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Colorado State University College of Veterinary Medicine and Biomedical Sciences College of Veterinary Medicine and Biomedical Sciences
Department of Biomedical Sciences

Rash Laboratory

Neurosciences 136: 65-86, 2005

Connexin-47 and Connexin-32 in Gap Junctions of Oligodendrocyte Somata, Myelin Sheaths, Paranodal Loops and Schmidt-Lanterman Incisures: Implications for Ionic Homeostasis and Potassium Siphoning

[click on images for a larger view]

Fig. 1  Immunofluorescence labeling of oligodendrocyte connexins in gray and white matter of adult rat brain and spinal cord.

(A) Confocal double immunofluorescence images of a pair of adjacent oligodendrocyte somata in spinal cord gray matter, showing an abundance of punctate labeling for both Cx47 (A1, arrows) and Cx32 (A2, arrows) around the periphery of the cells. Cx47 and Cx32 are generally co-localized in the overlay (A3, arrows). (B) Confocal double immunofluorescence images of an oligodendrocyte soma in spinal cord white matter, with a relatively greater abundance of punctate labeling for Cx47 (B1, arrows) than for Cx32 (B2, arrows). Co-localization of puncta for Cx47 occurs where Cx32 is also present (B3, arrows). (C) Low magnification micrographs showing oligodendrocyte-associated (arrows) as well as dispersed punctate labeling of Cx47 and Cx43 in the corpus callosum. (D, E) Confocal double immunofluorescence labeling shows considerable overlap of dispersed Cx47-positive and Cx43-positive puncta in the corpus callosum and spinal cord white matter. Areas of Cx43 (red) without Cx47 presumably represent astrocyte/astrocyte gap junctions. Scale bars=100 μm C; A, B, D, E, 10 μm.

Fig. 2  Thin-section immuno-localization of Cx47 in adult mouse brain.

(A) Oligodendrocyte somata in cerebral cortex immunolabeled with polyclonal anti-Cx47 antibody display large deposits of DAB reaction product in cytoplasmic compartments close to the plasma membrane (arrows), and smaller deposits in the cytoplasm closer to the nucleus (n), associated with presumptive rough endoplasmic reticulum (arrowheads). (B, C) Higher magnifications of DAB deposits near oligodendrocyte (o) plasma membranes in the thalamus (B) and cerebral cortex (C), showing close proximity of deposits to gap junctions (arrowheads) between oligodendrocytes and astrocytes (as), and localization of deposits exclusively on the oligodentrocyte side of junctions. (D) Silver-intensified immunogold labeling showing intracellular sites of Cx47 labeling at plasma membranes of oligodendrocytes (arrows). (E, F) Higher magnification image showing silver-intensified immunolabeling for Cx47 at O/A gap junctions (arrowheads). Scale bars=1 μm A; B, C, 0.1 μm; D, 0.5 μm; E, F, 0.1 μm.

Fig. 3  Stereoscopic images of gap junctions in oligodendrocyte somatic plasma membranes after double-labeling for Cx47 and Cx32.

(A) One large gap junction (three arrowheads delineating its edge) exhibits robust labeling for Cx47 (136 12nm gold) but little or no labeling for Cx32 (no 6nm and only one 18nm gold bead). N, nucleus; *, cytoplasm. The lower left quadrant of the image is not shadowed with platinum because of blockade by a large tissue fragment, but instead is replicated by carbon only. (B) Higher magnification stereoscopic image of the same gap junction. Carbon-replicated connexons are faintly delineated, whereas platinum-shadowed connexons (upper right) are clearly delineated. White arrowhead, 18nm gold bead for Cx32. Unlabeled scale bars=0.1 μm.

Fig. 4  Gap junctions on oligodendritic processes in sample of rat spinal cord after single-labeling for Cx47 (12nm gold beads).

(A) Cytoplasmic process (“oligodendrite”) linking an oligodendrocyte soma (not shown) to the outermost layer of myelin (M) of two different cross-fractured axons (Ax). Arrow indicates area of transition from dendritic process to flattened cytoplasmic myelin. Of seven gap junctions on this dendritic process (arrowheads), all were heavily labeled for Cx47. (B) Stereoscopic image of two closely-adjacent gap junctions (or one irregular gap junction), from the inscribed area in 4A; labeled by 20 12nm gold beads. (C) One or two small gap junctions on the same dendrite; labeled for Cx47 by eight 12nm gold beads. (D) Large (>1500 connexons), medium and small gap junctions on oligodendrite; labeled for Cx47. Two central “reciprocal patches,” which are composed of mixed IMPs and pits, are not labeled. Unlabeled scale bars=0.1 μm.

