Cell-Specific Expression of Connexins and Evidence of Restricted Gap Junctional Coupling between Glial Cells and between Neurons

Abstract

The transmembrane connexin proteins of gap junctions link extracellularly to form channels for cell-to-cell exchange of ions and small molecules. Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. There is consensus that gap junctions link neurons to neurons and astrocytes to oligodendrocytes, ependymocytes, and other astrocytes. However, unresolved are the existence and degree to which gap junctions occur between oligodendrocytes, between oligodendrocytes and neurons, and between astrocytes and neurons. Using light microscopic immunocytochemistry and freezefracture replica immunogold labeling of adult rat CNS, we investigated whether four of the best-characterized CNS connexins are each present in one or more cell types, whether oligodendrocytes also share gap junctions with other oligodendrocytes or with neurons, and whether astrocytes share gap junctions with neurons. Connexin32 (Cx32) was found only in gap junctions of oligodendrocyte plasma membranes, Cx30 and Cx43 were found only in astrocyte membranes, and Cx36 was only in neurons. Oligodendrocytes shared intercellular gap junctions only with astrocytes, with each oligodendrocyte isolated from other oligodendrocytes except via astrocyte intermediaries. Finally, neurons shared gap junctions only with other neurons and not with glial cells. Thus, the different cell types of the CNS express different connexins, which define separate pathways for neuronal versus glial gap junctional communication.Key words: astrocyte; connexin; connexon; gap junction; neuron; oligodendrocyte Astrocytes, ependymocytes, and oligodendrocytes, the macroglial cells of the adult CNS, are richly invested with gap junctions. Astrocytes, in particular, share gap junctions with all three macroglia, thereby creating a functional panglial syncytium (Mugnaini, 1986;Rash et al., 1997). In contrast, gap junctions involving neurons were reported to be rare (Brightman and Reese, 1969;Sotelo and Korn, 1978), with glial gap junctions greatly outnumbering neuronal gap junctions and neuron-to-glial junctions not detected (Wolff et al., 1998;Rash et al., 2000). The initial "restricted coupling partner" hypothesis that oligodendrocytes share intercellular gap junctions only with astrocytes and that neurons share gap junctions only with neurons (Massa and Mugnaini, 1982;Mugnaini, 1986;Rash et al., 1997) was supported by immunocytochemical data showing that neurons and glia express different connexins Condorelli et al., 1998;Nagy et al., 1999;Nagy and Rash, 2000;Rash et al., 2000). A quite different "shared-connexins/mixed-coupling" hypothesis, which arose from in situ hybridization, imaging of calcium waves, electrical and dye coupling, and immunocytochemistry, suggests that neurons and glia coexpress connexin32 (Cx32) and...

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

“…7G), the extracellular space was narrowed to 3 nm. When viewed from oligodendrocyte toward the underlying astrocyte, the gap junction oligodendrocyte E-faces and astrocyte Pfaces were labeled for Cx43, as previously demonstrated in other CNS regions (Rash et al, 2001b) …”

“…Unmapped are >50 Cx43-labeled ependymocyte gap junctions, >200 Cx43-labeled astrocyte gap junctions, and more than a dozen Cx43/Cx30 double-labeled astrocyte gap junctions. Many additional examples of labeled astrocyte gap junctions were found but not photographed because they were essentially confirmatory and did not constitute exceptions to the labeling patterns reported here and elsewhere (Rash et al, 2001b). …”

“…Goat anti-rabbit IgG conjugated to 10-nm and 20-nm gold beads was from Chemicon (Temecula, CA; no longer available), goat anti-rabbit IgG and goat anti-mouse IgG conjugated to the 6-nm, 12-nm, and 18-nm gold beads were from Jackson ImmunoResearch, and goat antirabbit IgG conjugated to 20-nm and 30-nm gold was from BBInternational, Ltd. (Ted Pella, Inc.). Controls included omission of primary antibodies to a target connexin, as well as doublelabeling some replicas for one connexin plus one non-connexin membrane protein [e.g., NMDA receptors (Rash and Yasumura, 1999;Rash et al, 2001bRash et al, , 2005 or aquaporin4 (AQP4) (Rash et al, 1998b;Furman et al, 2003)]. Labeled replicas were rinsed in distilled water, air dried at 60°C, and a stabilizing 20-nm coat of carbon was applied to the labeled side of the replica.…”

Modulation of connexin expression and gap junction communication in astrocytes by the gram‐positive bacterium S. aureus

“…Blots were probed using polyclonal rabbit anti-mouse Cx26, Cx30, and Cx43 antibodies (all from Zymed, San Francisco, CA; Cat nos. 51-2800, 71-2200, and 71-0700, respectively) (Penes et al, 2005;Rash et al, 2001) followed by a donkey anti-rabbit IgG-HRP conjugate (Jackson Immunoresearch, West Grove, PA). Blots were developed using the ChemiGlow West substrate (Alpha Innotech, San Leandro, CA) and visualized by exposure to X-ray film.…”

Loss-of-function GJA12/Connexin47 mutations cause Pelizaeus–Merzbacher-like disease

“…Astrocyte/astrocyte (A/ A) coupling is likely mediated by Cx43/Cx43 and Cx30/Cx30 homotypic channels, and astrocyte/oligodendrocyte (A/O) coupling is most likely mediated by Cx43/Cx47 and Cx30/ Cx32 heterotypic channels, with an uncertain contribution of Cx26/Cx32 channels (Rash et al 2001;Nagy et al 2003;Altevogt and Paul 2004;Li et al 2004). Cx32/Cx32 homotypic channels appear to form gap junctions between layers of CNS myelin sheaths (Kamasawa et al 2005), as previously described in PNS myelin sheaths (Bergoffen et al 1993;Scherer et al 1995;Balice-Gordon et al 1998;Meier et al 2004).…”