Perisynaptic surface distribution of multiple classes of nicotinic acetylcholine receptors on neurons in the chicken ciliary ganglion

Abstract

Most nicotinic ACh receptors (AChRs) studied to date in the embryonic chicken ciliary ganglion are recognized either by monoclonal antibody (mAb) 35 or by alpha-bungarotoxin (alpha-Bgt). Previous studies found that mAb 35-AChRs are found at both synaptic and extrasynaptic sites, while alpha-Bgt-AChRs are found exclusively at extrasynaptic sites. To gain a three-dimensional understanding of the distribution of these two AChR classes and their spatial relationship to synaptic sites, we have visualized mAb 35-AChRs and alpha-Bgt-AChRs immunofluorescently using laser scanning confocal microscopy and have compared their distribution with that of the synaptic vesicle antigen SV2. Both mAb 35-AChR and alpha-Bgt-AChRs are found in clusters that are widely distributed over the surface of embryonic chicken ciliary ganglion neurons. Some mAb 35-AChRs are located at synaptic sites, but the bulk of them are located extrasynaptically in well-defined patches measuring 1-4 micron in diameter. alpha-Bgt-AChRs are found almost exclusively in these extrasynaptic sites, which thus contain both AChR types. These sites are often surrounded by elements of the synaptic calyx but are themselves largely free of SV2 antigen. In 14 week chickens the relationship between mAb 35-AChRs, alpha-Bgt-AChRs, and synaptic sites is similar to that in embryos except that in this instance individual synaptic boutons are often surrounded by AChR-containing patches. These results suggest that most surface AChRs in both embryonic and mature chicken ciliary neurons are perisynaptic, which raises questions about the function of these AChRs.