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Complex sphingolipids are strongly expressed in neuronal tissue and contain ceramides in their backbone. Ceramides are synthesized by six ceramide synthases (CerS1-6). Although it is known that each tissue has a unique profile of ceramide synthase expression and ceramide synthases are implicated in several neurodegenerative disorders, the expression of ceramide synthase isoforms has not been investigated in the retina. Here we demonstrate CerS1, CerS2 and CerS4 expression in mouse retina and cornea, with CerS4 ubiquitously expressed in all retinal neurons and Müller cells. To test whether ceramide synthase deficiency affects retinal function, we compared electroretinograms and retina morphology between wild-type and CerS1-, CerS2- and CerS4-deficient mice. Electroretinograms were strongly reduced in amplitude in ceramide synthase-deficient mice, suggesting that signalling in the outer retina is affected. However, the number of photoreceptors and cone outer segment length were unaltered and no changes in retinal layer thickness or synaptic structures were found. Mass spectrometric analyses of ceramides, hexosyl-ceramides and sphingomyelins showed that C20 to C24 acyl-containing species were decreased whereas C16-containing species were increased in the retina of ceramide synthase-deficient mice. Similar but smaller changes were also found in the cornea. Thus, we hypothesize that the replacement of very long-chain fatty acyl residues by shorter C16 residues may affect the electrical properties of retina and cornea, and alter receptor-mediated signal transduction, vesicle-mediated synaptic transmission or corneal light transmission. Future studies need to identify the molecular targets of ceramides or derived sphingolipids in light signal transduction and transmission in the eye., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Gap junction coupling synchronizes activity among neurons in adult neural circuits, but its role in coordinating activity during development is less known. The developing retina exhibits retinal waves--spontaneous depolarizations that propagate among retinal interneurons and drive retinal ganglion cells (RGCs) to fire correlated bursts of action potentials. During development, two connexin isoforms, connexin 36 (Cx36) and Cx45, are expressed in bipolar cells and RGCs, and therefore provide a potential substrate for coordinating network activity. To determine whether gap junctions contribute to retinal waves, we compared spontaneous activity patterns using calcium imaging, whole-cell recording, and multielectrode array recording in control, single-knock-out (ko) mice lacking Cx45 and double-knock-out (dko) mice lacking both isoforms. Wave frequency, propagation speed, and bias in propagation direction were similar in control, Cx36ko, Cx45ko, and Cx36/45dko retinas. However, the spontaneous firing rate of individual retinal ganglion cells was elevated in Cx45ko retinas, similar to Cx36ko retinas (Hansen et al., 2005; Torborg and Feller, 2005), a phenotype that was more pronounced in Cx36/45dko retinas. As a result, spatial correlations, as assayed by nearest-neighbor correlation and functional connectivity maps, were significantly altered. In addition, Cx36/45dko mice had reduced eye-specific segregation of retinogeniculate afferents. Together, these findings suggest that although Cx36 and Cx45 do not play a role in gross spatial and temporal propagation properties of retinal waves, they strongly modulate the firing pattern of individual RGCs, ensuring strongly correlated firing between nearby RGCs and normal patterning of retinogeniculate projections.

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons., (© 2010 Wiley-Liss, Inc.)

Background: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown., Methods: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina., Results: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina., Conclusion: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

The gap junction protein connexin-45 (Cx45) is expressed in the conduction system of the heart and in certain neurons of the retina and brain. General and cardiomyocyte-directed deficiencies of Cx45 in mice lead to lethality on embryonic day 10.5 as a result of cardiovascular defects. Neuron-directed deletion of Cx45 leads to defects in transmission of visual signals. Connexin-36 (Cx36) is co-expressed with Cx45 in certain types of retinal interneurons. To determine whether these two connexins have similar functions and whether Cx36 can compensate for Cx45, we generated knock-in mice in which DNA encoding Cx45 was replaced with that encoding Cx36. Neuron-directed replacement of Cx45 with Cx36 resulted in viable animals. Electroretinographic and neurotransmitter coupling analyses demonstrated functional compensation in the retina. By contrast, general and cardiomyocyte-directed gene replacement led to lethality on embryonic day 11.5. Mutant embryos displayed defects in cardiac morphogenesis and conduction. Thus, functional compensation of Cx45 by Cx36 did not occur during embryonic heart development. These data suggest that Cx45 and Cx36 have similar functions in the retina, whereas Cx45 fulfills special functions in the developing heart that cannot be compensated by Cx36.

