The anterior portion of intrinsic buccal muscle (I3a) is innervated by two excitatory motor neurons, B3 and B38, and the newly identified inhibitory motor neuron, B47. We show that B47 is cholinergic while B3 and B38 are not. B3 and B38 have previously been shown to express the neuropeptides FMRFamide and the small cardioactive peptides (SCPs) A and B, respectively. We present evidence here that B47 synthesizes the neuropeptide myomodulin A (Mma). When placed in culture, B3, B38, and B47 continued to synthesize their respective peptides. These peptides were released in a stimulation- and Ca(2+)-dependent manner, suggesting that they are transmitters in these neurons. By using B3-evoked excitatory junction potentials (EJPs) and muscle contractions as assays, we next examined the modulatory effects of superfusion of peptides and stimulation of motor neurons B38 and B47. Superfusing the muscle with low concentrations of the SCPs, FMRFamide, or Mma enhanced B3-evoked EJPs and contractions. Stimulation of B47 simultaneously with B3 reduced the amplitude of B3-evoked contractions. However, when either B47 or B38 was stimulated in extended bursts designed to release their peptide transmitters, subsequent B3-evoked EJPs and contractions were enhanced. We believe that this modulation is due at least in part to the release of peptides from the terminals of B38 and B47. The SCPs potently increase cAMP levels in I3a muscle fibers. Likewise, stimulation of B38 in extended bursts increased cAMP levels in the muscle. This provides independent evidence that the SCPs are released from B38 terminals in the muscle. Therefore, we have described a neuromuscular preparation amenable to the study of both excitatory and inhibitory motor neurons that utilize a variety of conventional and peptide transmitters. Our results suggest that these motor neurons can function in two states. When stimulated in single brief bursts, they primarily release conventional transmitters. When stimulated in a series of prolonged bursts, they release both conventional transmitters and peptide cotransmitters. These dual states are most pronounced in the case of B47, which, depending on the stimulation paradigm, can act selectively to inhibit or enhance the effects of a second motor neuron innervating the same muscle.
In the present study, we have demonstrated that ACh is the predominant fast excitatory transmitter used by identified motor neurons innervating feeding muscles in Aplysia. A detailed study of ACh metabolism was then carried out in a well-characterized neuromuscular preparation, intrinsic muscle 5 (15). This neuromuscular system has a high-affinity uptake system for choline. The rate of uptake of choline was increased by motor neuron stimulation, and this increased uptake appears to be selectively targeted to motor neuron terminals. These properties appear similar to those observed in vertebrate neuromuscular preparations. However, we have made two observations that are surprising in light of our knowledge concerning the vertebrate neuromuscular junction where released ACh is rapidly hydrolyzed by acetylcholinesterase (AChE) to choline, which is then taken up by a high-affinity uptake system. This Aplysia neuromuscular system has limited endogenous AChE activity and contains a separate high-affinity uptake system for ACh itself that actually has a higher velocity than that for choline uptake. It is possible that the uptake system for ACh is involved in terminating the action of released transmitter in a manner similar to that previously described for noncholinergic transmitters. Using this preparation, we have demonstrated release of labeled ACh in response to intracellular stimulation of identified motor neurons. The release per spike appears to be highly plastic,increasing markedly with stimulation frequency. This preparation is amendable to study the regulation of release of peptide and conventional transmitters from the terminals of individual neurons.
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