Connexin36 mediates gap junctional coupling of alpha-ganglion cells in mouse retina

Alpha-ganglion cells are present in all vertebrate retinae and are subdivided into ON and OFF types according to their level of dendritic ramification within the inner plexiform layer. They have large dendritic fields and usually a good responsiveness to moving stimuli. They were the first ganglion cells in which tracer coupling was observed, suggesting the presence of gap junctions composed of unknown connexins. Here we show that ON-alpha-ganglion cells in the mouse retina are coupled to amacrine cells, whereas OFF-alpha-ganglion cells are coupled to other OFF-alpha-ganglion cells and to amacrine cells. These tracer coupling patterns were completely absent in mice deficient in connexin36 (Cx36). The expression of Cx36 protein in alpha-ganglion cells but not in coupled amacrine cells was confirmed in mice in which the Cx36 coding DNA was replaced by the lacZ reporter gene. The dendritic localization and the distribution pattern of Cx36 patches, analyzed in mice in which the enhanced green fluorescent protein (EGFP) was linked to the C-terminal region of the Cx36 protein, revealed a rather small number of fluorescent plaques and different patterns for ON- and OFF-alpha-ganglion cells. Furthermore, tracer coupling between OFF-alpha-ganglion cells could be inhibited by quinine, a gap junctional blocker with a slight preference for gap junctions formed by Cx36. These data strongly suggest that Cx36 gap junction channels are functional not only in interneurons but also in output neurons of the retina and are responsible for distinct coupling patterns of ganglion cells.     

Connexin50 couples axon terminals of mouse horizontal cells by homotypic gap junctions

Horizontal cells in the mouse retina are of the axon‐bearing B‐type and contribute to the gain control of photoreceptors and to the center‐surround organization of bipolar cells by providing feedback and feedforward signals to photoreceptors and bipolar cells, respectively. Horizontal cells form two independent networks, coupled by dendro‐dendritic and axo‐axonal gap junctions composed of connexin57 (Cx57). In Cx57‐deficient mice, occasionally the residual tracer coupling of horizontal cell somata was observed. Also, negative feedback from horizontal cells to photoreceptors, potentially mediated by connexin hemichannels, appeared unaffected. These results point to the expression of a second connexin in mouse horizontal cells. We investigated the expression of Cx50, which was recently identified in axonless A‐type horizontal cells of the rabbit retina. In the mouse retina, Cx50‐immunoreactive puncta were predominantly localized on large axon terminals of horizontal cells. Electron microscopy did not reveal any Cx50‐immunolabeling at the membrane of horizontal cell tips invaginating photoreceptor terminals, ruling out the involvement of Cx50 in negative feedback. Moreover, Cx50 colocalized only rarely with Cx57 on horizontal cell processes, indicating that both connexins form homotypic rather than heterotypic or heteromeric gap junctions. To check whether the expression of Cx50 is changed when Cx57 is lacking, we compared the Cx50 expression in wildtype and Cx57‐deficient mice. However, Cx50 expression was unaffected in Cx57‐deficient mice. In summary, our results indicate that horizontal cell axon terminals form two independent sets of homotypic gap junctions, a feature which might be important for light adaptation in the retina. J. Comp. Neurol. 523:2062–2081, 2015. © 2015 Wiley Periodicals, Inc.     

Horizontal cell receptive fields are reduced in connexin57-deficient mice

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.     

Expression pattern and functional characterization of connexin29 in transgenic mice

Using newly generated transgenic mice in which the coding region of the connexin29 (Cx29) gene was replaced by the lacZ reporter gene, we confirmed previous immunochemical results that Cx29 is expressed in Schwann cells, oligodendrocytes and Bergmann glia cells. In addition, we detected lacZ/Cx29 in Schwann cells of the sciatic nerve and in particular of the spiral ganglion in the inner ear, as well as at low abundance in the stria vascularis. Furthermore, we found lacZ/Cx29 expression in nonmyelinating Schwann cells of the adrenal gland, in chondrocytes of intervertebral discs and the epiphysis of developing bones. Electron microscopic analyses of myelin sheaths in the central and peripheral nervous system of Cx29-deficient mice detected no abnormalities. The nerve conduction in the sciatic nerve of adult Cx29-deficient mice and the auditory brain stem response as well as visually evoked potentials in 4- to 10-week-old Cx29-deficient mice were not different from wild-type littermate controls. Thus, in contrast to connexin32 and connexin47, which are also expressed in myelinating cells, Cx29 does not contribute to the function of myelin in adult mice.     

