Oligodendrocytes in vivo form heterologous gap junctions with astrocytes. These oligodendrocyte/ astrocyte (A/O) gap junctions contain multiple connexins (Cx), including Cx26, Cx30, and Cx43 on the astrocyte side, and Cx32, Cx29, and Cx47 on the oligodendrocyte side. We investigated connexin associations at A/O gap junctions on oligodendrocytes in normal and Cx32 knockout (KO) mice. Immunoblotting and immunolabeling by several different antibodies indicated the presence of Cx32 in liver and brain of normal mice, but the absence of Cx32 in liver and brain of Cx32 KO mice, confirming the specificity and efficacy of the antibodies, as well as allowing the demonstration of Cx32 expression by oligodendrocytes. Oligodendrocytes throughout brain were decorated with numerous Cx30-positive puncta, which also were immunolabeled for both Cx32 and Cx43. In Cx32 KO mice, astrocytic Cx30 association with oligodendrocyte somata was nearly absent, Cx26 was partially reduced, and Cx43 was present in abundance. In normal and Cx32 KO mice, oligodendrocyte Cx29 was sparsely distributed, whereas Cx47-positive puncta were densely localized on oligodendrocyte somata. These results demonstrate that astrocyte Cx30 and oligodendrocyte Cx47 are widely present at A/O gap junctions. Immunolabeling patterns for these six connexins in Cx32 KO brain have implications for deciphering the organization of heterotypic connexin coupling partners at A/O junctions. The persistence and abundance of Cx43 and Cx47 at these junctions after Cx32 deletion, together with the paucity of Cx29 normally present at these junctions, suggests Cx43/Cx47 coupling at A/O junctions. Reductions in Cx30 and Cx26 after Cx32 deletion suggest that these astrocytic connexins likely form junctions with Cx32 and that their incorporation into A/O gap junctions is dependent on the presence of oligodendrocytic Cx32.

The cellular localization, relation to other glial connexins (Cx30, Cx32, and Cx43), and developmental expression of Cx29 were investigated in the mouse central nervous system (CNS) with an anti-Cx29 antibody. Cx29 was enriched in subcellular fractions of myelin, and immunofluorescence for Cx29 was localized to oligodendrocytes and myelinated fibers throughout the brain and spinal cord. Oligodendrocyte somata displayed minute Cx29-immunopositive puncta around their periphery and intracellularly. In developing brain, Cx29 levels increased during the first few postnatal weeks and were highest in the adult brain. Immunofluorescence labeling for Cx29 in oligodendrocyte somata was intense at young ages and was dramatically shifted in localization primarily to myelinated fibers in mature CNS. Labeling for Cx32 also was localized to oligodendrocyte somata and myelin and absent in Cx32 knockout mice. Cx29 and Cx32 were minimally colocalized on oligodendrocytes somata and partly colocalized along myelinated fibers. At gap junctions on oligodendrocyte somata, Cx43/Cx32 and Cx30/Cx32 were strongly associated, but there was minimal association of Cx29 and Cx43. Cx32 was very sparsely associated with astrocytic connexins along myelinated fibers. With Cx26, Cx30, and Cx43 expressed in astrocytes and Cx29, Cx32, and Cx47 expressed in oligodendrocytes, the number of connexins localized to gap junctions of glial cells is increased to six. The results suggested that Cx29 in mature CNS contributes minimally to gap junctional intercellular communication in oligodendrocyte cell bodies but rather is targeted to myelin, where it, with Cx32, may contribute to connexin-mediated communication between adjacent layers of uncompacted myelin. Indexing termsbrain; glia; antibodies; intercellular communication; confocal microscopy; Schmidt-Lanterman incisures Cells in the central nervous system (CNS) communicate directly via gap junctions at close appositions between plasma membranes, thereby providing channels for movement of ions and small molecules from cell to cell. It appears that virtually every neural cell type in the brain has the capacity for gap junctional intercellular communication (GJIC), and evidence for several levels of complexity in this process is beginning to emerge. Each cell type exhibits Mugnaini, 1986;Rash et al., 1997Rash et al., , 2000 Nagy et al., 2003a). In addition, vast numbers of gap junctions have been reported to connect not only astrocyte processes of different cells but also those of the same cell, resulting in extensive autocellular coupling formed by autologous or reflexive gap junctions (Wolburg and Rohlmann, 1995;Nagy and Rash, 2000). At least 10 members of the family of connexin proteins that form gap junctions in mammalian tissues are expressed in the CNS, and individual cells and gap junctions often contain two or more different connexins (Nagy et al., 1999;Severs, 1999;Nagy and Dermietzel, 2000;Nagy and Rash, 2000). It is now established that A/A junctions contain Cx26, Cx30, and Cx43, and th...

Gap junctions between glial cells in mammalian CNS are known to contain several connexins (Cx), including Cx26, Cx30 and Cx43 at astrocyte-to-astrocyte junctions, and Cx29 and Cx32 on the oligodendrocyte side of astrocyte-to-oligodendrocyte junctions. Recent reports indicating that oligodendrocytes also express Cx47 prompted the present studies of Cx47 localization and relationships to other glial connexins in mouse CNS. In view of the increasing number of connexins reported to interact directly with the scaffolding protein zonula occludens-1 (ZO-1), we investigated ZO-1 expression and Cx47/ZO-1 interaction capabilities in brain, spinal cord and Cx47-transfected HeLa cells. From counts of over 9000 oligodendrocytes labeled by immunofluorescence in various brain regions, virtually all of these cells were found to express Cx29, Cx32 and Cx47. Oligodendrocyte somata displayed robust Cx47-immunopositive puncta that were co-localized with punctate labeling for Cx32 and Cx43. By freeze-fracture replica immunogold labeling, Cx47 was abundant on the oligodendrocyte-side of oligodendrocyte/astrocyte gap junctions. By immunofluorescence, labeling for Cx47 along myelinated fibers was sparse in most brain regions, whereas Cx29 and Cx32 were previously found to be concentrated along these fibers. By immunogold labeling, Cx47 was found in numerous small gap junctions linking myelin to astrocytes, but not within deeper layers of myelin. Brain subcellular fractionation revealed a lack of Cx47 enrichment in myelin fractions, which nevertheless contained an enrichment of Cx32 and Cx29. Oligodendrocytes were immunopositive for ZO-1, and displayed almost total Cx47/ZO-1 colocalization. ZO-1 was found to co-immunoprecipitate with Cx47, and pull-down assays indicated binding of Cx47 to the second PDZ domain of ZO-1.Our results indicate widespread expression of Cx47 by oligodendrocytes, but with a distribution pattern in relative levels inverse to the abundance of Cx29 in myelin and paucity of Cx29 in oligodendrocyte somata. Further, our findings suggest a scaffolding and/or regulatory role of ZO-1 at the oligodendrocyte side of astrocyte-to-oligodendrocyte gap junctions. Keywords connexin47; connexin32; connexin43; gap junctions; PDZ domains; glia Gap junctions are localized at specialized cell-to-cell contacts of plasma membranes in which connexin (Cx) proteins form bidirectional intercellular channels that allow passage of ions and *Corresponding author. Tel: +1-204-789-3767; fax: +1-204-789-3934. E-mail address: nagyji@ms.umanitoba.ca (J. I. Nagy).. 1 Present address: Department of Pathology, Weifang Medical College, PR China. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2007 March 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsmall molecules between cells (Goodenough et al., 1996;Kumar and Gilula, 1996;Willecke et al., 2002). In the CNS, astrocytes are extensively coupled by gap junctions (astrocyte-toastrocyte gap junctions [A/A junctions]) that, base...