Pretreatment with hypertonic solutions increases quantal size at the frog neuromuscular junction

Abstract

Miniature end-plate potentials (MEPPs) are increased in size by pretreatment in a hypertonic Ringer. This paper is about the treatments that increase quantal size and the evidence that it occurs by packing more acetylcholine (ACh) in the quanta. Sartorius muscles were soaked for 2 h in a hypertonic Ringer, containing 200 mM NaCl in place of the usual 120 mM, and then returned to Ringer. In the hypertonic solution the miniature frequency was above 100/s. The treatment increased the size of the miniatures, recorded with an intracellular electrode after the return to usual Ringer, by a factor of approximately 2.5, compared with paired muscles kept in usual Ringer throughout. When the hypertonic solution was made with 200 mM sodium gluconate, after the return to usual Ringer, the miniatures increased in size by a factor of approximately 3.6. The large miniatures produced by the treatment fit a log normal probability distribution function, except for a variable small fraction of the largest events that may represent multiple releases. Miniatures recorded from untreated end plates fit better to a log normal distribution function than to a normal distribution function. The effects of the hypertonic treatments on the acetylcholine receptor (AChR) were accessed by measuring, after the return to usual Ringer, ACh noise, alpha-bungarotoxin binding, and the reversal potential. None of these measurements revealed significant differences between experimental and control preparations. The increases occurred when neostigmine was present in the hypertonic and usual solutions and when it was not, so changes in acetylcholinesterase (AChE) do not appear to be involved. Both the MEPPs and miniature end-plate currents, recorded with the two electrode voltage clamp, increased in size, so the increase is not owing to an rise in the input resistance of the muscle fiber. The increase in quantal size was prevented by including 1-10 microM AH5183 in the hypertonic solution. This drug blocks ACh uptake into synaptic vesicles. Therefore, it seems likely that the treatments act by increasing the quantity of ACh/quantum. Cyclohexamide (10 micrograms/ml), an inhibitor of protein synthesis, did not affect the increase in size caused by hypertonic solutions. Size increases were detected after 15 min in hypertonic solution. Most of the increase was complete after 1 h and there was no further increase after 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)