Mammalian retinae express multiple connexins that mediate the metabolic and electrical coupling of various cell types. In retinal neurons, only connexin 36, connexin 45, connexin 50, and connexin 57 have been described so far. Here, we present an analysis of a novel retinal connexin, connexin 30.2 (Cx30.2), and its regulation in the mouse retina. To analyze the expression of Cx30.2, we used a transgenic mouse line in which the coding region of Cx30.2 was replaced by lacZ reporter DNA. We detected the lacZ signal in the nuclei of neurons located in the inner nuclear layer and the ganglion cell layer (GCL). In this study, we focused on the GCL and characterized the morphology of the Cx30.2-expressing cells. Using immunocytochemistry and intracellular dye injections, we found six different types of Cx30.2-expressing ganglion cells: one type of ON-OFF, three types of OFF, and two types of ON ganglion cells; among the latter was the RG A1 type. We show that RG A1 cells were heterologously coupled to numerous displaced amacrine cells. Our results suggest that these gap junction channels may be heterotypic, involving Cx30.2 and a connexin yet unidentified in the mouse retina. Gap junction coupling can be modulated by protein kinases, a process that plays a major role in retinal adaptation. Therefore, we studied the protein kinase-induced modulation of coupling between RG A1 and displaced amacrine cells. Our data provide evidence that coupling of RG A1 cells to displaced amacrine cells is mediated by Cx30.2 and that the extent of this coupling is modulated by protein kinase C.

Mammalian retinas comprise an enormous variety of amacrine cells with distinct properties and functions. The present paper describes a new interplexiform amacrine cell type in the mouse retina. A transgenic mouse mutant was used that expressed the gene for the enhanced green fluorescent protein (EGFP) instead of the coding DNA of connexin45 in several retinal cell classes, among which a single amacrine cell population was most prominently labelled. Staining for EGFP and different marker proteins showed that these amacrine cells are interplexiform: they stratify in stratum S4/5 of the inner plexiform layer and send processes to the outer plexiform layer. These cells were termed IPA-S4/5 cells. They belong to the group of medium-field amacrine cells and are coupled homologously and heterologously to other amacrine cells by connexin45. Immunostaining revealed that IPA-S4/5 cells are GABAergic and express GAT-1, a plasma-membrane-bound GABA transporter possibly involved in non-vesicular GABA release. To characterize the light responses of IPA-S4/5 cells, patch-clamp recordings in retinal slices were made. Consistent with their stratification in the ON sublamina of the inner plexiform layer, cells depolarized in response to light ON stimuli and transiently hyperpolarized in response to light OFF. Responses of cells to green (578 nm) and blue (400 nm) light suggest that they receive input from cone bipolar cells contacting both M- and S-cones, possibly with reduced S-cone input. A new type of interplexiform ON amacrine cell is described, which is strongly coupled and uses GABA but not dopamine as its neurotransmitter.

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.

Direction selectivity, a key feature of visual perception, originates in the retina and is transmitted by bistratified ganglion cells that, in the rabbit retina, exhibit a particular coupling pattern. We intracellularly labeled ganglion cells in different transgenic mouse lines, allowing a morphological classification of bistratified ganglion cells, an analysis of their coupling pattern, and the molecular identification of the connexins responsible for the coupling. Based on dendritic characteristics including co-fasciculation with the dendrites of cholinergic starburst amacrine cells, we were able to distinguish three types of bistratified ganglion cells. Two of these co-fasciculate with starburst amacrine cells and exhibit a specific homologous coupling pattern. Connexin45 (Cx45) appears to be the major component of the gap junctional channels because tracer coupling is absent in Cx45-deficient animals whereas it persists in Cx36-deficient animals. It is speculated that the transjunctional voltage dependence of Cx45 channels could support the transmission of direction selectivity., (Copyright (c) 2005 Wiley-Liss, Inc.)