Connexin expression in the retina

Here, we review recent results from our laboratory on connexin expression in mouse retina in the context of previous results with other vertebrate species. In mouse retina, four different connexin proteins were detected by immunoblot and immunofluorescence: connexin (Cx)-36, -37, -43 and -45. Cx36 and Cx45 immunoreactive signals were found in the inner and outer plexiform layer, both of which are known to show interneuronal gap junctions. Cx43 was detected in the ganglion cell layer, presumably in astrocytes, where it appeared to be colocalized with glial fibrillary acid protein. Cx37 was expressed in retinal endothelial cells. Additionally, Cx26, -31, -32 and -40 mRNAs were detected in retina by RT–PCR but none of the corresponding proteins were found. In order to exclude cross reactions of the corresponding antibodies, retinae from targeted connexin-deficient mice (Cx31 −/−, Cx32 −/− and Cx40 −/−) were used as negative controls for immunoblot and immunofluorescence analyses of wild-type retina. Further detailed investigation of cell type specific connexin expression in the mouse retina will be necessary for functional analyses of targeted mouse mutants with defects in connexins expressed in retinal neuronal cells.     

Endogenous dopaminergic regulation of horizontal cell coupling in the mammalian retina

Horizontal cells in an isolated wholemount preparation of the mouse retina were injected with Lucifer yellow and neurobiotin to characterize both the pattern of gap junctional connectivity and its regulation by dopamine. The injected horizontal cells had a uniform morphology of a round cell body, a compact dendritic tree, and an axon, which could sometimes be traced to an expansive terminal system. The dendro-dendritic gap junctions between neighboring cells mediated both weak Lucifer yellow dye coupling and strong neurobiotin tracer coupling. The extent of the tracer coupling was decreased by either exogenous dopamine (100 microM) or cyclic adenosine monophosphate (cAMP) analogs and was significantly increased by the D1 antagonist SCH 23390 (10 microM). These results provide the first evidence in the mammalian retina that the gap junctions between horizontal cells are endogenously regulated by dopamine, which acts through D1 receptors to increase the intracellular cAMP. It has been proposed that the gap junctional coupling between horizontal cells is mediated by connexin 32 (Cx32), but the pattern and dopaminergic regulation of horizontal cell coupling were unaffected in Cx32-knockout mice, ruling out the possible involvement of Cx32. Every tracer-coupled horizontal cell showed calbindin immunoreactivity, and vice versa, providing strong evidence that the horizontal cells in the mouse retina comprise a single cell type. Like the axonless horizontal cells in other mammalian retinas, the axon-bearing horizontal cells in the mouse retina are coupled by gap junctions that are permeable to Lucifer yellow and dopamine sensitive, suggesting that the mouse horizontal cells have hybrid properties to compensate for the absence of axonless horizontal cells.     

Connexin45 mediates gap junctional coupling of bistratified ganglion cells in the mouse retina

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.     

Connexin57 is expressed in dendro‐dendritic and axo‐axonal gap junctions of mouse horizontal cells and its distribution is modulated by light

Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flat‐mounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57‐deficient mice. Significantly more connexin57‐positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57‐immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions. J. Comp. Neurol. 513:363–374, 2009. © 2009 Wiley‐Liss, Inc.     

