ONTOGENY OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

促黄体激素释放激素（LHRH）神经元来源于 嗅觉基板并迁移到大脑，在那里它们成为整体 下丘脑-垂体-性腺轴的成员。研究 参与LHRH神经元向CNS移动的迁移机制， 我们使用正常和转基因动物，以及嗅觉外植体。在 此外，长期的器官型切片培养用于研究 LHRH的内在和跨突触调节机制 基因表达、肽合成和分泌 分化的LHRH神经元。 LHRH神经元在外周神经元上迁移的假设 从嗅窝到间脑的阳性（+）嗅轴突，我们 发现：（1）LHRH神经元不表达外周蛋白mRNA;（2）LHRH神经元不表达外周蛋白mRNA; 神经元不表达N-CAM mRNA，但嗅轴突为N-CAM+; （3）嗅小凹细胞差异表达外周蛋白mRNA， N-CAM mRNA，表明不同的人群。在胚胎移植中，我们 区分N-CAM+和peripherin+轴突，并证实LHRH 神经元通过peripherin+而不是N-CAM+轴突移动。审查 胚胎LHRH神经元上的电压门控和配体门控通道被揭示 高度分化的神经元的膜特征。 此外，本发明还提供了一种方法， 我们已经从转基因小鼠中产生了胚胎外植体， LHRH神经元中的荧光素酶。 当被注射了一种可检测到的信号 在来自这些外植体的裂解细胞中是可测量的。 我们检测了LHRH细胞和催产素（OT）的第二信使活性。 细胞维持在器官型切片外植体中。 毛喉素和/或佛波醇 12-肉豆蔻酸酯13-乙酸酯（PMA）处理显著降低LHRH mRNA 与此相反，毛喉素治疗显著增加了 使用放线菌素D（一种转录抑制剂）， 我们测定了神经肽mRNA的周转率：LHRH mRNA具有非常快的 周转率（约4小时），而OT mRNA则慢得多 （约40小时）。 我们认为第二信使主要起作用 增加OT细胞中OT mRNA的转录，但降低LHRH mRNA的转录 转录和/或增加LHRH神经元中的LHRH mRNA降解。 目前，我们正在确定：（1）细胞表面糖蛋白表达 在LHRH神经元和/或与它们相关的外周蛋白+轴突上; (2)表达N-CAM的细胞与表达外周蛋白的细胞的同一性 （3）LHRH神经元在培养液中是否释放LHRH， LHRH在脉动的方式;和（4）是否标记的LHRH神经元， 原位可视化以监测胚胎外植体和~或 确定出生后LHRH神经元的膜特性， 器官型切片

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. To study the migratory mechanism(s) involved in LHRH neuronal movement into the CNS, we use normal and transgenic animals, as well as olfactory explants. In addition, long-term organotypic slice cultures are used to study mechanisms underlying intrinsic and trans-synaptic regulation of LHRH gene expression, peptide synthesis and secretion in postnatal differentiated LHRH neurons. Working on the hypothesis that LHRH neurons migrate on peripherin positive (+) olfactory axons from the olfactory pit to diencephalon, we found that: (1) LHRH neurons do not express peripherin mRNA; (2) LHRH neurons do not express N-CAM mRNA, although olfactory axons are N-CAM+; and (3) olfactory pit cells differentially express peripherin mRNA and N-CAM mRNA, suggesting distinct populations. In embryonic explants, we distinguished N-CAM+ and peripherin+ axons, and verified that LHRH neurons moved via peripherin+ but not N-CAM+ axons. Examination of voltage- and ligand-gated channels on embryonic LHRH neurons revealed membrane characteristics of highly differentiated neurons. In addition, we have generated embryonic explants from transgenic mice expressing luciferase in LHRH neurons. When given luciferin, a detectable signal is measurable in lysed cells from these explants. We examined second messengers active in LHRH cells and oxytocin (OT) cells maintained in organotypic slice explants. Forskolin and/or phorbol 12-myristate 13-acetate (PMA) treatment significantly decreased LHRH mRNA levels at 4 hr. In contrast, forskolin treatment significantly increased OT mRNA levels by 8 hr. Using actinomycin D (a transcription inhibitor), we determined neuropeptide mRNA turnover rates: LHRH mRNA has a very fast turnover rate (approximately 4 hr), while OT mRNA is much slower (approximately 40 hr). We propose that second messengers act primarily to increase transcription of OT mRNA in OT cells, but decrease LHRH mRNA transcription and/or increase LHRH mRNA degradation in LHRH neurons. Currently, we are determining: (1) cell surface glycoproteins expressed on LHRH neurons and/or the peripherin+ axons with which they associate; (2) the identity of cells expressing N-CAM vs those expressing peripherin in nasal regions; (3) whether LHRH neurons maintained in cultures release LHRH in a pulsatile manner; and (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.