MAB3045 Sigma-AldrichAnti-Connexin 35/36 Antibody, clone 8F6.2

Retinal degeneration (RD) encompasses a family of diseases that lead to photoreceptor death and visual impairment. Visual decline due to photoreceptor cell loss is further compromised by emerging spontaneous hyperactivity in inner retinal cells. This aberrant activity acts as a barrier to signals from the remaining photoreceptors, hindering therapeutic strategies to restore light sensitivity in RD. Gap junctions, particularly those expressed in AII amacrine cells, have been shown to be integral to the generation of aberrant activity. It is unclear whether gap junction expression and coupling are altered in RD. To test this, we evaluated the expression and phosphorylation state of connexin36 (Cx36), the gap junction subunit predominantly expressed in AII amacrine cells, in two mouse models of RD, rd10 (slow degeneration) and rd1 (fast degeneration). Using Ser293-P antibody, which recognizes a phosphorylated form of connexin36, we found that phosphorylation of connexin36 in both slow and fast RD models was significantly greater than in wildtype controls. This elevated phosphorylation may underlie the increased gap junction coupling of AII amacrine cells exhibited by RD retina.

The Down syndrome cell adhesion molecule (Dscam) gene is required for normal dendrite arborization and lamination in the mouse retina. In this study, we characterized the developmental localization of the DSCAM protein to better understand the postnatal stages of retinal development during which laminar disorganization occur in the absence of the protein.Immunohistochemistry and colocalization analysis software were used to assay the localization of the DSCAM protein during development of the retina.We found that DSCAM was initially localized diffusely throughout mouse retinal neurites but then adopted a punctate distribution. DSCAM colocalized with catenins in the adult retina but was not detected at the active zone of chemical synapses, electrical synapses, and tight junctions. Further analysis identified a wave of colocalization between DSCAM and numerous synaptic and junction proteins coinciding with synaptogenesis between bipolar and retinal ganglion cells.Research presented in this study expands our understanding of DSCAM function by characterizing its location during the development of the retina and identifies temporally regulated localization patterns as an important consideration in understanding the function of adhesion molecules in neural development.

"Dye-coupling", whole-mount immunohistochemistry for gap junction channel protein connexin 35 (Cx35), and freeze-fracture replica immunogold labeling (FRIL) reveal an abundance of electrical synapses/gap junctions at glutamatergic mixed synapses in the 14th spinal segment that innervates the adult male gonopodium of Western Mosquitofish, Gambusia affinis (Mosquitofish). To study gap junctions' role in fast motor behavior, we used a minimally-invasive neural-tract-tracing technique to introduce gap junction-permeant or -impermeant dyes into deep muscles controlling the gonopodium of the adult male Mosquitofish, a teleost fish that rapidly transfers (complete in less than 20 mS) spermatozeugmata into the female reproductive tract. Dye-coupling in the 14th spinal segment controlling the gonopodium reveals coupling between motor neurons and a commissural primary ascending interneuron (CoPA IN) and shows that the 14th segment has an extensive and elaborate dendritic arbor and more gap junctions than do other segments. Whole-mount immunohistochemistry for Cx35 results confirm dye-coupling and show it occurs via gap junctions. Finally, FRIL shows that gap junctions are at mixed synapses and reveals that greater than 50 of the 62 gap junctions at mixed synapses are in the 14th spinal segment. Our results support and extend studies showing gap junctions at mixed synapses in spinal cord segments involved in control of genital reflexes in rodents, and they suggest a link between mixed synapses and fast motor behavior. The findings provide a basis for studies of specific roles of spinal neurons in the generation/regulation of sex-specific behavior and for studies of gap junctions' role in regulating fast motor behavior. Finally, the CoPA IN provides a novel candidate neuron for future studies of gap junctions and neural control of fast motor behaviors.

In contrast to the knowledge of chemical synapses, little is known regarding the properties of gap junction-mediated electrical synapses in developing zebrafish, which provide a valuable model to study neural function at the systems level. Identifiable "mixed" (electrical and chemical) auditory synaptic contacts known as "club endings" on Mauthner cells (2 large reticulospinal neurons involved in tail-flip escape responses) allow exploration of electrical transmission in fish. Here, we show that paralleling the development of auditory responses, electrical synapses at these contacts become anatomically identifiable at day 3 postfertilization, reaching a number of ∼6 between days 4 and 9. Furthermore, each terminal contains ∼18 gap junctions, representing between 2,000 and 3,000 connexon channels formed by the teleost homologs of mammalian connexin 36. Electrophysiological recordings revealed that gap junctions at each of these contacts are functional and that synaptic transmission has properties that are comparable with those of adult fish. Thus a surprisingly small number of mixed synapses are responsible for the acquisition of auditory responses by the Mauthner cells, and these are likely sufficient to support escape behaviors at early developmental stages.