Expression pattern of lacZ reporter gene representing connexin36 in transgenic mice

Targeted deletion of the connexin36 (Cx36) gene in the mouse genome leads to visual transmission defects, weakened synchrony of rhythmic inhibitory potentials in the neocortex, and disruption of gamma-frequency network oscillations. We have generated transgenic mice in which a reporter protein consisting of the exon1 coded N-terminal part of Cx36 fused to beta-galactosidase (N36-beta-gal) is expressed instead of Cx36. Here, we have used these mice for a detailed analysis of the reporter gene expression. By beta-gal staining of adult retina, we found expression of the lacZ reporter gene in the ganglion cell layer, in two rows of the inner nuclear layer, and in the photoreceptor layer. In the brain, beta-gal staining was present in gamma-aminobutyric acid (GABA)ergic neurons of the cerebellar nuclei, in non-GABAergic neurons of the inferior olive, in mitral cells of the olfactory bulb, and in parvalbumin-positive cells of the cerebral cortex. Outside the central nervous system, N36-beta-gal signals were detected in insulin producing beta-cells of the pancreas and in the medulla of the adrenal gland of adult Cx36(+/del[LacZ]) mice. This expression pattern suggests that Cx36 fulfills functional roles not only in several types of neurons in the retina and central nervous system but also in excitable cells of the pancreas and adrenal gland.     

Connexin36 is required for gap junctional coupling of most ganglion cell subtypes in the mouse retina

Converging evidence indicates that electrical synaptic transmission via gap junctions plays a crucial role in signal processing in the retina. In particular, amacrine and ganglion cells express numerous gap junctions, resulting in extensive electrical networks in the proximal retina. Both connexin36 (Cx36) and connexin45 (Cx45) subunits are widely distributed in the inner plexiform layer (IPL) and therefore are likely contribute to gap junctions formed by a number of ganglion cell subtypes. In the present study, we used the gap junction‐permeant tracer Neurobiotin to compare the coupling pattern of different ganglion cell subtypes in wild‐type (WT) and Cx36 knockout (KO) mouse retinas. We found that homologous ganglion‐to‐ganglion cell coupling was lost for two subtypes after deletion of Cx36, whereas two other ganglion cell subtypes retained homologous coupling in the KO mouse. In contrast, deletion of Cx36 resulted in a partial or complete loss of ganglion‐to‐amacrine cell heterologous coupling in 9 of 10 ganglion cell populations studied. Overall, our results indicate that Cx36 is the predominant subunit of gap junctions in the proximal mouse retina, expressed by most ganglion cell subtypes, and thereby likely plays a major role in the concerted activity generated by electrical synapses. J. Comp. Neurol. 518:911–927, 2010. © 2009 Wiley‐Liss, Inc.     

Morphology and tracer coupling pattern of alpha ganglion cells in the mouse retina

Alpha cells are a type of ganglion cell whose morphology appears to be conserved across a number of mammalian retinas. In particular, alpha cells display the largest somata and dendritic arbors at a given eccentricity and tile the retina as independent on- (ON) and off-center (OFF) subtypes. Mammalian alpha cells also express a variable tracer coupling pattern, which often includes homologous (same cell type) coupling to a few neighboring alpha cells and extensive heterologous (different cell type) coupling to two to three amacrine cell types. Here, we use the gap junction-permeant tracer Neurobiotin to determine the architecture and coupling pattern of alpha cells in the mouse retina. We find that alpha cells show the same somatic and dendritic architecture described previously in the mammal. However, alpha cells show varied tracer coupling patterns related to their ON and OFF physiologies. ON alpha cells show no evidence of homologous tracer coupling but are coupled heterologously to at least two types of amacrine cell whose somata lie within the ganglion cell layer. In contrast, OFF alpha cells are coupled to one another in circumscribed arrays as well as to two to three types of amacrine cell with somata occupying the inner nuclear layer. We find that homologous coupling between OFF alpha cells is unaltered in the connexin36 (Cx36) knockout (KO) mouse retina, indicating that it is not dependent on Cx36. However, a subset of the heterologous coupling of ON alpha cells and all the heterologous coupling of OFF alpha cells are eliminated in the KO retina, suggesting that Cx36 comprises most of the junctions made with amacrine cells.     

