Acetylcholine release at identified nerve terminals in the organ-cultured frog neuromuscular preparation

1. The frog cutaneous pectoris neuromuscular preparation was maintained in organ culture for a few days, at either 24 or 14 degrees C. The synaptic activity of identified nerve terminals was repeatedly examined in order to describe the sequence and time course of changes from normal synaptic activity to a complete synaptic silence. 2. We found that the 'transient stage' (stage II, according to Ko, 1981) consists of at least three distinct periods each characterized by a unique trend of change in synaptic activity. The initial change involved a simultaneous decay of both spontaneous and nerve stimulation-evoked release of acetylcholine (sub-stage II1). Subsequently, the frequency of spontaneous miniature endplate potentials (MEPPs) increased gradually, while the evoked release continued its monotonous decay (sub-stage II2). During the third sub-stage spontaneous MEPPs, but no evoked endplate potentials (EPPs), were observed (sub-stage II3). 3. Statistical properties of acetylcholine release in still-transmitting junctions at sub-stage II2 and in non-transmitting junctions at sub-stage II3 were investigated. MEPPs with skewed amplitude histograms and bursting behaviour were evident at both sub-stages. However, the incidence and the extent of these distortions were higher in the non-transmitting junctions. 4. An inverse relation between the quantal content of evoked release and the rate of spontaneous secretion was found in the transmitting junctions. 5. These results suggest that some of the deterioration of synaptic activity in organ culture is caused by an impairment of the release process itself. Possible cellular mechanisms are discussed.