R.J., Zhou, N., MacVicar, B.A., 2006. Ischemia opens neuronal gap junction hemichannels. Science 312, 924–927. Traub, R.D., Kopell, N., Bibbig, A., Buhl, E.H., LeBeau, F.E., Whittington, M.A., 2001. Gap junctions between interneuron dendrites can enhance synchrony of gamma oscillations in distributed networks. J. Neurosci. 21, 9478–9486. Traub, R.D., Pais, I., Bibbig, A., LeBeau, F.E., Buhl, E.H., Hormuzdi, S.G., Monyer, H., Whittington, M.A., 2003. Contrasting roles of axonal (pyramidal cell) and dendritic (interneuron) electrical coupling in the generation of neuronal network oscillations. Proc. Natl. Acad. Sci. U. S. A. 100, 1370–1374.

Traub, R.D., Bibbig, A., LeBeau, F.E., Buhl, E.H., Whittington, M.A., 2004. Cellular mechanisms of neuronal population oscillations in the hippocampus in vitro. Annu. Rev. Neurosci. 27, 247–278. Vida, I., Bartos, M., Jonas, P., 2006. Shunting inhibition improves robustness of gamma oscillations in hippocampal interneuron networks by homogenizing firing rates. Neuron 49, 107–117. Vogt, A., Hormuzdi, S.G., Monyer, H., 2005. Pannexin1 and Pannexin2 expression in the developing and mature rat brain. Brain Res. Mol. Brain Res. 141, 113–120. Ylinen, A., Bragin, A., Nadasdy, Z., Jando, G., Szabo, I., Sik, A., Buzsaki, G., 1995. Sharp wave-associated high-frequency oscillation (200 Hz) in the intact hippocampus: network and intracellular mechanisms. J. Neurosci. 15, 30–46.