ACETYLCHOLINESTERASE ACTIVITY IN THE FROG NEUROMUSCULAR-JUNCTION OBSERVED AT ELECTRON-MICROSCOPIC LEVEL AFTER VITAL HISTOENZYMATIC REACTION

The aim of this work was to observe at electron microscopic level acet ylcholinesterase (AChE) activity of frog neuromuscular junction reveal ed by an histoenzymatic reaction made on living tissues. In this purpo se, Karnovsky's histochemical medium was used for the detection of ACh E activity at concentrations ten times more diluted than in the origin al method. The medium was dissolved in Ringer solution containing citr ate buffer, a chelating agent of cupric ions, and additional calcium c hloride. Living nerve muscle preparation of the bullfrog was incubated in this histochemical Ringer solution. The muscle activity in respons e to electrical stimulation of motor nerve (0.1 Hz) was monitored by r ecording of muscular contraction. The incubation was not prolonged bey ond 15 min, since a blockade of the neuromuscular transmission steadil y occurred at that time. The tissue remained alive, however, since tra nsmission was recovered by washing with fresh Ringer solution containi ng citrate buffer and additional calcium chloride. The primary histoch emical precipitates endowed with oxidoreductase-like activity were rev ealed by secondary incubation with diaminobenzidine (DAB) and hydrogen peroxide. After osmification, osmium black of DAB gave fine localizat ion of AChE activity in the synaptic cleft under the electron microsco pe.