Synaptic and extrasynaptic distribution of two distinct populations of nicotinic acetylcholine receptor clusters in the frog cardiac ganglion.

We examined the distribution of neuronal nicotinic acetylcholine receptor clusters in relation to synaptic sites on autonomic neurons in the frog heart using immunofluorescence techniques and laser scanning confocal microscopy. Acetylcholine receptor clusters were visualized using the rat anti-Electrophorus acetylcholine receptor monoclonal antibody no. 22 and cyanine 3.18-labelled goat anti-rat secondary antibody. Synaptic boutons were labelled with the mouse anti-synaptic vesicle protein SV2, monoclonal antibody no. 10h and cyanine 5.18-labelled goat anti-mouse secondary antibody. Acetylcholine receptor clusters on the neuronal surface exist in two populations that vary in size, staining intensity, and surface distribution. The more prominent population consists of large, brightly stained clusters numbering 30 +/- 15 per cell, while the second class is smaller and less brightly stained and numbers over 100 per cell. The large clusters tend to be organized into groups of 2-6 members. This arrangement results from the fact that 80% of the large clusters colocalize at synaptic boutons and that single boutons can have several associated clusters. The remaining 20% of large/bright acetylcholine receptor clusters are extrasynaptic, but they, too, are clustered and are found in close proximity to synaptic boutons. The small/dim acetylcholine receptor clusters are randomly distributed over the cell surface. The large/bright synaptic acetylcholine receptor clusters presumably underlie fast excitatory synaptic transmission. The small/dim clusters and the large/bright extrasynaptic clusters may represent intermediates in the metabolism of large/bright synaptic clusters.