Gap junctions are membrane specializations that allow the passage of ions and small molecules from one cell to another. In vertebrates, connexins are the protein subunits that assemble to form gap junctional plaques. Connexin-35 (Cx35) is the fish ortholog of mammalian Cx36, which is enriched in the retina and the brain and has been shown to form neuronal gap junctions. As a first step toward understanding the role of neuronal gap junctions in central nervous system (CNS) development, we describe here the distribution of Cx35 in the CNS during zebrafish development. Cx35 expression is first seen at 1 day post fertilization (dpf) along cell boundaries throughout the nervous system. At 2 dpf, Cx35 immunoreactivity appears in commissures and fiber tracts throughout the CNS and along the edges of the tectal neuropil. In the rhombencephalon, the Mauthner neurons and fiber tracts show strong Cx35 immunoreactivity. As the larva develops, the commissures and fiber tracts continue to be immunoreactive for Cx35. In addition, the area of the tectal neuropil stained increases vastly and tectal commissures are visible. Furthermore, at 4-5 dpf, Cx35 is seen in the habenulae, cerebellum and in radial glia lining the rhombencephalic ventricle. This pattern of Cx35 immunoreactivity is stable at least until 15 dpf. To test whether the Cx35 immunoreactivity seen corresponds to functional gap junctional coupling, we documented the number of dye-coupled neurons in the hindbrain. We found several dye-coupled neurons within the reticulospinal network indicating functional gap junctional connectivity in the developing zebrafish brain.

Even though it has been known for some time that in many mammalian brain areas interneurons are electrically coupled, a quantitative description of the network electrical connectivity and its impact on cellular passive properties is still lacking. Approaches used so far to solve this problem are limited because they do not readily distinguish junctions among direct neighbors from indirect junctions involving intermediary, multiply connected cells. In the cerebellar cortex, anatomical and functional evidence indicates electrical coupling between molecular layer interneurons (basket and stellate cells). An analysis of the capacitive currents obtained under voltage clamp in molecular layer interneurons of juvenile rats or mice reveals an exponential component with a time constant of ~20 ms, which represents capacitive loading of neighboring cells through gap junctions. These results, taken together with dual cell recording of electrical synapses, have led us to estimate the number of direct neighbors to be ~4 for rat basket cells and ~1 for rat stellate cells. The weighted number of neighbors (number of neighbors, both direct and indirect, weighted with the percentage of voltage deflection at steady state) was 1.69 in basket cells and 0.23 in stellate cells. The last numbers indicate the spread of potential changes in the network and serve to estimate the contribution of gap junctions to cellular input conductance. In conclusion the present work offers effective tools to analyze the connectivity of electrically connected interneuron networks, and it indicates that in juvenile rodents, electrical communication is stronger among basket cells than among stellate cells.

Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibit adenylyl cyclase (AC) and reduce PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to coregulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately downregulated expression of D4R and AC type 1 mRNA. We conclude that adenosine and dopamine coregulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function.

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of single-gene administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo. Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.

Photoreceptors are coupled via gap junctions in many mammalian species. Cone-to-cone coupling is thought to improve sensitivity and signal-to-noise ratio, while rod-to-cone coupling provides an alternative rod pathway active under twilight or mesopic conditions (Smith et al., 1986; DeVries et al., 2002; Hornstein et al., 2005). Gap junctions are composed of connexins, and connexin36 (Cx36), the dominant neuronal connexin, is expressed in the outer plexiform layer. Primate (Macaca mulatta) cone pedicles, labeled with an antibody against cone arrestin (7G6) were connected by a network of fine processes called telodendria and, in double-labeled material, Cx36 plaques were located precisely at telodendrial contacts between cones, suggesting strongly they are Cx36 gap junctions. Each red/green cone made nonselective connections with neighboring red/green cones. In contrast, blue cone pedicles were smaller with relatively few short telodendria and they made only rare or equivocal Cx36 contacts with adjacent cones. There were also many smaller Cx36 plaques around the periphery of every cone pedicle and along a series of very fine telodendria that were too short to reach adjacent members of the cone pedicle mosaic. These small Cx36 plaques were closely aligned with nearly every rod spherule and may identify sites of rod-to-cone coupling, even though the identity of the rod connexin has not been established. We conclude that the matrix of cone telodendria is the substrate for photoreceptor coupling. Red/green cones were coupled indiscriminately but blue cones were rarely connected with other cones. All cone types, including blue cones, made gap junctions with surrounding rod spherules.

Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.