Proton NMR detection of acetylcholine status in synaptic vesicles

The storage granules of cholinergic nerve terminals, the synaptic vesicles, have a central role in the mechanism of cholinergic transmission, and direct evidence as to whether acetylcholine is stored in them in a free or partially immobilized form is therefore of interest. In some secretory systems it has become evident that low molecular weight secretion products are stored within the granules as complexes with proteins or proteoglycans. The application of high resolution NMR spectroscopy to isolated granules has recently elucidated the internal structure of secretory granules. We report here that proton NMR analysis of isolated cholinergic synaptic vesicles from Torpedo marmorata allows the identification of acetylcholine within the organelle. The spectrum shows that all the acetylcholine is dissolved in an essentially fluid phase within the core. Vesicular and uncompartmentalized acetylcholine can be distinguished from each other and in addition, any hydrolysis to choline and acetate can be monitored. The results open the possibility of studying dynamics of acetylcholine pools and their breakdown in cholinergic tissue by a direct and non-perturbing method.