Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons.

Abstract

Monoclonal antibodies raised against purified acetylcholine receptor from muscle and electric organ were tested for cross-reaction with surface components on chicken ciliary ganglion neurons. Indirect immunofluorescence indicated that antibodies to a determinant in the "main immunogenic region" of the receptor bind to the neurons in culture. Ultrastructural studies on 16-day embryonic ganglia, using horseradish peroxidase-conjugated monoclonal antibody, revealed that most of the conjugate labeling was associated with synaptic membrane on the neurons. A lesser amount of labeling was associated with the short processes extending from the neuronal somata in the region of preganglionic innervation. The labeling was blocked by coincubation with unlabeled antibodies of appropriate specificity and not by nonimmune serum. The pattern of labeling was clearly different from that previously found for a horseradish peroxidase conjugate of a-bungarotoxin: the toxin conjugate bound extensively to the short processes but not to synaptic membrane on the neurons. The synaptic antigen identified here by the crossreacting antibodies is a candidate for the synaptic acetylcholine receptor on chicken ciliary ganglion neurons.Chicken ciliary ganglion neurons have nicotinic acetylcholine (AcCho) receptors that mediate chemical synaptic transmission through the ganglion (1, 2). Efforts to study the regulation and distribution of such receptors on neurons have been limited by the absence of suitable probes. a-Bungarotoxin has been a very useful probe for studying nicotinic AcCho receptors in vertebrate skeletal muscle and electric organ, where the toxin binds tightly and specifically to the receptor and blocks its function (3). Ciliary ganglion neurons also have high-affinity binding sites for a-bungarotoxin, but in this case the binding sites appear to be distinct from synaptic AcCho receptors. Ultrastructural studies demonstrate that toxin binding sites on the neurons are located in the immediate vicinity of presynaptic terminals but are not present in the postsynaptic membrane (4), as would be expected for synaptic receptors. Moreover, a-bungarotoxin does not block AcCho receptor function on the neurons (5, 6), and, when the levels of AcCho sensitivity and toxin binding are compared for the neurons grown under various conditions in cell culture, no simple correlation is found between the two properties (7), as would be expected if they represented the same membrane component.An alternative approach is to use antibodies against the AcCho receptor as probes. Monoclonal antibodies (mAbs) against AcCho receptors from skeletal muscle and electric organ have been useful in studying the structure and synthesis of these receptors (8)(9)(10)(11)(12). Recent studies have shown that mAbs specific for each of the four subunits of AcCho receptors from muscle and electric organ bind to neurons in the lateral spiriform nucleus of chicken brain (13). These neurons do not bind a-bungarotoxin. Best cross-reaction was obtained with mAbs to the "...

Nicotinic α7 receptors: Synaptic options and downstream signaling in neurons

“…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). The fact that both the spines and PSDs are overlaid by a large vesicle-filled presynaptic calyx engulfing the ciliary neuron suggests that even "perisynaptic" ␣7-nAChRs, i.e.…”

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.…”

Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture

“…Cross-linking studies with a photoaffinity derivative of Bgt 3.1 demonstrate that Bgt 3.1 and mAb 35 recognize the same neuronal component Berg, 1986b, 1987). The AChR identified by both Bgt 3.1 and mAb 35 is clearly distinct from the membrane component of unknown function on the neurons that binds a-bungarotoxin (Jacob and Berg, 1983;Smith et al, 1983Smith et al, , 1985Jacob et al, 1984).…”

“…One of these is a monoclonal antibody, mAb 35, to the "main immunogenic region" (MIR) of AChR a-subunits from muscle and electric organ. MAb 35 cross-reacts with a component on chick ciliary ganglion neurons that has the ultrastructural location, biochemical properties and cholinergic modulation expected for the neuronal AChR (Jacob et al, 1984;Smith et al, 1985Smith et al, , 1986. Antisera raised against a similar component immunopurified from chick brain with mAb 35 specifically block the ACh response of ciliary ganglion neurons (Stollberg et al, 1986).…”