DEVELOPMENT AND REGULATION OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

促黄体生成素释放激素(LHRH)神经元来源于 嗅觉定位并迁移到大脑中，在那里它们成为完整的 下丘脑-垂体-性腺轴的成员。我们研究了 LHRH神经元迁移至中枢神经系统的机制 转基因动物，以及在鼻部外植体中。内在的和 跨突触调节LHRH基因表达、多肽合成和蛋白合成 出生后LHRH神经元(中枢神经系统)分泌与胚胎LHRH的比较 用长期器官培养法研究神经元(中枢神经系统外) 和鼻腔外植体。 基于LHRH神经元在外周迁移的假设 细胞表面黏附的(P+)嗅觉阳性轴突 分子，我们正在检测特定的碳水化合物部分，可能 “突出显示”这些粘合剂分子。利用凝集素细胞化学，我们 发现：1)鼻外植体中的嗅轴突表达几种糖 2)体外碳水化合物的表达模式相似 3)在体外，D-N-乙酰氨基葡萄糖齐聚物 在与LHRH神经元相关的P+轴突上检测到 定位于LHRH神经元，这些神经元从外植体中迁移出来。vbl.使用 衣霉素，我们发现在早期的时间窗口中，抑制N- 糖基化可阻止P+轴突的生长，但不影响 LHRH神经元与这些纤维的联系。使用LHRH启动子 融合到荧光素酶记者，我们试图监测迁移的LHRH 原位神经元。然而，荧光素酶信号不够强劲，不足以 侦测到。我们现在已经将LHRH启动子与Lac Z报告程序融合在一起，并 目前正在使用这个标签优化荧光信号。 使用两种不同的转录抑制物，我们扩大了我们的 LHRH信使核糖核酸稳定性和转化率的研究。使用第二个信使 类似的，我们正在检查(刺激后1-24小时)LHRH的变化 确定a)在LHRH细胞中活跃的第二信使系统；以及 B)监测LHRH mRNA水平变化的最佳时间点 用神经递质刺激。 目前，我们正在确定：1)N-糖基化的机制 “引导”P+轴突生长；2)表达D-N-的分子的作用 乙酰氨基葡萄糖低聚物对LHRH神经元运动和/或嗅觉的影响 轴突生长；3)培养的LHRH神经元是否在 4)标记的LHRH神经元是否可以在 以监测胚胎外植体中的运动和/或确定 器官型脑片中出生后LHRH神经元的膜特性； 5)GABA对胚胎和出生后LHRH基因表达的影响 LHRH神经元。

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.