Connexin45 (Cx45) is known to be expressed in the retina, but its functional analysis was problematic because general deletion of Cx45 coding DNA resulted in cardiovascular defects and embryonic lethality at embryonic day 10.5. We generated mice with neuron-directed deletion of Cx45 and concomitant activation of the enhanced green fluorescent protein (EGFP). EGFP labeling was observed in bipolar, amacrine, and ganglion cell populations. Intracellular microinjection of fluorescent dyes in EGFP-labeled somata combined with immunohistological markers revealed Cx45 expression in both ON and OFF cone bipolar cells. The scotopic electroretinogram of mutant mice revealed a normal a-wave but a 40% reduction in the b-wave amplitude, similar to that found in Cx36-deficient animals, suggesting a possible defect in the rod pathway of visual transmission. Indeed, neurotransmitter coupling between AII amacrine cells and Cx45-expressing cone bipolar cells was disrupted in Cx45-deficient mice. These data suggest that both Cx45 and Cx36 participate in the formation of functional heterotypic electrical synapses between these two types of retinal neurons that make up the major rod pathway.

Horizontal cells are interneurons of the vertebrate retina that exhibit strong electrical and tracer coupling but the identity of the channel-forming connexins has remained elusive. Here we show that horizontal cells of the mouse retina express connexin57 (Cx57). We have generated Cx57-deficient mice by replacing the Cx57 coding region with a lacZ reporter gene, expressed under control of the endogenous Cx57 promoter. These mice were fertile and showed no obvious anatomical or behavioural abnormalities. Cx57 mRNA was expressed in the retina of wild-type littermates but was absent from the retina of Cx57-deficient mice. Previously reported results that the Cx57 gene was very weakly expressed in several other mouse tissues turned out to be unspecific. Cx57 mRNA is abundantly expressed in the retina and weakly in the thymus of adult mice but absent in all other adult tissues tested, including brain. Furthermore, Cx57 is expressed in embryonic kidney at E16.5 to E18.5 days post-conception, as indicated by the pattern of lacZ expression. Within the retina, lacZ signals were assigned exclusively to horizontal cells based on co-localization with cell-type-specific marker proteins. Microinjection of Neurobiotin into horizontal cells of isolated retinae revealed less than 1% of tracer coupling in Cx57-deficient retinae compared with wild-type controls. Cx57 is the first connexin identified in mammalian horizontal cells and the first connexin whose expression is apparently restricted to only one type of neuron.

Transgenic technology, immunocytochemistry, electrophysiology, intracellular injection techniques, and reverse transcription PCR were combined to study the expression of neuronal connexin36 (Cx36) in the outer plexiform layer of the mouse retina. Transgenic animals expressed either a fusion protein of full-length Cx36 with enhanced green fluorescent protein (EGFP) attached at the C terminus or exon 2 of Cx36 was replaced bybeta-galactosidase (beta-gal). In the outer nuclear layer,beta-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin. Cx36-EGFP puncta colocalized with cone pedicles, which were visualized by intracellular injection. In reverse transcriptase PCR experiments, Cx36 mRNA was never detected in samples of rods harvested from the outer nuclear layer. These results strongly suggest expression of Cx36 in cones but not in rods. In vertical sections, Cx36 expression in the vitreal part of the outer plexiform layer was characterized by a patchy distribution. Immunocytochemistry with antibodies against the neurokinin-3 receptor and the potassium channel HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel) displayed clusters of the Cx36 label on the dendrites of OFF-cone bipolar cells. In horizontal sections, these clusters of Cx36 appeared as round or oval-shaped groups of individual puncta, and they were always aligned with the base of cone pedicles. Double-labeling experiments and single-cell reverse transcriptase PCR ruled out expression of Cx36 in horizontal cells and rod bipolar cells. At light microscopic resolution, we found close association of Cx36-EGFP with the AMPA-type glutamate receptor subunit GluR1 but not with GluR2-GluR4, the kainate receptor subunit GluR5, or the metabotropic glutamate receptor mGluR6.