Fig. 5  Gap junctions on the outer surface of myelin that had been double-labeled for Cx47+Cx32.

(A–F) Myelin is characterized by broad expanses of particle free membranes. Gap junctions are distinguished from “reciprocal patches” (RP in A) by having 100% of IMPs on P-faces and 100% of pits on E-faces. Plasma membranes of astrocytes were identified by the presence of E-face images of AQP4 square arrays (C; white arrow). (C) Enlarged image of B. (B) M, cross-fractured myelin. (A–C) Most gap junctions (66%) were immunogold labeled for Cx47 (12nm gold) but did not label for Cx32. A few (21%), particularly the smallest gap junctions (D), were double-labeled for Cx47 (12nm gold beads) plus Cx32 (white arrowhead, 6nm gold bead; black arrowhead, 18nm gold bead), and fewer still (13%) on outer myelin (E, F) were labeled for Cx32, only. Scale bars = 0.1 μm.

Fig. 6  Laser scanning confocal double immunofluorescence images of caspr and Cx32 labeling in white matter of adult rat spinal cord.

(A) Low magnification images of dorsolateral white matter showing intermittent labeling for caspr along myelinated fibers (A1, arrowheads), intermittent (A2, arrowheads) as well as continuous (A2, arrows) labeling for Cx32 along these fibers, and occasional regions of overlap (A3, arrowheads). (B) Higher magnification images from regions similar to those in (A) showing granular appearance of labeling for Cx32 along some fibers (B2, arrows), and intense immunofluorescence for caspr (B1, arrowheads) and Cx32 (B2, arrowheads) on each side of a node of Ranvier. Overlay shows overlap of labeling (B3, arrowheads). (C–E) Double immunofluorescence labeling showing co-localization of caspr and Cx32 (arrowheads) in other white matter regions of spinal cord, including ventrolateral (C), ventral (D), and dorsal columns (E). Scale bars = 25 μm A; B–E, 5 μm.

Fig. 7  FRIL images of gap junctions at Schmidt-Lanterman incisure; between the innermost tongue of cytoplasmic myelin and the second innermost layer of partially-compacted myelin; immunogold labeled for Cx32 (6nm and 18nm gold) and Cx47 (12nm gold, none present).

(A) M, myelin; E, E-face; P, P-face; white arrow, astrocyte process containing bundle of GFAP filaments; black arrow, cytoplasm of innermost tongue of myelin. Inscribed areas shown at higher magnification, below. (B) *, cytoplasm of 2nd and 3rd innermost layers of myelin. (C–E) High magnification stereoscopic images of three gap junctions labeled for Cx32 (from small inscribed boxes in A). (C′–E′) Reverse stereoscopic images of C–E allow recognition of 6nm gold beads against the equally electron-dense platinum replica. White arrowhead, 6nm gold beads for Cx32; black arrowhead, 18nm gold bead for Cx32. (C″–E″) Reversed contrast (i.e. black shadows) versions of C–E, which more clearly reveal the hexagonal arrays of E-face pits (black pits or holes representing where connexons had been removed; black shadows according to the original imaging convention of Steere 1957). Nearby E-face IMPs cast distinctive black shadows, as if the IMPs had been illuminated with white light. However, immunogold beads appear anomalously white. Unlabeled scale bars = 0.1 μm.

Fig. 8 Stereoscopic FRIL images of “string” gap junctions linking paranodal loops of myelin after double-labeling for Cx32 (6 and 12nm) and Cx29 (18nm; none found in paranodes).

