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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

The Journal of Neuroscience 23(20):7489 –7503 (2003)

Connexin35 Mediates Electrical Transmission at Mixed Synapses on Mauthner Cells

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Fig. 1 Western blot analyses with various anti-connexin and anti-glutamate receptor antibodies. A, Polyclonal anti-Cx36 Ab298 recognizes a complex of bands at 31–33 kDa in goldfish retina (Ret) and hindbrain (Brn) and bands at 60 –70 kDa possibly representing a dimer form of Cx35 (left). In bacterial extracts (right), Ab298 detects the 36 kDa Cx35 C-terminal GST fusion protein (35CT) and its lower molecular weight cleavage products, but not Cx34.7 C-terminal fusion protein (34.7CT) or GST alone. B, Monoclonal anti-Cx35 also detects the 31–33 kDa bands in hindbrain and retina (left). In crude bacterial extracts (right), only the Cx35 intracellular loop GST fusion protein is recognized. C, D, Both monoclonal ( C) and polyclonal ( D) anti-Cx43 antibodies recognize two to three prominent bands at 40–44 kDa in goldfish hindbrain.
These bands comigrate with Cx43 from rat brain ( D), and both membranes (lanes a) and supernatant fractions (lanes b) contain Cx43. E, Monoclonal anti-NR1 recognizes a single 100 kDa band in retina and hindbrain.

Fig. 2 Anatomical and physiological identification of large myelinated club endings.

A, Confocal image of Mauthner cell and its axon cap, soma, and lateral dendrite visualized after intracellular injection of Lucifer yellow. Club endings are segregated to the distal portion of the lateral dendrite (boxed area), and these endings exhibit faint labeling by dye transfer from the dendrite. B, Distal portion of the lateral dendrite (LD) labeled by peroxidase–DAB reaction after intracellular injection with Neurobiotin tracer. Some club ending afferents are labeled as a result of gap junction-mediated tracer transfer to presynaptic terminals. C, Normarski optics view of the lateral dendrite surface where club endings are readily identified among other terminals by their large size (dotted circle). D, Experimental design for intracranial simultaneous recordings. Club endings forming mixed synapses (asterisk) were penetrated while simultaneously recording from a Mauthner cell lateral dendrite. E, Mixed synaptic transmission at club endings demonstrated by intracranial simultaneous recordings. A presynaptic depolarizing pulse triggers a presynaptic action potential (Pre spike) and depolarization. Both the spike and subthreshold pulse depolarization are recorded as the electrical component in the postsynaptic lateral dendrite. A glutamate-mediated slower response (chemical, yellow area) follows the electrical potential produced the presynaptic spike.

Fig. 3  Laser scanning confocal immunofluorescence showing Cx35 at large myelinated club endings with polyclonal anti-Cx36 antibody Ab298.

A, Confocal projection (30 sections; 20 mm) of the distal portion of the Mauthner cell lateral dendrite, which was physiologically identified and intracellularly injected with Neurobiotin for cell identification during confocal microscopy. Cx35 was visualized by Texas Red secondary antibody (red), and Neurobiotin was visualized by streptavidin FITC (green). B, Stack of three z-sections (2 m) through the surface of the Mauthner cell lateral dendrite. Several club endings, identified by their large size and delineated by red immunofluorescence, exhibit multiple sites of punctate or patchy labeling for Cx35 (magnified in inset, asterisk). C, Lateral view of club ending showing restriction of labeling at lateral dendrite– club ending interface. D, Laser scanning confocal immunofluorescence showing Cx35 at large myelinated club endings using monoclonal anti-Cx35 antibody. Images derived from three confocal z-sections (2 mm) through the surface of the distal portion of a Mauthner cell lateral dendrite show multiple labeled club endings.

Fig. 4   Laser scanning confocal immunofluorescence of Cx35 associated with Mauthner cell somata and proximal dendrite.
A, View of the Mauthner cell soma showing intense labeling for Cx35 with polyclonal Ab298 (red). The Mauthner cell was injected with Neurobiotin (green) as in Figure 3. Labeling is seen on both soma and small somatic dendritic processes (inset). Small “cap dendrites” projecting inside the axon cap (arrowhead) are also faintly labeled with Neurobiotin. B, View of a most proximal region of the lateral dendrite where a club ending in the boxed area (magnified in inset, arrowhead) appears among other terminals of smaller size, likely corresponding to anterior eighth nerve or lateral line inputs to the Mauthner cell, or both. C–E, Labeling of Cx35 (Ab298) associated with neurons in various regions of goldfish hindbrain. Image shows a confocal projection of large vestibulospinal (C) and two reticulospinal (D, E) neurons. Punctate labeling is seen dispersed throughout the surface of these neurons.

