ONTOGENY OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

黄体生成素释放激素系统的个体发育

• 批准号: 3760315
• 负责人: S WRAY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3760315
• 关键词: Xenopus; cell migration; central neural pathway /tract; developmental neurobiology; embryo /fetus tissue /cell culture; forskolin; gene expression; genetic transcription; genetically modified animals; gonadotropin releasing factor; laboratory mouse; messenger RNA; neural cell adhesion molecules; neurofilament proteins; olfactory lobe; oxytocin; peptide hormone biosynthesis; second messengers; secretion

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.

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。为了研究 迁移机制(S)，促肾上腺皮质激素释放激素神经元进入中枢神经系统的机制， 我们使用正常和转基因动物，以及嗅觉外植体。在……里面 此外，长期器官型切片培养用于研究 促性腺激素释放激素的内源性和跨突触调节机制 出生后基因表达、多肽合成和分泌 分化的LHRH神经元。 基于LHRH神经元在外周迁移的假设 从嗅窝到间脑的阳性(+)嗅觉轴突 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 虽然嗅觉轴突为N-CAM+，但神经元不表达N-CAM mRNA； (3)嗅窝细胞差异表达外周蛋白mRNA和 N-CAM信使核糖核酸，提示不同的种群。在胚胎外植体中，我们 区分N-CAM+和外周蛋白+轴突，并验证LHRH 神经元通过外周蛋白+移动，而不是N-CAM+轴突。审查 胚胎LHRH神经元电压门控和配基门控通道的研究 高分化神经元的细胞膜特性。此外, 我们已经从转基因小鼠中获得了胚胎外植体，表达了 LHRH神经元中的荧光素酶。当给予荧光素时，可检测到的信号 在这些外植体的裂解细胞中是可以测量到的。 我们检测了LHRH细胞和催产素(OT)中活跃的第二信使 细胞保持在器官类型的切片外植体中。佛斯可林和/或佛波尔 12-肉豆蔻酸13-乙酸酯(PMA)处理显著降低LHRH基因的表达 在4小时的水平。相比之下，Forsklin治疗组显著增加 Ot m RNA水平降低8小时。使用放线菌素D(一种转录抑制物)， 我们测定了神经肽信使核糖核酸的周转率：LHRH信使核糖核酸有非常快的 周转速度(大约4小时)，而OT mRNA要慢得多 (约40小时)。我们建议第二个信使主要扮演 增加OT细胞中OT mRNA的转录，但降低LHRH mRNA 转录和/或增加LHRH神经元中LHRH mRNA的降解。 目前，我们正在测定：(1)细胞表面糖蛋白的表达 LHRH神经元和/或它们所联系的外周蛋白+轴突； (2)表达N-CAM的细胞与表达外周蛋白的细胞的一致性 鼻区；(3)培养中的LHRH神经元是否释放 以及(4)标记的LHRH神经元是否可以 可视化原位监测胚胎外植体和~或 新生大鼠LHRH神经元膜特性的测定 器官切片。