Rash Laboratory

Fig. 1 Confocal images of Mauthner cells labeled for Cx35 and glutamate receptor NR1, with companion FRIL immunogold labeling of gap junctions in goldfish Mauthner cells. (A) Confocal immunofluorescence image (three Z sections of 2 µm) showing Cx35 (MAB 3045) at a Club Ending on distal portion of a Mauthner Cell lateral dendrite. These terminals, identified by their large size, exhibit multiple sites of punctate labeling for Cx35. (B and C) Cx35 (Ab298) labeling of conventional “plaque” gap junctions (red overlay) in MC/CE synapses. Both images are from the same goldfish MC/CE synapse, showing primarily postsynaptic P-face (B) and presynaptic E-face (C). In both images, all labeling is on postsynaptic connexins. (D) Laser scanning immunofluore-scence image (three Z sections of 2 µm) of a Club Ending on a Mauthner-cell lateral dendrite after labeling with anti-NR1 antibody. (E–H) Simultaneous co-localization of Cx35 at gap junctions and NR1 glutamate receptors in E-face particles arrays (yellow overlay) in goldfish Mauthner cell. (E) E-face view of the postsynaptic membrane of goldfish Mauthner cell. E-face image of gap junction in postsynaptic membrane, but with unfractured and unreplicated presynaptic plasma membrane beneath. It is these unreplicated presynaptic connexins that are labeled (18 nm gold). In contrast, the two arrays of E-face IMPs represent glutamate receptor clusters, which are immunogold labeled for NR1 (12 nm gold beads in the extracellular space; see Fig. 2B–E for explanatory diagram). Inscribed areas are shown at higher magnification (F–H). (F) Stereoscopic (left pair) and reverse stereoscopic images (right pair) of a neuronal gap junction labeled for Cx35. (G and H) Portions of two PSDs after immunogold labeling for glutamate receptor NR1 by two gold beads (G) and one gold bead (H). The gap junction and the PSD on the right are separated by~50 nm, or less than the limit of resolution of light microscopy. For confocal images A and D, calibration bars = 5 µm. In all FRIL replicas, calibration bars = 0.1 µm, unless otherwise designated.

Fig. 2 Immunogold labeling of glutamate receptor PSDs and nearby gap junctions. (A) Portions of three Cx35-immunogoldlabeled gap junctions (red overlays) surrounding a P-face imprint of a PSD (yellow overlay), corresponding to the arrays of IMPs on E-faces (see Fig. 1G and H). The PSD P-face pits are devoid of NR-1 labeling. (B–E) Drawing of fracture plane through two Club Ending synapses on a Mauthner cell. (B and C) From left to right, the fracture plane (blue line) sequentially fractures within the postsynaptic plasma membrane, through a glutamate receptor PSD (lavender) and through a postsynaptic gap junction (left side of image), across the Mauthner cell cytoplasm (center of image), then to within the Mauthner cell plasma
membrane (right side), exposing the postsynaptic membrane E-face particles corresponding to glutamate receptors, then the gap junction E-face pits (postsynaptic membrane) and, finally, gap junction P-face particles (presynaptic membrane of lower club ending). (B and C) PSD P-face pits are not labeled because no glutamate receptor proteins remain with the P-face pits
(left side). However, P-face connexon IMPs (orange connexons, left side) in the adjacent gap junction are labeled. Glutamate receptor E-face particles are labeled on their extracellular determinants (right side), whereas connexons of presynaptic gap junctions are labeled on their cytoplasmic epitopes (D and E). In the gap junction on the right (2C, box) and in closer view (D), postsynaptic E-face pits and presynaptic P-face particles are replicated, but in both cases, it is the presynaptic connexons (yellow connexons) of the underlying Club Ending that are labeled. (E) Diagram showing immunogold labeling of epitope in
the cytoplasmic carboxy terminus of a single connexin molecule by Ab298 (Pereda et al., 2003). (F) Stereoscopic and reverse stereoscopic image of portion of mixed synapse in adult rat inferior olive labeled for Cx36 (20 nm gold; red overlay) and NR1 glutamate receptors (10 nm gold, arrow; yellow overlay). Reverse stereoscopy (right pair) is helpful for discerning the 10 nm gold bead superimposed on the equally electron-dense platinum replica, as well as for discriminating between the two layers of replica that arose when a portion of replica was displaced (darker area at top).