Fig. 5  Figure 5. FRIL images of Cx35 localization at gap junctions between a Mauthner cell lateral dendrite and large myelinated club endings.

A, Confocal micrograph of a FRIL replica containing a Lucifer yellow-injected Mauthner cell bonded to a gold index grid. Fracture plane traverses obliquely through the Mauthner cell, revealing views from club ending toward Mauthner cell (B, C) and from Mauthner cell toward club ending (Fig. 7). Yellow arrow indicates approximate location of club ending illustrated in B and C. At this light microscopic magnification, the yellow arrowhead covers ~2000 mm2 of the sample, whereas the endplate in Figure 5B covers ~12 m2. B, FRIL image showing one club ending with 85 gap junctions (redareas), 83 of which are immunogold labeled for Cx35. CE, Club ending; MC, Mauthner cell; SV, synaptic vesicles; blue areas, labeled Mauthner cell gap junctions at adjacent club ending. The area shown is equivalent to 2% of the area of a single grid opening in Figure 7A, or less than one pixel of the image. C, Higher magnification of boxed area in B showing E-face view of club ending (CE) surrounded by P-face of Mauthner cell plasma membrane(MC). In this area,19 of 19 gapjunctions are labeled with10 nm gold beads. Blue arrow, Gap junction with ~30 connexons labeled by two immunogold beads. White arrows, Exocytotic vesicle remnants in the club ending plasma membrane; black arrow, rosette of E-face IMPs typically seen in nerve terminal plasma membranes. Scale bars (in FRIL images): 0.1 mm unless designated otherwise.

Fig. 7  FRIL double labeling of Cx35 in gap junction plaques within the presynaptic membrane and of NR1 subunit of NMDA glutamate receptors in nearby IMPs in postsynaptic particle clusters (designated PSDs) in the postsynaptic membrane.

A, Gap junctions and PSDs in postsynaptic membrane of a Mauthner cell. Cx35 labeling (Ab298) is approximately as dense in the presynaptic membrane as in postsynaptic membranes elsewhere in the same cell (Fig. 5C). B, Simultaneous double labeling for Cx35 (6 nm gold beads) and for glutamate receptor NR1 (18 nm gold beads) in a PSD of an identified Mauthner cell club ending in a different replica. The PSD (yellow) contains 10 nm IMPs, some of which were damaged by scraping during the fracturing event (right side of PSD). C, Four of five Cx35-labeled gap junctions (10 nm gold beads) in a small portion of a club ending on the same Mauthner cell as shown in Figures 5 and 7A. A nearby small PSD is labeled for NR1 (18 nm gold bead). Because membrane continuity was interrupted, identification as a club ending was on the basis of synapse size, number of closely spaced gap junctions, and FRIL mapping criteria. D, NR1-labeled PSD in a non-club ending synapse in the same Mauthner cell illustrated in Figures 5 and 7A. As above, Cx35 is labeled by 10 nm gold (none present here), whereas NR1 is labeled by 6 nm (arrow) and 18 nm gold beads. The PSD is shown in stereoscopic (left pair) and reverse stereoscopic perspective (right pair). In reverse stereoscopic perspective, the smallest gold beads (6 nm) are readily discerned as if suspended above the replicated 6-10 nm IMPs. E, Laser scanning immunofluorescence showing NR1 at large myelinated club endings with anti-AptNR1 antibody. The image represents a stack of three z-sections (2 µm) through the surface of the M-cell lateral dendrite. Two club endings, identified by their large size and location, are delineated by green immunofluorescence. Each exhibits multiple punctate labeling that, in contrast with Cx35 (Fig. 3), is restricted to the perimeter of the contacts.

Fig. 6 Basis for FRIL identification of membrane proteins.