(A) In rare cross-fracture through paranodal myelin (M), low magnification images reveal that the outer layers of myelin are compacted, whereas the paranodal loops (Pn) are widely separated and “bowl-shaped,” forming a concave toroidal spiral. (Ax, axon). Inscribed areas shown at higher magnification, below. Within the plasma membrane, two gap junctions (B, C) and several tight junctions (D) are depicted. (B) At higher magnification, linear rows of 20 8nm E-face pits (E between white arrows) having a center-to-center spacing of 10nm, and C, 38 9nm P-face IMPs (P) having center-to-center spacings of 10nm are labeled for Cx32 by 12nm gold beads (black arrowhead), one gold bead in each image, yielding combined labeling efficiencies (Rash and Yasumura, 1999) of ca. LE=1:30. (D) Formaldehyde-fixed tight junctions (TJ) consist of discontinuous rows of partially-fused particles and partially-fused pits (furrows) on both E- and P-faces. (E) String gap junction, with step from E-face (E) to P-face (P), showing continuity of rows of E-face pits with P-face IMPs. The gap junction consists of ca. 16 connexons and is labeled for Cx32 by five 6nm gold beads (white arrowhead) (LE=1:3). Unshadowed area has E-face pits (black arrow) faintly delineated by the carbon replica film. Unlabeled scale bars = 0.1 μm.

Fig. 9  Laser scanning confocal double-immunofluorescence images of caspr and Cx47 labeling in white matter of adult rat spinal cord.

(A) Low magnification of lateral white matter showing labeling of caspr at paranodal segments of nodes of Ranvier (A1, arrowheads) and linearly arranged labeling of Cx47 along myelinated fibers, with occasional co-association of immunolabeling (A3, arrowheads). (B–D) Higher magnifications of caspr-positive paranodes along large diameter fibers in lateral (B1, arrows) and ventral (C1, arrows) spinal cord white matter, and along a narrower fiber in lateral white matter (D1, arrows). In the same fields labeled for Cx47 (B2–D2, respectively), Cx47-positive puncta are seen closely apposed to the region of caspr-immunostaining immediately adjacent to the node of Ranvier (B3–D3, arrowheads). (E) Triple immunofluorescence showing labeling of caspr at a node of Ranvier (E1, arrows), labeling of Cx43 adjacent to a caspr-positive segment (E2, arrowhead) as well as localized within the nodal area (E2 and E4, double arrowhead), and of Cx47 (E3, arrowhead), also adjacent to caspr and overlapping with Cx43 (E4, arrowhead). Scale bars = 25 μm A; B–E, 5 μm.

Fig. 10  Stereoscopic FRIL images of gap junctions on everted loops of paranodal plasma membrane after immunogold labeling for Cx47.

(A) Low magnification stereoscopic image revealing the outer surface of myelin (left side), including a small area where the fracture plane steps between three outermost layers of myelin (arrowhead). Four paranodal loops (arrows) extend beyond the outermost layer of myelin, there to contact astrocytes (As) in the nodal gap. The boxed area is shown at higher magnification and after tilting ca. 50°, thereby allowing en face viewing of an immunogold-labeled gap junction on the shoulder of a paranodal loop. (B) At higher magnification, the gap junction is seen to be immunogold labeled for Cx47 (three 12nm gold beads). Arrows delineate the edge of the gap junction. Although insufficient area of the contacting cell is visible for positive identification, the image is consistent with coupling to one of the astrocyte processes that are common in this area (see Results for references). Ax, axon; M, cross-fractured myelin. Unlabeled scale bars = 0.1 μm.

Fig. 12  Diagram of locations of heterologous O/A gap junctions on oligodendrocyte cell body, dendrites, outer surface of myelin and everted paranodal loops, and autologous (i.e. interlamellar) gap junctions at Schmidt-Lanterman incisures and between paranodal loops (A), and pathways for “potassium siphoning” (B). (A) Astrocyte endfeet contacting capillaries and forming the glia limitans release excess K + ions (uppermost red arrows) and associated osmotic water (blue arrows) via Kir4.1 potassium channels (Hibino et al., 2004) and AQP4 water channels that are concentrated in the endfoot plasma membranes (Nielsen et al 1997 and Rash et al 2004a). (B) Summary of movement of positive charge following an axon action potential. Red-to-yellow gradient=charge gradient from areas of excess positive charge/positive membrane potential to lowest positive charge/highest negative membrane potential. Initial positive potential from entry of Na+ (arrow #1); excess positive charge in axon results in movement of K + into the inter-perinodal space (arrow #2). K + returns to the axon (arrow #3a) or enters the innermost cytoplasmic layer of myelin (arrow #3b), passes though successive paranodal loops (arrow #4), either circumferentially or through Cx32-containing gap junctions to the outermost cytoplasmic layer of myelin, then through Cx47/Cx32-containing gap junctions into the astrocyte syncytium (arrow #5), to astrocyte endfeet at capillaries and the glia limitans (arrow #6). Blue connexons, Cx47; green connexons, Cx32.

Neuroscience 2005

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