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. 2001 Mar 15;21(6):1983-2000.
doi: 10.1523/JNEUROSCI.21-06-01983.2001.

Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons

J E Rash  1 , T Yasumura, F E Dudek, J I Nagy
Affiliations

Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons

J E Rash et al. J Neurosci. .

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 freeze-fracture 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.

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Figures

Fig. 1.
Fig. 1.
Western blots showing connexin detection in the CNS with the various anti-connexin antibodies used for LM immunofluorescence and FRIL. A, Blots of whole-brain homogenate probed with polyclonal (lane 1) and monoclonal (lane 2) anti-Cx30 antibodies. The top two intense bands detected by the monoclonal antibody may correspond to Cx30 dimer and trimer forms. B, Blots of whole-brain homogenate probed with polyclonal (lane 1) and monoclonal (lane 2) anti-Cx43 antibodies.C, Blots of liver and spinal cord homogenates probed with anti-Cx32 antibodies. Lanes 1, 2, Monoclonal 7C7;lane 3, Zymed monoclonal 2C2; lane 4, Sigma polyclonal; lane 5, Chemicon polyclonal;lane 6, Zymed polyclonal. Each of the antibodies detects the monomer form of Cx32 and to varying degrees its dimer form. The Sigma and Chemicon antibodies detect a lower molecular weight band of uncertain identity. Numbers on the left of each blot correspond to molecular weight markers.
Fig. 2.
Fig. 2.
Photomicrographs showing patterns of immunolabeling obtained with various anti-connexin antibodies used in double-immunofluorescence and FRIL studies. A, Low magnification of immunoperoxidase labeling (arrows) with a monoclonal anti-Cx30 antibody. The section shows dense punctate labeling in the globus pallidus (GP) and weaker labeling in the striatum (St). B, Cerebral cortex showing a high concentration of punctate immunofluorescence with anti-Cx43 antibody 18A. Dark ovals represent unstained neuronal cell bodies. C, Cx32 labeling of myelinated fibers (arrows) as well as oligodendrocyte cell bodies (arrowheads; cells barely discernable at this magnification) using antibody 7C7. D, E, Higher magnifications showing Cx32-positive puncta with antibody 7C7 along the surface of oligodendrocytes in the cerebral cortex (D; arrows) and ventral lateral nucleus of the thalamus (E; arrows). F, Through-focus confocal micrograph of Cx32-immunopositive puncta distributed on an oligodendrocyte soma (arrows) and its processes (arrowheads). G, Double immunofluorescence of the oligodendrocyte marker CNPase (G1, green) and Cx32 (G2,red) in the same field of cerebral cortex. CNPase-positive cells are also immunopositive for Cx32 as seen by theoverlay of images (G3,yellow). H, Higher magnification double-immunofluorescence confocal micrographs showing a CNPase-positive oligodendrocyte in cerebral cortex (H1) decorated with numerous Cx32-immunopositive puncta (red puncta in H2 and red andyellow puncta in the overlay of images inH3). Scale bars: A, 200 μm; B, C, 50 μm; D, E, G, 20 μm; F, H, 5 μm.
Fig. 3.
Fig. 3.
Confocal microscope images showing double-immunofluorescence labeling for connexins in various brain regions. A1–A3, Labeling for Cx30 (A1) and Cx43 (A2) in the subthalamic nucleus with theoverlay (A3) showing colocalization inyellow. B–D, Labeling for Cx43 (green in B1, C1, D1) and Cx32 (red in B2, C2, D2) with theoverlay showing colocalization (yellow in B3, C3, D3) of immunofluorescent puncta surrounding an oligodendrocyte (OL,arrow) in the cerebral cortex (B), the ventral lateral thalamic nucleus (C), and the lateral hypothalamus (D). Scale bars, 5 μm.
Fig. 4.
Fig. 4.
