Synapses on the mauthner cell of the goldfish: Thin section, freeze‐fracture, and immunocytochemical studies

Abstract

Large myelinated club ending and small-vesicle bouton synapses on the distal part of the lateral dendrite of the goldfish Mauthner cell were investigated with thin section, freeze-fracture, and immunocytochemical electron microscopic methods. Large myelinated club endings form mixed synapses, having both gap junctions and chemical synaptic junctions. The correlation of the number of gap junction particles (connexons) and the data from electrophysiological studies of single large myelinated club ending synapses suggest that only a small fraction of gap junction channels are open at any given time during electrical synaptic transmission. The chemical synaptic junctions at the large myelinated club ending synapse have large, round synaptic vesicles, indicating that they are excitatory. This result is in agreement with electrophysiological data demonstrating the excitatory nature of this chemical synapse. Freeze-fracture of these excitatory chemical synaptic junctions reveals the presence of the intramembrane particle aggregates in the postsynaptic E face.Small-vesicle boutons form chemical synaptic junctions with small, flat or oval synaptic vesicles. These structural data, in combination with previous electrophysiological studies, suggest that the small-vesicle bouton synapses are inhibitory. In support of this theory, the cytoplasmic side of the postsynaptic membrane of some of these synapses show positive immunocytochemical reaction to monoclonal antibodies against the rat glycine receptor. Freezefracture data reveal intramembrane particle aggregates in the postsynaptic P face of some small-vesicle bouton synapses which could possibly represent glycine receptor aggregates.

Heterotypic gap junctions at glutamatergic mixed synapses are abundant in goldfish brain

“…9C,D; 11B, left side), revealing stacks of widely-spaced Nissl substance (distinctive rough endoplasmic reticulum characteristic of neurons) or Golgi membranes, which are found in neuronal somata and dendrites but not in axons; 5 ) distinctive clusters of 10-nm E-face particles in the postsynaptic membrane within 0.03 to 0.15 μm of the gap junction and confirmed to be within the same synaptic contact (Figs. 10B-C; 11A,B; yellow overlays), with these distinctive particle clusters previously shown to correspond to glutamate receptors in both fish and mammals (Tuttle et al, 1986; Nakajima et al, 1987; Rash et al, 2005; Masugi-Tokita and Shigemoto, 2007; Hamzei-Sichani et al, 2012)]; 6 ) in appropriately-labeled replicas, these E-face particle clusters were positively identified by immunogold labeling (Fig. 11A, 18-nm gold without 6-nm or 12-nm gold) as containing at least a few NMDAR1 glutamate receptors (Pereda et al, 2003); 7) equally distinctive clusters of pits in postsynaptic P-faces, corresponding to the locations where glutamate receptors particles had been removed during fracturing (Figs.…”

“…7B,C and 11C), and 8) E- or P-face views of one or more active zones in the presynaptic plasma membrane [(Fig. 12B, upper right; see also Nakajima at al. (1987) and Tuttle et al (Tuttle et al, 1986)].…”

“…By freeze fracture, LMCE/M-cell synapses exhibit up to 160 dense clusters of hexagonally-packed 9-nm i ntra m embrane p articles (IMPs, or simply “particles”) in the protoplasmic leaflet or “P-face” (Tuttle et al, 1986; Nakajima et al, 1987), representing the freeze-fracture correlate of gap junctions (Fig. 6).…”

“…8C-G] corresponding to the LMCE that arise from glutamatergic axons of the eight cranial nerve (Diamond and Huxley, 1968), 2) axon terminals containing >10 uniform-diameter “large” (50-nm) round synaptic vesicles (SV) in the cross-fractured axoplasm (Figs. 9C-D) (Nakajima et al, 1987), identifying them as excitatory chemical synapses and distinguishing them from inhibitory (GABAergic and glycinergic) axon terminals, which contain small (20- to 40-nm) flattened or “pleomorphic” SVs (Nakajima et al, 1987; Peters et al, 1991; Legendre, 2001; Peters, 2014), as further described below; 3) clear impression of the axon terminal into the somatic or dendritic plasma membrane (Figs. 8-10; 11C), with the gap junction within the delineated synaptic contact; 4) fracture from axon terminal into the postsynaptic neuron soma or proximal dendrite cytoplasm (Figs.…”

“…LMCE's of adult goldfish co-express specializations for both chemical and electrical transmission, having 60-260 tightly-clustered gap junctions surrounded by and interspersed among variable numbers of “active zones” in presynaptic membranes, apposed by equal numbers of distinctive glutamate-receptor-containing postsynaptic densities (PSDs) (Tuttle et al, 1986; Nakajima et al, 1987). Collectively, LMCE/M-cell gap junctions consist of up to 106,000 intercellular ion channels per synaptic contact (Tuttle et al, 1986), thereby providing the ultrastructural basis for the first example of electrical coupling observed in the vertebrate central nervous system (CNS) (Robertson et al, 1963; Furshpan, 1964).…”