1998 Volume 74 Issue 6 Pages 145-148
The aim of this work was to observe at electron microscopic level acetylcholinesterase (AChE) activity of frog neuromuscular junction revealed by an histoenzymatic reaction made on living tissues. In this purpose, Karnovsky's histochemical medium was used for the detection of AChE activity at concentrations ten times more diluted than in the original method. The medium was dissolved in Ringer solution containing citrate buffer, a chelating agent of cupric ions, and additional calcium chloride. Living nerve muscle preparation of the bullfrog was incubated in this histochemical Ringer solution. The muscle activity in response to electrical stimulation of motor nerve (0.1Hz) was monitored by recording of muscular contraction. The incubation was not prolonged beyond 15min, since a blockade of the neuromuscular transmission steadily occurred at that time. The tissue remained alive, however, since transmission was recovered by washing with fresh Ringer solution containing citrate buffer and additional calcium chloride. The primary histochemical precipitates endowed with oxidoreductase-like activity were revealed by secondary incubation with diaminobenzidine (DAB) and hydrogen peroxide. After osmification, osmium black of DAB gave fine localization of AChE activity in the synaptic cleft under the electron microscope.