Connexin 57 is expressed by the axon terminal network of B-type horizontal cells in the rabbit retina

In the rabbit retina there are two types of horizontal cell (HC). A-type HCs (AHC) are axonless and extensively coupled via connexin (Cx)50 gap junctions. The B-type HC (BHC) is axon-bearing; the somatic dendrites form a second network coupled by gap junctions while the axon terminals (ATs) form a third independent network in the outer plexiform layer (OPL). The mouse retina has only one type of HC, which is morphologically similar to the B-type HC of the rabbit. Previous work suggested that mouse HCs express Cx57 (Hombach et al. [2004] Eur J Neurosci 19:2633-2640). Therefore, we cloned rabbit Cx57 and raised an antibody to determine the distribution of Cx57 gap junctions among rabbit HCs. Dye injection methods were used to obtain detailed fills for all three HC networks for analysis by confocal microscopy. We found that Cx57 was associated with the B-type AT plexus. Cx57 plaques were anticorrelated with the B-type somatic dendrites and the A-type HC network. Furthermore, there was no colocalization between Cx50 and Cx57. We conclude that in the rabbit retina, Cx57 is only found on BHC-AT processes. Thus, in species where there are two types of HC, different connexins are expressed. The absence of Cx57 labeling in the somatic dendrites of B-type HCs suggests the possibility of an additional unidentified HC connexin in the rabbit.     

Role of astroglial connexin30 in hippocampal gap junction coupling

The impact of connexin30 (Cx30) on interastrocytic gap junction coupling in the normal hippocampus is matter of debate; reporter gene analyses indicated a weak expression of Cx30 in the mouse hippocampus. In contrast, mice lacking connexin43 (Cx43) in astrocytes exhibited only 50% reduction in coupling. Complete uncoupling of hippocampal astrocytes in mice lacking both Cx30 and Cx43 suggested that Cx30 participates in interastrocytic gap junction coupling in the hippocampus. With comparative reporter gene assays, immunodetection, and cre/loxP-based reporter approaches we demonstrate that Cx30 is more abundant than previously thought. The specific role of Cx30 in interastrocytic coupling has never been investigated. Employing tracer coupling analyses in acute slices of Cx30 deficient mice here we show that Cx30 makes a substantial contribution to interastrocytic gap junctional communication in the mouse hippocampus.     

Analysis of connexin expression during mouse Schwann cell development identifies connexin29 as a novel marker for the transition of neural crest to precursor cells

Connexins are transmembrane proteins forming gap junction channels for direct intercellular and, for example in myelinating glia cells, intracellular communication. In mature myelin-forming Schwann cells, expression of multiple connexins, i.e. connexin (Cx) 43, Cx29, Cx32, and Cx46 (after nerve injury) has been detected. However, little is known about connexin protein expression during Schwann cell development. Here we use histochemical methods on wildtype and Cx29lacZ transgenic mice to investigate the developmental expression of connexins in the Schwann cell lineage. Our data demonstrate that in the mouse Cx43, Cx29, and Cx32 protein expression is activated in a developmental sequence that is clearly correlated with major developmental steps in the lineage. Only Cx43 was expressed from neural crest cells onwards. Cx29 protein expression was absent from neural crest cells but appeared as neural crest cells generated precursors (embryonic day 12) both in vivo and in vitro. This identifies Cx29 as a novel marker for cells of the defined Schwann cell lineage. The only exception to this were dorsal roots, where the expression of Cx29 was delayed four days relative to ventral roots and spinal nerves. Expression of Cx32 commenced postnatally, coinciding with the onset of myelination. Thus, the coordinated expression of connexin proteins in cells of the embryonic and postnatal Schwann cell lineage might point to a potential role in peripheral nerve development and maturation.     

Subcellular distribution of connexin45 in OFF bipolar cells of the mouse retina

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.