SPECIFICITY OF IONIC FIXATION WITH SILICOTUNGSTIC ACID FOR CYTOCHEMICAL LOCALIZATION AND IDENTIFICATION OF ACETYLCHOLINE IN SYNAPTIC VESICLES

Silicotungstic acid (STA), as well as phosphomolybdic acid (PMA) and phosphotungstic acid (PTA), are known to exist as a large molecular complex of heteropolyanion in aqueous solution, and to be a potent precipitating agent of quaternary ammonium compounds, like acetylcholine (ACh) and choline (ionic fixation). In most cytochemical detection of acetylcholine in synaptic vesicles, STA is preferentially employed, because of the stability of the heteropolyanion structure in a wide pH range. The specificity of the ionic fixation for ACh was examined by a model experiment in vitro. Among various transmitter substances, ACh can be precipitated in the lowest concentration by STA. After the treatment of nervous tissue by STA, a bulk of ACh (more than 1/3) was recovered from the STA-precipitate. STA applied to nerve fibers in the sinus venosus of frog heart formed a punctiform precipitate in clear vesicles, but not in large cored vesicles in varicosities. It is suggested that electron-dense punctiform precipitates in synaptic vesicles in the present study and in previous cytochemical observations on neuromuscular junction represent ACh-silicotungstate precipitates. A number of staining methods with phosphotungstic acid (PTA) are known for cellular constituents such as proteins and carbohydrates insolubilized in aldehyde-fixed tissue (5, 8, 9). PTA was also used for cytochemical staining of synaptic ultrastructure (2). However, neither PTA nor its analogous compounds such as phosphomolybdic acid (PMA) and silicotungstic acid (STA) have been used to fix the synaptic ultrastructure and to preserve acetylcholine (ACh)-like cation. 2T0 whom correspondence should be sent Recently we devised a cytochemical technique of ionic fixation for synaptic ACh which is localized as a punctiform precipitate in the synaptic vesicle (12-17). The technique is based on a rapid ionic interaction between ACh and molybdic or tungstic heteropolyanions (formation of insoluble salts). Since the heteropolyanions decompose in neutral pH (10), they are used directly as aqueous solutions. In spite of a strong acidity of the solution, the ultrastructure of the tissue is well preserved. The term ionic fixation was proposed to designate the preservation of membrane ultrastructure and ACh-like cation in the synaptic vesicle. However, it was difficult