DEVELOPMENT AND REGULATION OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

点击翻译按钮获取中文摘要

Luteinizing hormone releasing hormone (LHRH) neurons are derived from the olfactory placode and migrate into the brain, where they become integral members of the hypothalamic-pituitary-gonadal axis. We study the mechanisms underlying LHRH neuronal migration into the CNS in normal and transgenic animals, as well as in nasal explants. The intrinsic and transsynaptic regulation of LHRH gene expression, peptide synthesis and secretion in postnatal LHRH neurons (in the CNS) vs embryonic LHRH neurons (outside the CNS) is studied using long-term organotypic cultures and nasal explants, respectively. Working on the hypothesis that LHRH neurons migrate on peripherin positive (P+) olfactory axons via adhesion between cell surface molecules, we are examining specific carbohydrate moieties which might "highlight" these adhesive molecules. Using lectin cytochemistry, we found that: 1) olfactory axons in nasal explants express several sugar moieties; 2) the pattern of carbohydrate expression in vitro is similar to that in vivo; and 3) in vitro, D-N-acetyl-glucosamine oligomers are detected on P+ axons with which LHRH neurons are associated and are localized to LHRH neurons which migrated out of the explant. Using tunicamycin, we found that during an early time window, inhibition of N- glycosylation prevents outgrowth of P+ axons, but does not affect the association of LHRH neurons with these fibers. Using the LHRH promoter fused to a luciferase reporter, we attempted to monitor migrating LHRH neurons in situ. However, the luciferase signal was not robust enough to detect. We have now fused the LHRH promoter to the Lac Z reporter and are currently working on optimizing a fluorescent signal using this tag. Using two different transcription inhibitors, we have broadened our studies on stability and turnover rates of LHRH mRNA. Using 2nd messenger analogs, we are examining (1-24 hr after stimulation) changes in LHRH mRNA to determine a) the 2nd messenger systems active in LHRH cells; and b) an optimal timepoint to monitor changes in LHRH mRNA levels after stimulation with neurotransmitters. Currently, we are determining: 1) the mechanism by which N-glycosylation "directs" outgrowth of P+ axons; 2) the role of molecules expressing D-N- acetyl-glucosamine oligomers on LHRH neuronal movement and/or olfactory axon outgrowth; 3) whether LHRH neurons in cultures release LHRH in a pulsatile manner; 4) whether tagged-LHRH neurons can be visualized in situ to monitor movement in embryonic explants and/or determine the membrane properties of postnatal LHRH neurons in organotypic slices; and 5) the effect of GABA on LHRH gene expression in embryonic and postnatal LHRH neurons.