Acetylcholine turnover in an autoactive molluscan neuron

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Abstract
We have studied acetylcholine (ACh) turnover at the cholinergic synapse between an identified motoneuron, the salivary burster (SB), and the muscle cells of the salivary duct (SD) in the terrestrial molluskLimax maximus. Electrophysiological recordings were made of the SB action potentials and the SB-elicited junction potentials (JPs) on the SD. The amplitude of the JP was used as a measure of ACh release by the SB. The SB is an autoactive neuron that discharges 1 to 12 bursts of action potentials per min. During sustained bursting activity, the SB is able to maintain transmitter release for 18 hr even in the absence of exogenous choline. The size of SB-elicited JPs does not vary during 18 hr of activity. If the choline uptake blocker, hemicholinium-3 (HC-3; 20µM), is present in the saline, transmitter release and JP size are depressed by about 30% after 14 hr of activity. Thus, the SB is partially dependent upon choline reuptake for maintained ACh synthesis and release. In high (9.45 mM)-potassium (K+) saline, the SB fired tonically at twice its average spike frequency. JP amplitude initially increased, then declined to an amplitude which was 60% of the initial level. The addition of 20µM HC-3 to the high-K+ saline caused a 75 to 100% decrease in JP size within 30 min. Thus, during high-frequency tonic firing, the SB was primarily dependent on choline reuptake for ACh synthesis and release. After JP size had been reduced in high-K+ saline containing HC-3, the SB-SD synapse was returned to normal choline-free saline. The SB resumed bursting activity. JP amplitude gradually increased over the next 30 min. Thus, high-frequency firing in HC-3 had not depleted the SB of its entire endogenous store of choline or ACh. If the synapse was fatigued in high-K+ saline containing HC-3 and then placed in saline enriched with 300µM choline, JP size increased within minutes. Thus, uptake of choline for ACh synthesis and release may be a more rapid process than mobilization of an endogenous transmitter store. Finally, the SB-SD synapse was fatigued in high-K+ saline containing HC-3. HC-3 was then removed from the saline. The SB maintained high-frequency tonic activity. JP size did not increase unless choline was added to the saline. We conclude that the SB possesses two releasable pools of ACh, a “readily releasable” and a “reserve” transmitter store. The readily releasable pool is made up of ACh newly synthesized from choline taken up from the saline. During high-frequency firing, the SB discharges all ACh from this store. During normal bursting activity, the SB may discharge ACh from both the readily releasable and the reserve transmitter pool. The proportion of ACh released from each pool may vary with the total transmitter stores and the spiking activity of the SB neuron.