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1. Surplus acetylcholine (ACh) is the extra ACh that accumulates in cholinergic nerve endings when they are exposed to an anticholinesterase agent. The synthesis and turnover of this ACh was examined in the cat's superior cervical ganglion.

2. Surplus ACh did not accumulate in chronically decentralized ganglia perfused with eserine-choline-Locke solution, and this shows that it is stored in presynaptic nerve terminals.

3. Surplus ACh accumulated more rapidly in ganglia perfused with eserine than in ganglia perfused with neostigmine or with ambenonium; accumulation was delayed by 45-60 min when a quaternary anticholinesterase was used. However, the release of ACh upon preganglionic nerve stimulation was the same during perfusion with eserine, neostigmine or ambenonium. It is concluded that intracellular acetylcholinesterase normally destroys surplus ACh, whereas extracellular enzyme destroys released ACh.

4. When ganglia were perfused with [³H]choline and eserine, the surplus ACh that accumulated was labelled but its specific radioactivity was only 38% of that of the choline added to the perfusion fluid.

5. Surplus ACh was not released by nerve stimulation and was not mobilized for release during, or after, prolonged nerve stimulation. It is concluded that ACh released by nerve impulses is replaced by synthesis at the site of ACh storage and not by movement of ACh from the surplus pool.

6. The accumulation of surplus ACh no more than doubled the total ACh content of ganglia, but turnover of ACh continued when the total amount was constant. Surplus ACh may contribute to spontaneous ACh output from eserinized preparations.

7. When ganglia were perfused with a medium containing high K⁺ (56 mM), surplus ACh was released.