Effects of denervation on acetylcholine receptor clusters on frog cardiac ganglion neurons as revealed by quantitative laser scanning confocal microscopy

Abstract

We examined the effects of denervation on clusters of nicotinic acetylcholine receptors (AChRs) on autonomic neurons in the frog heart using immunofluorescence techniques and laser scanning confocal microscopy. We showed previously that normally innervated neurons have both large, brightly stained AChR clusters and small, dim AChR clusters. A majority (80%) of the large/bright AChR clusters are located at synaptic sites, whereas the small/dim clusters are distributed widely over the cell surface. Here, we use image analysis to identify these two classes of clusters on images generated from stacks of optical sections through neuronal cell bodies and to examine the effects of denervation on their number, size, and brightness (pixel intensity). Denervation reduces the number of large/ bright AChR clusters per cell to < 10% of sham-operated values and increases the number of small/dim clusters per cell by two- to threefold. These changes occur at 4 d of denervation, the earliest time examined, and are sustained for 6 weeks. The size of large/bright AChR clusters is decreased compared with sham-operated controls, and their brightness is unchanged. The size of small/dim AChR clusters is unchanged by denervation, but their brightness is increased. Denervation results in a shift in the contribution of each AChR cluster class to the total measurable AChR pool-from one dominated by large/bright clusters to one dominated by small/dim clusters. These results show that the nerve terminals on cardiac ganglion neurons appear to exert a continual and reversible influence on the organization of the postsynaptic membrane.