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however,howhorizontal cell signals also affect the temporal, spatial,andcontrast tuning in retinal output neurons, the ganglion cells.Tostudy this,wegenerated a genetically modifiedmouseline inwhichweeliminated the lightdependencyof feedbackbydeleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actualmodeof feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-+ retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. [ABSTRACT FROM AUTHOR]

Mammalian retinae express multiple connexins that mediate the metabolic and electrical coupling of various cell types. In retinal neurons, only connexin36, connexin45, connexin50, and connexin57 have been described so far. Here, we present an analysis of a novel retinal connexin, connexin30.2 (Cx30.2), and its regulation in the mouse retina. To analyze the expression of Cx30.2, we used a transgenic mouse line in which the coding region of Cx30.2 was replaced by lacZ reporter DNA. We detected the lacZ signal in the nuclei of neurons located in the inner nuclear layer and the ganglion cell layer (GCL). In this study, we focused on the GCL and characterized the morphology of the Cx30.2- expressing cells. Using immunocytochemistry and intracellular dye injections, we found six different types of Cx30.2-expressing ganglion cells: one type of ON-OFF, three types of OFF, and two types of ON ganglion cells; among the latter was the RGA1 type. We show that RGA1 cells were heterologously coupled to numerous displaced amacrine cells. Our results suggest that these gap junction channels may be heterotypic, involving Cx30.2 and a connexin yet unidentified in the mouse retina. Gap junction coupling can be modulated by protein kinases, a process that plays a major role in retinal adaptation. Therefore, we studied the protein kinase-induced modulation of coupling between RGA1 and displaced amacrine cells. Our data provide evidence that coupling of RGA1 cells to displaced amacrine cells is mediated by Cx30.2 and that the extent of this coupling is modulated by protein kinase C. [ABSTRACT FROM AUTHOR]

Background: The visual system can adjust itself to different visual environments. One of the most well known examples of this is the shift in spatial tuning that occurs in retinal ganglion cells with the change from night to day vision. This shift is thought to be produced by a change in the ganglion cell receptive field surround, mediated by a decrease in the coupling of horizontal cells. Methodology/Principal Findings: To test this hypothesis, we used a transgenic mouse line, a connexin57-deficient line, in which horizontal cell coupling was abolished. Measurements, both at the ganglion cell level and the level of behavioral performance, showed no differences between wild-type retinas and retinas with decoupled horizontal cells from connexin57-deficient mice. Conclusion/Significance: This analysis showed that the coupling and uncoupling of horizontal cells does not play a dominant role in spatial tuning and its adjustability to night and day light conditions. Instead, our data suggest that another mechanism, likely arising in the inner retina, must be responsible. [ABSTRACT FROM AUTHOR]

Horizontal cells are coupled by gap junctions; the extensive coupling of the horizontal cells is reflected in their large receptive fields, which extend far beyond the dendritic arbor of the individual cell. In the mouse retina, horizontal cells express connexin57 (Cx57). Tracer coupling of horizontal cells is impaired in Cx57-deficient mice, which suggests that the receptive fields of Cx57-deficient horizontal cells might be similarly reduced. To test this hypothesis we measured the receptive fields of horizontal cells from wildtype and Cx57-deficient mice. First, we examined the synaptic connections between horizontal cells and photoreceptors: no major morphological alterations were found. Moreover, horizontal cell spacing and dendritic field size were unaffected by Cx57 deletion. We used intracellular recordings to characterize horizontal cell receptive fields. Length constants were computed for each cell using the cell's responses to concentric light spots of increasing diameter. The length constant was dependent on the intensity of the stimulus: increasing stimulus intensity reduced the length constant. Deletion of Cx57 significantly reduced horizontal cell receptive field size. Dark resting potentials were strongly depolarized and response amplitudes reduced in Cx57-deficient horizontal cells compared to the wildtype, suggesting an altered input resistance. This was confirmed by patch-clamp recordings from dissociated horizontal cells; mean input resistance of Cx57-deficient horizontal cells was 27% lower than that of wildtype cells. These data thus provide the first quantification of mouse horizontal cell receptive field size and confirm the unique role of Cx57 in horizontal cell coupling and physiology. [ABSTRACT FROM AUTHOR]

