The ultrastructural localization of alpha-bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons

Abstract

The distribution of alpha-bungarotoxin binding sites on chick ciliary ganglion neurons was examined at the ultrastructural level by incubating ganglia with horseradish peroxidase-conjugated toxin and examining the peroxidase-stained and thin-sectioned ganglia with the electron microscope. Both in embryonic and in adult ganglia heavy labeling was restricted to the surface membrane of short processes emerging from the ciliary and choroid cell somata in the region of preganglionic innervation. Less dense labeling occasionally was present on the smooth surface membrane of the soma in the same region. In contrast, the pre- and postsynaptic membranes of most synapses were clearly not labeled even in the immediate vicinity of heavily labeled processes. The labeling represented specific binding of the toxin conjugate since it could be prevented by d-tubocurarine and hexamethonium or by unconjugated toxin. The conjugated toxin was not excluded from the synaptic cleft on the basis of size because a substantially larger protein conjugate, a horseradish peroxidase-labeled monoclonal antibody, was able to enter the cleft and heavily label synaptic membranes as well as soma membranes. Even neurons in adult ganglia had very little synaptic labeling after exposure to the conjugated toxin. These results strongly suggest that the high affinity alpha-bungarotoxin binding sites on chick ciliary ganglion neurons are different from the synaptic ACh receptors which would be expected to be concentrated in the postsynaptic membrane. Clustering of the alpha-bungarotoxin binding sites in the vicinity of synapses, however, may reflect a related synaptic function.

Nicotinic α7 receptors: Synaptic options and downstream signaling in neurons

“…This is most clear in the case of chick ciliary neurons where ␣7-nAChRs are largely confined to somatic spines emanating from the neurons and are absent from PSDs (Fig. 1) either on the soma or on the spines (Jacob and Berg, 1983;Loring et al, 1985;Wison Horch and Sargent, 1995;Shoop et al, 1999Shoop et al, , 2002. PSDs on the neurons contain either heteromeric nicotinic receptors composed of ␣3, ␤4, ␣5, and sometimes ␤2 subunits (Jacob et al, 1984;Loring and Zigmond, 1987;Vernallis et al, 1993) or, alternatively, glycine receptors (Tsen et al, 2000).…”

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

“…GlyR clusters localize to separate but proximal postsynaptic membrane regions, all under one presynaptic terminal (Tsen et al, 2000). In contrast, α7-nAChRs are excluded from the synapse and localize perisynaptically on somatic spines (Jacob and Berg, 1983;Coggan et al, 2005). For the present studies, we developed a new dominant negative construct to selectively block endogenous APC binding to EB1 during synapse formation in vivo.…”

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…In particular, the ␣-bgt-sensitive receptors have higher calcium permeability, and in some neuron populations faster kinetics of activation and desensitization, but a slowly desensitizing response in others (Seguela et al, 1993;Ullian et al, 1997;Chang and Berg, 1999;Cuevas et al, 2000). Within one neuron, the diverse nAChR subtypes are spatially segregated relative to one another and target to discrete synapse-associated sites: the presynaptic terminal, the specialized postsynaptic membrane, and the perisynaptic dendritic surface membrane (Jacob and Berg, 1983;Jacob et al, 1984Jacob et al, , 1986Loring et al, 1985;Loring and Zigmond, 1987;Moss and Role, 1993;Horch and Sargent, 1995;McGehee and Role, 1995;Gray et al, 1996;Shoop et al, 1999). The spatial segregation and distinct biophysical properties of the diverse nAChR subtypes are likely to create functionally specialized synapse-associated microregions and establish distinct spatial and temporal patterns of calcium influx that locally target different downstream signaling events (see review by D.K.…”

“…The ␣3-nAChRs, composed of ␣3, ␣5, and ␤4 (occasionally ␤2) subunits, are largely concentrated in the postsynaptic membrane (Jacob et al, 1986;Loring and Zigmond, 1987;Vernallis et al, 1993;Horch and Sargent, 1995). In contrast, the ␣7-nAChRs, composed of ␣7 subunits, are excluded from the synapse and restricted to perisynaptic regions on somatic spines (Jacob and Berg, 1983;Loring et al, 1985;Vernallis et al, 1993;Horch and Sargent, 1995;Shoop et al, 1999;Conroy and Berg, 2000). The ␣3-nAChRs mediate fast excitatory synaptic transmission through the ganglion at all developmental stages, while the ␣7-nAChRs contribute to fast throughput signaling, but only at early embryonic ages (Chang and Berg 1999).…”