Short-range functional interaction between connexin35 and neighboring chemical synapses

Abstract

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication--chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.

Figure 1

Cx35 mediates electrical transmission at Large Myelinated Club endings. (A) Auditory afferents (Club endings) terminate on the ipsilateral M-cell lateral dendrite as mixed, electrical and chemical, synaptic contacts known as Large Myelinated Club endings (*, Mixed synapse). (B) Confocal microscopy reveals the presence of Cx35 and NMDA receptors in Club endings. (B1) Laser scanning confocal immunofluorescence showing Cx35 at a Club Ending using monoclonal anti-Cx35 antibody. These terminals, identified by their large size and delineated by immunofluorescence, exhibit multiple sites of punctate labeling for Cx35. Image derived from 3 confocal z sections (2 μm) through the surface of the distal portion of a M-cell lateral dendrite. (B2) Laser scanning immunofluo-rescent image of a Club Endings labeled with anti-NR1 antibody. The image also represents a stack of 3 z sections (2 μm) through the surface of the M-cell lateral dendrite. Calibration bars: 5 μm. (C) Freeze fracture immunogold labeling (FRIL) confirms the presence of Cx35 and NMDA receptors. 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 of a Club ending. Image shows five gap junctions, delineated by the gray areas, labeled for Cx35 (10 nm gold beads, Ab298 antibody) in a small portion of a Club Ending. A nearby small PSD is labeled for NR1 (18-nm gold bead, monoclonal NR1 antibody). Calibration bar: 0.1 μm. interpretations of these images, are described in a previous report (9).

Figure 2

Electrical synapses belonging to neighboring Club endings co-exist at different degrees of conductance. (A) Electrical component of mixed synaptic potentials (electrical) evoked by stimulation of different afferent fibers differ dramatically in amplitude. Mixed unitary excitatory synaptic potentials (EPSPs, a–d) evoked by stimulation of four afferents obtained sequentially while recording in the same fish at the same dendritic site. The electrical component of the unitary synaptic potentials show much greater variability in amplitude than the presynaptic action potentials (not shown, see reference [9]). Only one of these four unitary synaptic potentials exhibited a clear chemical component (chemical, trace a). (B) Transfer of Neu-robiotin at neighboring Club Endings differs dramatically, indicating that they differ in permeability. View of the bifurcation of the lateral dendrite (dark branches) obtained with Nomarski optics and revealing differences of labeling between two neighboring Club Endings. Labeled (bottom) and unlabeled (top) fibers terminate in the same area of lateral dendrite. Only a small number of Club Endings were detected by transfer following injection of the tracer Neurobiotin into the M-cells (5–25% of the estimated population) suggesting that gap junction permeability at Club Endings is generally low and that most fibers did not accumulate enough Neurobiotin to produce a visible reaction product. Calibration bar: 20 μm.