Acetylcholine release evoked by single or a few nerve impulses in the electric organ of Torpedo.

1. The acetylcholine (ACh) store in the Torpedo electric organ was partially labelled with choline and acetate at the same molar concentration but with different isotopes. Under these conditions the two precursors were incorporated into ACh in a ratio 1 to 1. 2. After a single electrical stimulus, or a brief burst of stimuli, the compound electroplaque potential (e.p.p.) was recorded and the radioactive choline and/or acetate counted in the perfusion fluid, providing a sensitive assay for ACh release in the absence of anticholinesterase drugs. 3. The so-called depression of transmission was found to be due to progressive impairment of ACh release in the successive impulses evoked by repeated stimuli. 4. In a pair of impulses separated by 50 ms interval, less ACh was released by the second than by the first impulse; this explained why the size of the second e.p.p. was depressed, using a direct measurement of ACh. 5. In repetitive stimulations of longer duration, the maximum rate of release declined as the activity was prolonged. Thus the tissue progressively lost its ability to ensure release at high frequencies. 6. An unexpected finding was that anticholinesterases like eserine or pre-treatment with fluostigmine (DFP) greatly reduced ACh release even by a single impulse. 7. Evoked ACh release and e.p.p. amplitude were both maximum between 10 and 20 degrees C. At higher temperatures, the evoked release decreased as the spontaneous release increased. 8. Changes in external Ca2+ and Mg2+ produced similar changes in the e.p.p. and evoked ACh release. The dose--response curve for Ca dependency of ACh release was very steep with a Hill's coefficient of 3.2. 9. With a single stimulus in the presence of 4-aminopyridine, there was a dramatic enlargement of the e.p.p. and a still larger potentiation of the evoked ACh release. 10. It has been possible with this approach to avoid the inconveniences often encountered in simliar studies, i.e. repetitive stimulation, low Ca solutions and cholinesterase inhibition. This permitted a good correlation between electrophysiological and biochemical estimates of transmitter release even by a single nerve impulse.