A, B, Diagram showing portions of a mixed synapse before (A) and after freeze fracturing and immunogold labeling (B). The fracture plane (A, green line) separates connexons at the point of contact in the extracellular space, leaving either intramembrane particles (IMPs) in the P-face (P) or arrays of pits in the E-face (E). All gap junctions exposed by fracturing are separated into two hemiplaques; one hemiplaque remains associated with each of the two freeze-fractured cells. Whether E-face pits or P-face particles are visualized by FRIL, antibody labeling of connexins and secondary immunogold beads bind only to connexins in the hemiplaque of the cell with a cytoplasmic membrane leaflet that remains beneath the replica. For E-face images, all connexons of the upper cell are removed, but connexons remain within the unsplit membrane of the underlying, nonvisualized cell. For intramembrane proteins other than connexins, however, immunogold labeling sites are present only beneath replicated IMPs, on either E-faces or P-faces. Proteins comprising PSDs of glutamatergic synapses consist of distinctive clusters of E-face IMPs (B), which are available for immunogold labeling of determinants exposed in the residual extracellular space. C, Diagram of fracture plane through an individual Mauthner cell, including views from the presynaptic side toward the postsynaptic side (top left) and from the postsynaptic side toward the presynaptic side (bottom right). Glutamate receptor PSDs are clusters of E-face IMPs that are immunogold labeled on their extracellular determinants.

Fig. 9 FRIL single labeling for Cx35 (A) and double labeling for Cx35 and NR1 (B) in mixed synapses between unidentified neurons in goldfish hindbrain. A, Two gap junctions (inscribed areas, magnified in inset at top right) linking nerve terminals (NT1, NT2) to postsynaptic membrane. One gap junction is unlabeled (top inset), and the other (bottom inset) is labeled by five 20 nm gold beads. Also evident is a PSD at the bottom right. B, Double-labeled mixed synapse with NR1 in the PSD labeled by 10 nm gold beads (white arrows) and the gap junction labeled for Cx35 by 20 nm gold beads (black arrow). Immunogold beads up to 50 nm from the margins of gap junctions presumably represent labeling of connexins in dissolving membrane blebs, as illustrated in Rash and Yasumura (1999), their Figure 9.

Fig. 8 FRIL labeling of Cx35 in gap junctions at mixed synapses of unidentified neurons in goldfish hindbrain.

A, Freeze-fracture view of the postsynaptic E-face and of two nerve terminal P-faces (NT1, NT2). Gap junctions are labeled with three sizes of gold beads. B, Higher magnification view of left boxed area in A, shown as stereoscopic images (left pair) and reverse stereoscopic images (right pair). Cx35 is labeled with 19 6 nm gold beads (arrow), 3 18 nm gold beads, and 3 20 nm gold beads. The field also contains a small unlabeled gap junction (arrowhead). C, Area of Mauthner cell with closely adherent astrocyte processes. Astrocyte gap junctions were not labeled for Cx35.

Fig. 10 Localization of Cx43 near Mauthner cell lateral dendrites.

A, Presynaptic and postsynaptic simultaneous unitary recording (A) from the labeled club ending and dendrite illustrated in B. The presynaptic electrode was inserted into afferent fibers of the posterior eighth nerve root and therefore differs from the recording in Figure 2 E, where the presynaptic electrode was inserted in the presynaptic terminal, allowing detection of subthreshold responses. No chemical component was detected at this contact, a common observation at these terminals during unitary recordings (Lin and Faber, 1988). Data represent the average of 20 individual traces. B, Confocal immunofluorescence labeling for Cx43 (red) after simultaneous intracellular injection of Mauthner cells and eighth nerve fibers with Neurobiotin (green). Punctate staining for Cx43 (arrowheads) is seen around the periphery of the Mauthner cell lateral dendrite (LD) but not at the junction between Neurobiotin-labeled club endings (arrow) and the lateral dendrite (inset, arrow). C, FRIL labeling of Cx43 at astrocyte gap junction displaying 33 20 nm gold beads. The astrocyte process was identified as such on the basis of the presence of GFAP filaments (GFAP) in the cytoplasm and by the characteristic high density of E-face particles (bottom right). D, Gap junction located between two PSDs in the E-face of an unidentified neuron in the same replica as illustrated in C is devoid of labeling for Cx43.

Journal of Neuroscience 2003

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