The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation

Abstract The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 μ M choline, washed, resuspended, and then incubated for 10 min in choline‐free buffer containing 4.74 (Krebs‐Ringer bicarbonate, KRB) or 25 m M KC1. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 μ M choline did not lead to a further increase, but preincubation with 25 or 50 μ M choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 μ M choline. When tissues were subsequently incubated for 10 min with either KRB or 25 μ M KC1, ACh release from slices preincubated with 50 μ M choline was greater than from slices preincubated with 0, 10, or 25 μ M choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 m M KC1, the ACh content of slices preincubated with 0 or 10 μ M choline decreased significantly, whereas that of slices preincubated with 25 or 50 μ M choline did not. Although ACh synthesis by slices incubated with KRB did not differ significantly among groups, synthesis by slices incubated with 25 mM KC1 was significantly greater for tissues preincubated with 50 μ M choline than for those preincubated with 0, 10, or 25 μ M choline. Preincubation in the absence of choline decreased choline levels by 43%, whereas preincubation with 50 μ M choline increased levels by 55%; choline levels were unaltered by preincubation with 10 μ M choline. Following incubation with 25 m M KCl, the choline content of slices preincubated with 50 μ M choline decreased by 0.59 nmol, whereas the content of slices preincubated with 10 μ M choline decreased by 0.33 nmol, suggesting that the former used more of its tissue choline to support ACh synthesis; choline levels did not change in slices preincubated in the absence of choline. These results suggest that prior exposure of hippocampal slices to choline provides precursor that is used during a subsequent incubation with 25 m M KCl to support the synthesis of ACh.