Connexin32 in oligodendrocytes and association with myelinated fibers in mouse and rat brain

Abstract

The distribution and cellular localization of connexin32 (Cx32) in the brain and spinal cord of the mouse and rat was investigated by light microscope (LM) and electron microscope (EM) immunohistochemistry by using several different antibodies against Cx32. By double immunofluorescence staining for Cx32 and either the oligodendrocyte markers cyclic nucleotide phosphodiesterase (CNPase) or Rip, Cx32 was consistently found in oligodendrocyte cell bodies and proximal processes. Cx32 immunoreactivity was also clearly visualized along CNPase- and Rip-positive myelinated fibers. Both immunopositive cells and fibers were heterogeneously distributed and were often more intensely labeled when dispersed in or associated with regions of gray matter than when concentrated in major white matter tracts. Labeling of myelin sheaths along fibers was restricted to subpopulations of myelinated axons. In the cerebellar cortex, for example, it was selectively localized to sheaths around Purkinje cell axons. Punctate staining, distinct from that corresponding to cells or fibers, was evident in the olfactory bulb and hippocampus. By EM, oligodendrocytes exhibited cytoplasmic labeling associated with rough endoplasmic reticulum and Golgi apparatus. Their processes were intermittently stained, most intensely when surrounding myelinated fibers and occasionally in paranodal loops. Cx32-immunoreactive gap junctions with symmetric labeling (staining on both junctional membranes) were observed between oligodendrocytic somata and processes as well as between presumptive oligodendrocytic processes. Unidentifiable elements forming asymmetrically labeled gap junctions (staining only one side of junctional membranes) were less frequently encountered. Western blot analysis confirmed anti-Cx32 antibody detection of Cx32 in whole brain homogenates and an enrichment of the protein in isolated myelin fractions. These results are consistent with earlier ultrastructural studies showing the occurrence of inter-oligodendrocytic gap junctions, but indicate that these may be more prevalent than previously thought. Furthermore, the results suggest a specialized role of gap junctions composed of Cx32 along myelinated fibers belonging to subpopulations of neurons.

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

“…Early on, it was proposed that neurons share gap junctions only with neurons, and that glia share intercellular gap junctions only with glia (Brightman and Reese, 1969;Mugnaini, 1986). This "restricted coupling partner" hypothesis gained support based on TEM and FRIL immunocytochemical data showing that neuronal, astrocytic and oligodendrocytic gap junctions each express different complements of connexins Nagy and Dermietzel, 2000;Rash et al, 2000Rash et al, , 2001bKamasawa et al, 2005;Fukuda et al, 2006), with Cx43, Cx30 and (in limited areas) Cx26 in astrocytes and leptomeninges (Li et al, 1997;Nagy et al, 1999Nagy et al, ,2001Rash et al, 2001b); Cx29, Cx32 and Cx47 in oligodendrocytes (Rash et al, 2001a(Rash et al, ,2001bNagy et al, 2004;Kamasawa et al, 2005); and Cx36 exclusively in neurons (Rash et al, 2001b;Fukuda et al, 2006), extending data from immunofluorescence analysis and thin-section immunocytochemistry (Yamamoto et al, 1990a(Yamamoto et al, ,1990bScherer et al, 1995;Mercier and Hatton, 2001;Altevogt et al, 2002;Kleopas et al, 2004;Rash et al, 2005).…”

“…Third, in contrast to purported abundance Cx32 in astrocytes (Alvarez-Maubecin et al, 2000), Cx32 was not found by FRIL in astrocytes at any age, paralleling observations in all other brain regions (Dermietzel et al, 1989;Nelles et al, 1996;Kunzelmann et al, 1997;Dermietzel, 1998;Rash et al, 2001b;Nagy et al, 2003b;Kamasawa et al, 2005). Fourth, consistent with our reports that detection of Cx26 and Cx32 is particularly sensitive to tissue fixation conditions (Li et al, 1997;Nagy et al, 2001Nagy et al, , 2003a, we found that the stronger 3.75% acrolein/2% formaldehyde fixative of AlvarezMaubecin (2000) applied under our conditions of immunohistochemistry abolished all labeling of Cx32 and Cx26 in brain parenchyma and greatly reduced Cx26 labeling in leptomeninges. These data suggest that the reported detection of hyper-abundant Cx32 in neuronal and astrocyte plasma membranes may have resulted from creation of extraneous silver product, possibly due to overdevelopment of the silver intensification reaction in the absence of positive controls to calibrate time of silver development.…”

Connexin47, connexin29 and connexin32 co-expression in oligodendrocytes and cx47 association with zonula occludens-1 (zo-1) in mouse brain

“…A number of other studies have demonstrated Cx29 and Cx32 along myelinated fibers, with a range of immunolabelling densities associated with varying proportions of fibers (Spray and Dermietzel, 1995;Li et al, 1997;Pastor et al, 1998;Scherer et al, 1995;Altevogt et al, 2002;Nagy et al, 2003a). In the most extreme case, Cx29 and Cx32 were reported to be localized to mutually exclusive subpopulations of fibers, which at face value appeared to be consistent with the reported expression of these Cxs by separate populations of oligodendrocytes (Altevogt et al, 2002).…”

Human oligodendrocytes express Cx31.3: Function and interactions with Cx32 mutants

“…For Cx32 staining, a rabbit antiserum against the intracellular loop (Chemicon; diluted 1:500), and a mouse monoclonal antibody (7C6.C7) against the carboxy-terminus (diluted 1:2) (Li et al 1997) were used. The rabbit antisera against the carboxy-terminus of Cx31.3 (1:800) were used to label cells expressing Cx31.3.…”