The Release of Labelled Acetylcholine and Choline From Cerebral Cortical Slices Stimulated Electrically

Abstract

1 In order to establish the origin of the increased efflux of radioactivity caused by electrical stimulation of cerebral cortical slices which had been incubated with [(3)H]-choline, labelled choline and acetylcholine (ACh) collected by superfusion were separated by gold precipitation.2 In the presence of physostigmine electrical stimulation (1 Hz, 10 min) increased the release of only [(3)H]-ACh which was greatly enhanced by the addition of atropine.3 Continuous stimulation in the presence of physostigmine resulted in an evoked release of [(3)H]-ACh which declined asymptotically. This evoked release appeared to follow first-order kinetics with a rate constant which remained stable over the course of prolonged stimulation.4 The rate constant for the evoked release of [(3)H]-ACh with 1 Hz stimulation was three times greater in the presence of physostigmine and atropine than in the presence of physostigmine alone, while the size of the store from which [(3)H]-ACh was released was nearly identical under these two conditions.5 In the absence of physostigmine and atropine, stimulation caused the appearance of only [(3)H]-choline in the samples.6 Reduction of [(3)H]-ACh stores before the application of physostigmine resulted in a reduced evoked release of total radioactivity, both in the absence or presence of physostigmine and atropine, and decreased the evoked release of [(3)H]-ACh without affecting the release of [(3)H]-choline.7 Results suggest that electrical stimulation of cortical slices which had been incubated with [(3)H]-choline causes the release of only [(3)H]-ACh, both in the presence or absence of an anticholinesterase. The evoked increase in the efflux of total radioactivity is therefore a good measure of the release of [(3)H]-ACh.

The Output Per Stimulus of Acetylcholine From Cerebral Cortical Slices in the Presence or Absence of Cholinesterase Inhibition

“…The preparation of cortical slices and the technique of superfusion and stimulation were similar to that described in the previous paper (Richardson & Szerb, 1974) (Dudar & Szerb, 1970). The leech muscle was bathed in dilute Krebs solution containing physostigmine 10-4 M but, instead of using water as a diluent as described previously, the Krebs solution was diluted with an equal volume of NaH2PO4 (1.45 mM) and CaCl2 (0.18 mM).…”

“…The method of preparation, incubation and superfusion of the cortical slices (0.4 mm wide strips) was the same as described in the previous paper (Richardson & Szerb, 1974). Incubation fluid always contained 50MM, 2,Ci/ml [3 HI-choline.…”

“…This method necessitated the use of a cholinesterase inhibitor which, as previous observations have shown (Szerb & Somogyi, 1973), depresses the release of ACh. The second method, in which the evoked release of labelled choline was measured (Somogyi & Szerb, 1972;Richardson & Szerb, 1974), avoided the use of a cholinesterase inhibitor, but gave information on the output of preformed ACh only. Results indicate that under physiological conditions the output of ACh per stimulus from the cerebral cortex is frequency-dependent over a wide range of frequencies in a similar way to the release of ACh from the cholinergic terminals of the guineapig ileum.…”

The effects of atropine on [3H]acetylcholine secretion from guinea‐pig myenteric plexus evoked electrically or by high potassium

“…Fig. 1 B) (Szerb, 1975(Szerb, , 1976Wikberg, 1977 & Szerb, 1974 (n = 8), and that of added [3H]choline in the aqueous phase 98-6±0-6 % (n = 6). After re-extraction of the organic phase with 1 M-hydrochloric acid, and freeze drying, samples were analysed by high-voltage electrophoresis (Heilbronn & Carlsson, 1960).…”

Effect of 1,3-Di-n-Butyl-7-(2-oxopropyl)-Xanthine (denbufylline) on metabolism and function of cerebral cholinergic neurons.

“…Therefore, the alteration in this uptake process is also unlikely to be involved in the cause of denbufylline-induced ACh decline in the brain. ACh release from brain slices has been determined mainly by the efflux of [3H]ACh formed from prelabeled [3H]choline (20)(21)(22), because endogenously released ACh is readily hydrolyzed by AChE. Recent observations, however, have indicated that the mechanism of endogenous ACh release from brain slices is different from that of preloaded [3H]ACh (23).…”