Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation

Summary Two different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors.When the whole nerve-muscle preparation (110–180 mg) was incubated with [³H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow.Incubation of the end-plate preparation (16–29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [³H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [³H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [³H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [³H]acetylcholine tissue content per pulse.It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [³H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used. Removal of most of the muscle fibres reduces the possibility for [³H]choline to be captured by them and then more radioactive choline can enter the end-plate region. From this end-plate preparation a calcium-dependent release of radioactive transmitter can be measured in the absence of cholinesterase inhibitors.