Demonstration of Acetylcholine Release by Measuring Efflux of Labelled Choline From Cerebral Cortical Slices

To demonstrate release of ACh in the absence of inhibition of cholinesterase, slices of cerebral cortex were incubated with [3H]choline, after which they were placed in a tissue bath for superfusion. Hemicholinium (HC‐3) increased the spontaneous efflux of [3H]choline. Electrical stimulation at 4/s increased the efflux of [3H]choline to the same extent whether the slices were stimulated early or late during superfusion. The effect of stimulation on efflux of [3H]choline was abolished by tetrodotoxin and by the absence of calcium. The extent of choline efflux resulting from stimulation, as calculated from the specific radioactivity of the incubation medium, was the same when the slices were incubated with 0.1 or 1.0mM choline, but was less with lower concentrations of choline. We conclude that the increased efflux of [3H]choline evoked by stimulation probably originates from stores of [3H]ACh synthetized during incubation.