Morphological changes related to reconstituted acetylcholine release in a release-deficient cell line

The membrane changes accompanying Ca2+-dependent acetylcholine release were investigated by comparing release-competent and release incompetent clones of mouse neuroblastoma N18TG-2 cells. No release could be elicited in nati ve N18 cells or in a N18-choline acetyltransferase clone in which acetylcho line synthesis was induced by transfection with the gene for rat choline ac etyltransferase. However, acetylcholine release was operative in a To/9 clo ne which was co-transfected with complementary DNAs from rat choline acetyl transferase and Torpedo mediatophore 16,000 mel. wt subunit. In thin sectio ns, the aspect of resting N18 and To/9 cells was identical: a very dense cy toplasm with practically no vesicle-like organelles. Cells were chemically fixed at different times during a stimulation using A-23187 and Ca2+, and e xamined following both freeze-fracture and thin section. Stimulation of To/ 9 cells induced a marked change affecting the intramembrane particles. The number of medium-sized particles (9.9-12.38 nm) increased, while that of th e small particles decreased. This change was not observed in control, relea se-incompetent cell lines. In the To/9 clone (but not in control clones), t his was followed by occurrence of a large new population of pits which init ially had a large diameter, but subsequently became smaller as their number decreased. Coated depressions and invaginations became abundant after stim ulation, suggesting an endocytosis process. By considering the succession o f events and by comparison with data from experiments performed on synapses in situ, it is proposed that a particle alteration was the counterpart of acetylcholine release in co-transfected To/9 cells; this was followed by a massive endocytosis. (C) 1999 TBRO. Published by Elsevier Science Ltd.