Regulation of acetylcholine release by presynaptic nicotinic receptors at developing neuromuscular synapses

Autoregulation of synaptic transmission in the nervous system is one of the homeostatic processes by which the transmission can be regulated according to varied physiological conditions. The neuromuscular cocultures of Xenopus laevis embryos were used to investigate the role of presynaptic nicotinic receptors in the autoregulation of developing motoneurons. The bath application of 2 microM nicotine had no significant effect on the frequency of spontaneous synaptic currents (SSCs). However, nicotine markedly increased the SSC frequency in the presence of low concentrations of glutamate (2 microM) or ATP (0.15 mM) or high K+ (8 mM), which only slightly increased the frequency of spontaneous acetylcholine (ACh) secretion. Carbachol but not oxotremorine was similar to nicotine in the positive regulation of spontaneous ACh release. Treatment with alpha-bungarotoxin, hexamethonium, d-tubocurarine, or mecamylamine, which only slightly inhibited the SSC amplitude, effectively antagonized the increasing effect of nicotine plus glutamate on SSC frequency. Local perfusion of isolated neurons with nicotine induced an inward current at nerve terminal but not at soma, suggesting that nicotinic receptors localize at nerve terminals. Both d-tubocurarine and hexamethonium, which produced tetanic fade in adult neuromuscular preparations, did not show tetanic fade at embryonic neuromuscular junction. The bath application of alpha-bungarotoxin or hexamethonium but not 6-cyano-2,3-dihydroxy-7-nitroquinoxaline inhibited the frequency of SSCs at high-activity (>3 Hz) synapses. A P2-purinoceptor antagonist, suramin, or desensitizing P2-purinoceptor with alpha, beta-methylene ATP also reduced the frequency of SSCs at these high-activity synapses. These results suggest that nicotinic receptors, P2-purinoceptors and glutamate, receptors coexist at nerve terminals of developing motoneurons. The activation of presynaptic nicotinic receptors, which cooperates with either P2-purinoceptors or glutamate receptors, may greatly increase the spontaneous ACh secretion. Endogenously released ACh and ATP are both involved in the positive regulation of spontaneous transmitter secretion at developing neuromuscular synapses.