Acetylcholine receptor-like molecules are found in both synaptic and extrasynaptic clusters on the surface of neurons in the frog cardiac ganglion

Abstract

Sixty monoclonal antibodies (mAbs) made against nicotinic acetylcholine receptors (AChRs) from electric organ were tested for their ability to cross-react at synaptic sites in the frog cardiac ganglion, where transmission is mediated by ACh via nicotinic receptors. Forty-one of the mAbs tested were known to bind to AChRs in frog skeletal muscle (Sargent et al., 1984). As determined by double-label immunofluorescence microscopy, 8 of the 60 mAbs bound to small, punctate sites that lay within areas of synaptic contact, marked by an anti-synaptic vesicle antibody. One of the 8 cross-reacting antibodies that produced particularly intense staining (mAb 22) was chosen for further study and was found to bind to the postsynaptic membrane beneath active zones, as determined by peroxidase immunocytochemistry and electron microscopy. This suggests that mAb 22 crossreacts with AChRs on the ganglion cell surface. While most mAb 22 immunoreactivity was located in the postsynaptic membrane, about 20% of the peroxidase-stained patches were extrasynaptic (327 patches analyzed from 8 ganglia). Virtually no peroxidase-stained patches were observed when an isotype-matched control mAb was used in place of mAb 22. In frog skeletal muscle peroxidase-stained patches obtained with mAb 22 were found exclusively at synaptic sites (179 patches examined from 6 muscles). These results suggest that extrasynaptic patches of AChRs are present in innervated autonomic neurons but not in innervated skeletal muscle.