Diagrammatic representation of FRIL showing relationships between visualized freeze–fracture faces and the location of immunogold-labeled connexins. A, A gap junction between two cells is shown where the fracture plane (blue dashed line) skips from the membrane bilayer of one cell to that of the other cell but always separates apposed connexons at the point of contact in the extracellular space. B, C, Where the membrane of the upper cell is fractured, the external leaflet (E-face) remains attached to the tissue fragment that contains the lower cell. The E-face pits delineate the sites from which the connexons were removed. Curved arrow indicates separation and removal of upper fragment. Pt arrow indicates direction of platinum shadowing. D–F, Where the fracture plane splits the membrane of the lower cell, the external leaflet is removed, exposing connexons as intramembrane particles in the protoplasmic leaflet (P-face). In all cases, regardless of whether the E-face of the upper cell or the P-face of the lower cell is replicated, only the connexons of the lower cell remain for potential labeling by FRIL.
Fig. 5.
Fig. 5.
Stereoscopic images of Cx32 labeling of gap junctions in oligodendrocyte plasma membranes in the cerebellum, with explanatory diagrams. A, Oligodendrocyte cell body with a broad expanse of plasma membrane P-face (P) enclosing the cross-fractured cytoplasm (asterisk).Arrows point to two Cx32-labeled gap junctions in the oligodendrocyte P-face (white arrows) and one unlabeled gap junction in the nearby astrocyte P-face (black arrow). An additional gap junction with ∼280 connexons was labeled by 12 gold beads (boxed area; enlarged in B). On theright of each stereo pair is an interpretivedrawing, in which each cell type is delineated by different shading and a stylized complement of IMPs or pits in each membrane fracture face is shown. B, Cx32-labeled gap junction linking a confirmed astrocyte finger that was fractured from its P-face (As P), through its cytoplasm (asterisk), to its E-face (As E). Air drying of the labeled replicas resulted in collapse of the labels to the underside of the replica. Ol P, oligodendrocyte P-face. C, Cx32-labeled gap junction consisting of 1280 connexons that was labeled by 30 gold beads (black dots). A nearby gap junction consisting of <40 IMPs was unlabeled. The reciprocal patch (arrow) and paucity of IMPs are characteristic of oligodendrocyte P-faces. D, Cx32-labeled gap junction (enlarged in inset) in the P-face of the outer layer of myelin, directly adjacent to the outer tongue (OT). The outer tongue of myelin terminates at a tight junction consisting of several rows of P-face furrows (arrow) and IMP ridges. Three gold beads were bound to the oligodendrocyte side of the gap junction. In all FRIL images (in this and subsequent figures), scale bars are 0.1 μm unless otherwise indicated.
Fig. 6.
Fig. 6.
Stereoscopic images of astrocyte gap junctions in the suprachiasmatic nucleus after labeling for Cx43 and AQP4 (A), in the supraoptic nucleus after labeling for Cx30 (B), in the supraoptic nucleus after double labeling for Cx43 and Cx30 (C), and in the paraventricular nucleus after triple labeling for Cx30, Cx32, and Cx43 (D). A, Two astrocyte-to-astrocyte gap junctions labeled for Cx43 (20 nm beads;large arrows) beneath their E-faces (E) and P-faces (P). Square arrays in P-faces were labeled for AQP4 (10 nm gold beads; small arrow and inset). B, Astrocyte-to-astrocyte gap junction labeled for Cx30 (10 nm gold).C, A gap junction linking two astrocyte processes after double labeling for Cx30 (10 nm gold beads) and Cx43 (20 nm gold beads). Astrocytes were positively identified by the presence of AQP4 square arrays in both P-faces (white arrows) and E-faces (black arrows), as well as by the high density of IMPs on both P- and E-faces. D, Two astrocyte gap junctions triple-labeled for Cx30 (20 nm gold), Cx43 (30–40 nm gold), and Cx32 (10 nm gold; none present). Cx32 was not detected in astrocyte-to-astrocyte gap junctions but was detected in astrocyte-to-oligodendrocyte gap junctions (Fig. 5).
Fig. 7.
Fig. 7.
Stereoscopic images of paired oligodendrocytes linked by tight junctions in the suprachiasmatic nucleus (A–E) after labeling for Cx43 and AQP4 and in the inferior olive (F) after double labeling for Cx43 (10 nm gold) and Cx36 (20 nm gold; none present in any oligodendrocyte gap junction). A, Low-magnification image of the large expanse of oligodendrocyte concave plasma membrane E-face contacting the cell body of a second oligodendrocyte, which has cross-fractured cytoplasm (asterisk).Boxes delineate areas of tight junctions (B) and 2 of the 12 Cx43-labeled gap junctions seen in this oligodendrocyte E-face (C).B, Higher magnification image of