The Removal of Acetylcholine by Diffusion at Nicotinic Synapses in the Rat Otic Ganglion

1. We have examined the clearance of synaptically released acetylcholine in the otic ganglion when acetylcholinesterase was blocked with eserine. 2. Intracellular recordings were made from otic ganglion neurones, in vitro. The decay of the excitatory postsynaptic potential (EPSP), in response to stimulation of afferent fibres, was greatly prolonged in the presence of eserine. Low frequency (0.05-4 Hz) repetitive synaptic stimulation led to a slow depolarization of the postsynaptic cell that persisted throughout the period of stimulation. This slow depolarization was blocked by the nicotinic antagonists mecamylamine (100 microM) or (+)tubocurarine (100 microM), but was unaffected by atropine (1 microM), indicating that the response was due to the activation of nicotinic receptors. 3. Following 2 Hz synaptic stimulation (30 s), the rate of rise of the slow depolarization had a time constant of 3.1 +/- 0.4 s and a peak amplitude of 12 +/- 1 mV. Upon cessation of stimulation, the depolarization decayed to resting levels with a time constant of 18.3 +/- 1.5 s (n = 23). At increasing stimulation frequencies the rate of rise of the depolarization increased. Lowering the probability of release, by adding cadmium to the perfusing solution or by lowering extracellular calcium, slowed the rise time of the response. 4. Both the onset and decay kinetics of the slow depolarization had a low temperature sensitivity, indicating that they reflect diffusional processes. 5. Repetitive stimulation (2 Hz) of the afferent nerve supplying the ganglion, in the presence of eserine, also caused a slow depolarization in cell in which we could not demonstrate a synaptic input. This indicates that synaptically released acetylcholine can spill over onto nearby neurones. 6. We conclude that at parasympathetic synapses, under physiological conditions, transmitter action is terminated by the enzymatic degradation of acetylcholine. When acetylcholinesterase is blocked, acetylcholine accumulates because its removal by diffusion is slow.