Separation of synaptic vesicles of different functional states from the cholinergic synapses of the Torpedo electric organ

Abstract

Synaptic vesicles were isolated on sucrose zonal gradients from perfused tissue blocks of Torpedo electric organ. They give rise to a coincident peak in the concentrations of acetylcholine and adenosine 5′-triphosphate. On low-frequency stimulation (0.1 Hz) of the nerve attached to the tissue block a distinct population of synaptic vesicles is found that sediments further into the density gradient forming a second (denser) vesicle peak. When dextran is added to the perfusate, only these denser vesicles contain electron-dense granules. This second (denser) peak contains about 25% of all vesicular acetylcholine and about 30% of the adenosine 5′-triphosphate and most of the newly synthesized acetylcholine as shown by incorporation of radiolabelled acetate. The specific radioactivity of acetylcholine in the denser vesicles after 1800 impulses is on average 16.5 times higher than that of the vesicles sedimenting at the original density and 9.2 times higher than the average of all vesicles isolated. The specific radioactivity of total tissue acetylcholine is lower than the average for all vesicles. It is concluded that stimulation makes apparent metabolic and morphological heterogeneity of synaptic vesicles. The increase in density of the vesicles containing newly synthesized acetylcholine could be due to endocytotic uptake of sucrose contained in the perfusate after the vesicle has undergone exocytosis. The results suggest that synaptic vesicles can be reloaded with transmitter and re-used even after uptake of extracellular marker.