the region linked by tight junctions. No gap junctions were within the sealed compartments enclosed by tight junction strands, but gap junctions were numerous in the nearby plasma membrane. C, Higher magnification image of two Cx43-labeled gap junctions whose connexons were in the plasma membrane of the oligodendrocyte coupling partner (boxed areas D, E, shown at higher magnification). D, E, Gap junctions labeled for Cx43 by 10 nm gold. “Reciprocal patches” of IMPs (arrows) were present in the margins of both gap junctions. Cx36 labeling (20 nm gold) was not present in any oligodendrocyte coupling partner. F, Tight junction strands linking two oligodendrocytes, with nearby gap junctions in the oligodendrocyte coupling partner labeled for Cx43.
Fig. 8.
Fig. 8.
Identification of astrocytes as oligodendrocyte coupling partners in suprachiasmatic nucleus (A, B) and spinal cord (C). A, Stereoscopic image of an oligodendrocyte linked to the same astrocyte by one unlabeled gap junction (top left box, enlarged intop right inset) and by one Cx43-labeled gap junction (20 nm gold; bottom left box, enlarged in bottom right inset). The coupled astrocyte contained a small bundle of GFAP filaments in the cross-fractured cytoplasm (asterisk), and a square array was labeled for AQP4 (10 nm gold beads; white arrow). In oligodendrocyte E-faces, reciprocal patches (black arrow) contain both IMPs and pits. B, Stereoscopic image of one Cx43-labeled gap junction and one unlabeled mixed gap junction/reciprocal patch (arrow) in an oligodendrocyte E-face. GFAP filaments are in the cross-fractured cytoplasm (asterisk).C, Stereoscopic image of a gap junction in a spinal cord oligodendrocyte E-face in a sample that was double-labeled for Cx32 (10 nm gold; none present) and Cx30 (20 nm gold). Only Cx30 labeling was present in the plasma membrane of the oligodendrocyte coupling partner. Gap junctions often abut or intermingle with reciprocal patches (arrow).
Fig. 9.
Fig. 9.
Oligodendrocyte-to-astrocyte gap junctions in myelin from suprachiasmatic nucleus after labeling for Cx43 (A) and in oligodendrocyte plasma membrane from supraoptic nucleus after double labeling for Cx43 and Cx30 (B). A, Small gap junction on outer myelin plasma membrane E-face labeled for Cx43 by four 20 nm gold beads (enlarged in adjacent inset). Continuity of the E-face (arrow labeled E) may be traced from the outer layer of myelin (My) to the gap junction. Outer myelin membrane had IMP-free areas and reciprocal patches. Tight junction strands (white arrows) linked a small patch of the second layer of myelin to the outer layer of myelin.B, Four gap junctions (1–4) in somatic plasma membrane of an oligodendrocyte after double labeling for Cx43 (20 nm gold beads) and Cx30 (10 nm gold beads). Two small gap junctions (2, 3) were not labeled, and one (4) was labeled by only two 10 nm gold beads (Cx30). Oligodendrocyte myelin E-faces are almost devoid of IMPs.
Fig. 10.
Fig. 10.
Stereoscopic images of Cx36-labeled neuronal gap junctions in inferior olive (A) and retina (B). A, Neuronal gap junction labeled for Cx36 by three 20 nm gold beads. Postsynaptic density (arrow) is a useful marker for identifying neuronal plasma membranes in freeze–fracture replicas (Rash et al., 1997,2000). C, Two Cx36-labeled gap junctions in a nerve terminal in rat retina. Postsynaptic density (arrow) is indicated.
Fig. 11.
Fig. 11.
Diagram illustrating cellular coupling partners and connexin constituents in their gap junctions, as identified by FRIL. The cells linked within the glial syncytium are indicated by light gray shading. Astrocytes (A) share gap junctions with ependymocytes (E), oligodendrocytes (O), and other astrocytes. Ependymocyte-to-ependymocyte (E) gap junctions contain Cx43 but not Cx30, Cx32, or Cx36. Astrocyte gap junctions contain Cx43 and Cx30 but not Cx32 or Cx36. Oligodendrocytes (O) share gap junctions only with astrocytes; the oligodendrocyte sides of these junctions contain Cx32 but not Cx30, Cx36, or Cx43. Neurons (N) share gap junctions with other neurons and not with astrocytes or oligodendrocytes. Neuronal gap junctions contain Cx36 but not Cx30, Cx32, or Cx43.
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