Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

Abstract

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructuraly-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocytc gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytcs, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in Ieptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.

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

“…We show that neuronal gap junctions in SCN were present in two major classes -a few large "conventional" gap junctions vs. more abundant miniature ("mini") gap junctions, most of which were smaller than 50 connexons. Our data also support the hypothesis of cell-type-specific expression of the other three connexins tested, with Cx32 confined to oligodendrocyte gap junctions; Cx30 and Cx43 in astrocyte gap junctions; and Cx43 (but none of the other connexins) in ependymocyte junctions, extending similar data from other regions of the CNS (Rash et al, 2001a(Rash et al, , 2001bNagy et al, 2003aNagy et al, ., 2003bNagy et al, , 2004Kamasawa et al, 2005Kamasawa et al, , 2006. The physiological data in the current study also demonstrate that the proportion of cell pairs that show evidence for electrotonic or tracer coupling or spike-to-spike synchrony is quite low (i.e., no spike synchrony in our study and only one pair of tracer-coupled neurons).…”

Characterization of astrocyte‐specific conditional knockouts

“…Finally, transgenic lines were crossed to a line carrying a floxed Connexin43 locus (Cx43) (Liao et al, 2001) to analyze recombination of an endogenous floxed gene. Since astrocytes are the main cell type expressing Cx43 in the brain (Nagy and Rash, 2000;Rash et al, 2001;Theis et al, 2003), the level of Cx43 remaining following Cre induction was measured by Western blotting and utilized as a measure of the extent of recombination.…”

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

“…With this alternative fixative, labeling for TH in the LC was robust, comparable to that observed with our standard fixative. However, immunolabeling for Cx26 and Cx32 was absent in the LC as well as most other brain regions (negative data not shown), except for very weak labeling for Cx26 observed in leptomeninges, which normally is intensely fluorescent for Cx26 following our standard fixation protocol (Rash et al, 2001a;Nagy et al, 2003b). …”

“…In addition to coupling between LC neurons, recent studies (Alvarez-Maubecin et al, 2000;Van Bockstaele et al, 2004) suggested widespread but weak gap junctional coupling between LC neurons and astrocytes in neonatal and adult rats, including strong dye-transfer from astrocytes to some neurons. Using silver-intensified immunogold-labeling electron microscopic immunocytochemistry, they described detection of connexin32 (Cx32) and connexin26 (Cx26) protein in a very high percentage of dendro-dendritic profiles and proposed even greater abundance of these two connexins at membrane appositions between dendrites and glial cells (primarily astrocytes).While clear evidence exists for coupling between neurons in developing LC, several challenging issues remain concerning documentation of gap junctional coupling in this nucleus: 1) No ultrastructural data exist for the presence of connexin-containing membrane appositions between neurons in the LC that meet accepted criteria for designation as gap junctions (Brightman and Reese, 1969;Sotelo and Korn, 1978;Rash et al, 1998); 2) Unlike the reported expression of abundant Cx26 and Cx32 in both neurons and astrocytes in LC, all other locations in the CNS examined by freeze-fracture replica immunolabeling (FRIL) electron microscopy have revealed cell-type-specific expression of connexin proteins, with Cx32 found only in gap junctions formed by oligodendrocytes (Rash et al, 2001a;Nagy et al, 2003a;Li et al, 2004a;Kamasawa et al, 2005), Cx26 only in gap junctions formed by pia mater and by subpopulations of astrocytes [(Nagy et al, 2001[(Nagy et al, ,2003bRash et al, 2001a); also see Mercier and Hatton (2001) and Altevogt et al, (2004)], and Cx36 only in neuronal gap junctions (Rash et al, ,2001bKamasawa et al, 2006) [also see Fukuda et al (2006)]; 3) Although proposals for coupling of neurons to astrocytes based on propagation of "calcium waves" (Nedergaard, 1994) were extended to dye coupling between astrocytes and neurons in LC slice preparations (Alvarez-Maubecin et al, 2000;Van Bockstaele et al, 2004), other nongap-junctional mechanisms appear to account for initiation and propagation of calcium waves (Charles, 1994(Charles, ,1996Parpura et al, 1994;Hassinger et al, 1995); and 4) ...…”

“…While clear evidence exists for coupling between neurons in developing LC, several challenging issues remain concerning documentation of gap junctional coupling in this nucleus: 1) No ultrastructural data exist for the presence of connexin-containing membrane appositions between neurons in the LC that meet accepted criteria for designation as gap junctions (Brightman and Reese, 1969;Sotelo and Korn, 1978;Rash et al, 1998); 2) Unlike the reported expression of abundant Cx26 and Cx32 in both neurons and astrocytes in LC, all other locations in the CNS examined by freeze-fracture replica immunolabeling (FRIL) electron microscopy have revealed cell-type-specific expression of connexin proteins, with Cx32 found only in gap junctions formed by oligodendrocytes (Rash et al, 2001a;Nagy et al, 2003a;Li et al, 2004a;Kamasawa et al, 2005), Cx26 only in gap junctions formed by pia mater and by subpopulations of astrocytes [(Nagy et al, 2001[(Nagy et al, ,2003bRash et al, 2001a); also see Mercier and Hatton (2001) and Altevogt et al, (2004)], and Cx36 only in neuronal gap junctions (Rash et al, ,2001bKamasawa et al, 2006) [also see Fukuda et al (2006)]; 3) Although proposals for coupling of neurons to astrocytes based on propagation of "calcium waves" (Nedergaard, 1994) were extended to dye coupling between astrocytes and neurons in LC slice preparations (Alvarez-Maubecin et al, 2000;Van Bockstaele et al, 2004), other nongap-junctional mechanisms appear to account for initiation and propagation of calcium waves (Charles, 1994(Charles, ,1996Parpura et al, 1994;Hassinger et al, 1995); and 4) Despite extensive studies of numerous brain regions (Brightman and Reese, 1969;Sotelo and Korn, 1978;Kosaka, 1985;Rash et al, 1998Rash et al, ,2001bKosaka and Kosaka, 2005), no ultrastructural evidence has been found for the existence of neuron-to-astrocyte gap junctions. These divergent reports raise questions about the reported uniqueness of the LC, parti...…”