Development of neuromuscular transmission in a larval tunicate

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Abstract
The time sequence of the development of acetylcholinesterase (AChE), acetylcholine (ACh) receptors and functional synapses on the embryonic muscle membrane in a tunicate larva (Halocynthia roretzi) was investigated in vivo. The fertilized tunicate egg was incubated in natural sea water at 9.degree. C. Free-swimming larva were hatched [which had 6 striated muscle fibers in the tail], 68 h after fertilization. The developmental stage of the embryo was indicated by the developmental hours after fertilization. The transmitter at the neuromuscular junction in the hatched larva was ACh. Neuromuscular transmission was completely blocked by D-tubocurarine (1-5 .times. 10-5 M). Eserine (5 - 10 .times. 10-7 M) approximately doubled the time constant of the falling phase of miniature excitatory junctional currents (ejc). The reversal potential of the membrane response to iontophoretically applied ACh was -10 mV and similar to that of ejc. AChE was present on the muscle membrane surface. AChE activity was visible histochemically on the embryonic cell membrane in the presumptive muscle region as early as the late gastrula stage (27 h after fertilization, 12 h before the ACh response appeared). The response to iontophoretically applied ACh was present at 39 h after fertilization but was not evoked at 38 h. Between 39 - 41 h after fertilization, the ACh responses increased rapidly, then remained relatively unchanged until larval hatching. The stage of the initial appearance of the ACh response corresponded to the stage when the Ca current abruptly increased in the muscle membrane. The 1st sign of neuromuscular transmission was appearance of a giant ejp with uniform amplitude (about 15-20 mV) and slow time course (time constant of the falling phase of a giant ejc was 23.4 .+-. 6.9 ms, mean and SD, at -60 mV and 11.degree. C). Within a few hours, these giant ejp disappeared and were successively replaced by medium-sized ejp and then ejp similar to those seen in hatched larvae (time constant of the falling phase of a miniature ejc was 8.5 .+-. 1.8 ms at -60 mV and 11.degree. C).