The long term goal of the proposed research is to understand the mechanisms that underlie the formation of functionally appropriate synaptic connections during development. An important aspect of this process is determination of the transmitter phenotypes of individual neurons. Many neurons contain one or more neuropeptides in addition to a classical neurotransmitter. Neuropeptides function as transmitters or neuromodulators in the mature nervous system and evidence suggests that they also play important roles in development. These findings raise the question of what developmental mechanisms are responsible determining the complement of peptides a particular neuron expresses. We found that acquisition by sympathetic neurons of their adult peptide phenotype is a complex process, distinct for each peptide examined, and involves both induction and restriction of expression. Glucocorticoids, presynaptic innervation and target interactions contribute. We also found that after axotomy leukemia inhibitory factor (LIF), produced in the ganglion, induces novel peptide phenotypes in mature neurons. The present experiments seek to define the role(s) of candidate factors in regulating peptide expression and to learn more about how peptide content is regulated. Cell culture studies provide evidence that LIF and ciliary neurotropic factor (CNTF) modulate peptide expression in sympathetic neurons. We will characterize the possible roles of LIF and CNTF in vivo by analyzing mutant mice singly or doubly deficient in these differentiation factors. We will examine the expression of cytokine receptor subunits and/or downstream signalling components in developing and axotomized neurons to determine if expression is correlated with the ability of cytokines to alter peptide expression. The cells responsible for producing LIF in axotomized ganglia will be identified and the functional significance of peptide induction by axotomy will be explored by analysis of peptide receptor mRNAs. A second class of molecules that could contribute to peptide determination are the neurotrophins (NT). To obtain evidence for a direct role, the effects of NTs on peptide expression will be examined in cell culture. NTs could also play an indirect role by controlling the distribution of axons within a complex target tissue with multiple sources of differentiation signals. To examine this possibility, footpads of transgenic mice in which the normal balance of NT production has been perturbed will be analyzed. Data suggest that enkephalin (Enk) content is regulated by post- transcriptionally. We will determine when proenkephalin mRNA levels and gene transcription are correlated with Enk-IR and if the expression of candidate processing enzymes is regulated in several experimental paradigms. The studies proposed will provide new important information concerning the mechanisms that determine neuropeptide phenotype during development and that regulate plasticity of neuropeptide expression. They may elucidate the pathogenesis of development disorders of the nervous system and of neurogenerative diseases involving peptide dysfunction.
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