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 FRIL images of outer myelin layers after immunogold labeling for Cx32.

A, Low-magnification image of myelin membrane 1exP, which is characterized by abundant caveolae. D, Layer 2exP, the outer membrane of the underlying Schwann cell wrapping, is characterized by the presence of rivulets containing residual cytoplasm, as documented at different tilt angle, 5D, adjacent. Two gap junctions are visible (inscribed areas B and C). B, A gap junction located where the fracture plane stepped from layer 2exP to1inE consists of both P-face IMPs and E-face pits. The extracellular space (*) is narrowed to <3 nm within the area of the gap junction. C, Gap junction in membrane 1inE. The E-face pits are immunogold labeled for Cx32. D, At a high tilt angle, rivulets at the margin of cross-fractured myelin (M) are seen to contain cytoplasm (blue shading). E, Removal of the top rivulet membrane (1exP) reveals a view of the E-face of the underlying membrane 1inE. Gap junctions frequently were found on rivulet membranes (inscribed area F ). F, Cx32 immunogold-labeled gap junction (yellow arrowhead) in particle-rich myelin membrane 2inE. ECM, Extracellular matrix; 1exP, P-face of the outer membrane of the outermost Schwann cell wrapping; 1inE, E-face of the inner membrane of the outermost (first) Schwann cell wrapping; 2exP, P-face of the outer membrane of the second Schwann cell wrapping.

Fig. 6  High-magnification FRIL images of immunogold-labeled gap junctions.

A, Cx32-labeled gap junction located at the step from P- to E-face. Note the narrowing of extracellular space (*) at the area of junctional contact. B, Two gap junctions, each composed of two connexons (arrows), with each gap junction labeled by one immunogold bead. C, Gap junction composed of 15 E-face pits, labeled by two immunogold beads. D, Gap junction composed of two connexons, with 12 nm immunogold beads indicating the presence of Cx32. E, Rare orphaned gap junction located on smooth myelin membrane deep within the stack of compact myelin. F, Gap junction at an indeterminate location in myelin. Gap junction P-face IMPs are double-labeled for Cx32 by one 18-nm and 13 6-nm gold beads.

Fig. 7  Figure 7. Diagrams of cross-sectional view versus view of unrolled outer layers of myelin in an internodal segment of peripheral nerve.

A, Perspective view of a partially unrolled outer myelin layer, showing the outer tongue (OT) of myelin and the relative locations of tight junctions (red) and gap junctions (blue). Tight junctions and gap junctions link the inner membrane of the outer tongue to the outer membrane of the second wrapping of myelin. B, View of outer membrane of the first two unrolled wrappings of myelin (1ex and 2ex ). Tight junctions are located away from the tip of the myelin tongue a distance equal to or slightly greater than the circumference of the outermost layer of myelin (p x D). The pattern of tight junction strands and gap junction hemichannels in 2ex is identical to the distribution of their pairing partners in the inner surface of the first turn of the outer wrapping (1in ), as seen in the right panel of B. Inverted view of the same Schwann cell, revealing the inner membrane of the first (1in ) and second (2in ) Schwann cell wrappings. Few or no caveolae are present in the zone of partial compaction inside the borders of tight junctions in layer 1in. Rivulets (R) of cytoplasm are present in the second wrapping but extend into the first wrapping, past the gap junctions, almost to the tight junctions. Additional myelin layers are not visible in this partially unrolled segment of myelin, because each successive myelin layer is wider than the previous layer, and each successive paranodal loop covers the previous loop (*).

View entire journal article by clicking on the left hightlighted column. You will need Adobe Acrobat Reader to access the PDF files.