In the experiments carried out to demonstrate humoral transmission at the neuromuscular junction, Dale, Feldberg & Vogt (1936) showed that the stimulation of the motor nerve while the muscle was perfused with eserinized Ringer's solution was -followed by the appearance of acetylcholine (ACh)
EXPERIMENTAL METHODSFreshly excised rabbit hearts were perfused at 35-37°C with Locke's solution (containing 0 05 % NaHCO3 and 0-1 % dextrose) until all traces of blood were removed and the beat was well established. The reservoir in the water-bath above the heart, having been emptied of Locke's solution, was then refilled with about 30 ml. Locke's solution containing eserine sulphate 4 x 10-6g/ ml. From the reservoir the solution ran down a jacketed tube to a cannula tied in the aorta. The heart was enclosed by a glass cup, in the bottom of which the perfusate collected and ran out through a tube which then turned horizontal for a distance of about 17 cm. At the end of the horizontal tube the perfusate was carried vertically upwards by a stream of oxygen, and so back to the reservoir. The perfusing fluid was thus circulated through the heart and back to the reservoir for 40 min or longer during which samples were withdrawn to test on the dorsal muscle of the leech. In some experiments when the fluid was withdrawn it was replaced by fresh solution for a second period of perfusion.To obtain the active material in the perfusate in a more concentrated form, it was taken to dryness by the process of 'freeze-drying'. The residue was extracted with absolute ethanol saturated with NaCl, using 7-5 ml. ethanol for the residue from 30 ml. perfusate. The alcoholic extract was then taken to dryness at a temperature not exceeding 400 C under reduced pressure.The residue was dissolved in 2-5 ml. of a mixture of 1-65 ml. frog saline (0-7 % NaCl solution) and 0-85 ml. distilled water.The extracts so prepared were tested on cat blood pressure, frog heart and frog rectus muscle.For the blood pressure cats were anaesthetized with chloralose; when small amounts of ACh did