Autoradiographic demonstration of the sodium-dependent high-affinity choline uptake system

Mouse phrenic nerve-hemidiaphragm preparations were incubated for 10 min at 37 degrees C in an oxygenated medium containing all the constituents reported as essential for the sodium-dependent high-affinity choline uptake system, including [3H]choline chloride, 3.1 Ci/mmol, 2.5 microM. The phrenic nerve was stimulated by a suction electrode with square waves of 4-msec duration, 3 V, at 10 Hz; some preparations were not stimulated. Similar preparations in which 25 microM hemicholinium was included in the medium served as controls. After exposure of emulsion-coated coverslips apposed to the preparations for 53 days, development, and staining of the motor endplates for acetylcholinesterase, there was a marked accumulation of silver grains over the motor endplates of stimulated preparations. Unstimulated preparations showed considerably lighter deposits; accumulation was blocked by hemicholinium.