Connexin45 (Cx45) is known to be expressed in the retina, but its functional analysis was problematic because general deletion of Cx45 coding DNA resulted in cardiovascular defects and embryonic lethality at embryonic day 10.5. We generated mice with neuron-directed deletion of Cx45 and concomitant activation of the enhanced green fluorescent protein (EGFP). EGFP labeling was observed in bipolar, amacrine, and ganglion cell populations. Intracellular microinjection of fluorescent dyes in EGFP-labeled somata combined with immunohistological markers revealed Cx45 expression in both ON and OFF cone bipolar cells. The scotopic electroretinogram of mutant mice revealed a normal a-wave but a 40% reduction in the b-wave amplitude, similar to that found in Cx36-deficient animals, suggesting a possible defect in the rod pathway of visual transmission. Indeed, neurotransmitter coupling between AII amacrine cells and Cx45-expressing cone bipolar cells was disrupted in Cx45-deficient mice. These data suggest that both Cx45 and Cx36 participate in the formation of functional heterotypic electrical synapses between these two types of retinal neurons that make up the major rod pathway. [ABSTRACT FROM AUTHOR]

Investigating the spatial and temporal expression of connexin36 (Cx36) protein in neuronal tissue is of prime importance to understand the molecular mechanisms underlying extensive electrical coupling. Although Cx36 mRNA was shown to be expressed in neurons of the central nervous system in different studies, only the determination of Cx36 protein expression allows a correlation between localization and its functional role in gap junction-mediated neuronal coupling. After the initial use of antibodies recognizing the skate connexin35 protein, antibodies directed to the mammalian Cx36 sequence allowed the detailed investigation of Cx36 cellular localization. However, results on Cx36 protein distribution still remained controversial in some areas of the central nervous system. In the present study, we have investigated: (a) the distribution of Cx36 protein in various areas of the central nervous system and (b) determined the specificity in the immunohistochemical staining of two polyclonal antibodies comparing wildtype and Cx36-deficient mice. In some areas of the central nervous system, for example in the retina and the inferior nuclear olivary complex, Cx36 antibodies were highly specific, and in the cerebellar cortex, Cx36 protein expression was partly specific. In other regions, particularly in pyramidal cells of the hippocampal formation, non-specific staining was prevalent, indicating that Cx36 antibodies also recognize proteins other than Cx36 in these tissues. The present results argue for a re-evaluation of many documented immunohistochemical protein distribution patterns and require, not only in connexin research, their assessment using null-mutant animals. [ABSTRACT FROM AUTHOR]

Abstract: Connexin36 (Cx36) is the main connexin isoform expressed in neurons of the central nervous system (CNS) and in pancreatic β-cells, i.e. two types of excitable cells that share – in spite of their different origins – a number of common features. Previous studies on Cx36 deficient mice have documented that loss of Cx36 resulted in phenotypic abnormalities in both the CNS and the pancreas which, however, could not be attributed to specific cell types due to the general deletion nature of the animal model used. Attempts to address this limitation using cell type specific deletions generated by the Cre/loxP strategy have so far been complicated by the lack of Cx36 expression from the floxed allele. We have now generated a conditional Cx36 deficient mouse mutant in which the coding region of Cx36 is flanked by loxP sites, followed by a cyan fluorescent protein (CFP) reporter gene. Here we show that Cx36 was still expressed from the floxed allele in neurons and pancreatic β-cells. In these cells, a 30–60% decrease of this protein, relative to the expression level of the wildtype allele, did not significantly perturb cell coupling. The deletion of Cx36 by ubiquitously and cell type specifically expressed Cre recombinases revealed that CFP functions as a reliable reporter for Cx36 expression in brain neurons and to some extent in retina neurons, but not in pancreas. Loss of Cx36 by Cre-mediated recombination was documented at transcript and protein levels. Cell type specific deletion of Cx36 in the endocrine pancreas revealed major alterations in the basal as well as the glucose-induced insulin secretion, hence specifically attributing to pancreatic Cx36 an important regulatory role in the control of β-cell function. Cell type specific deletion of Cx36 in the CNS by suitable Cre recombinases should also help to elucidate the functional role of Cx36 in different neuronal subtypes. [Copyright &y& Elsevier]