The aim of this project is to study the functional significance of additional transmitter substances in motoneurons. We have shown that, in insects and crustacea, the pentapeptide neurotransmitter candidate proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is present in individually identifiable skeletal motoneurons. Our studies will focus on a neuromuscular system in crayfish which contains just five identified excitatory motoneurons. We have shown that three of these neurons contain the neuropeptide proctolin, in addition to their 'conventional' transmitter, which is thought to be L-glutamate. We propose to examine the relative contributions of the two transmitter substances to generating tension in the muscle. We shall demonstrate release of proctolin and shall examine the factors that lead to the release. We shall measure tension and correlate transmembrane potential and conductance changes produced by application of proctolin and L-glutamate, alone and in combination. The results will be compared to the effects of selective stimulation of the motoneurons that contain proctolin and those that do not contain proctolin. We will further elucidate the role of proctolin by eliminating it's effect(s) with either a proctolin-specific antiserum or by selectively depleting the peptide from terminals by removing the motoneuron's cell body. If time permits, we will examine the ultrastructure of nerve terminals depleted of the peptide and compare them and terminals of non-proctolin containing axons to normal proctolin-containing terminals. This could provide insight into the packaging of multiple transmitters at the neuromuscular junction. Finally, we will examine biochemically and physiologically the levels of the two transmitters in these neurons during the course of two behavioral changes, which involve an altered use of these muscles, in order to examine, at a cellular level, potential regulatory roles of additional transmitters. This research has considerable health relevance to the extent that specific neuromuscular disorders result from abnormal levels and functioning of 'extra' transmitters.
