NEUROTENSIN SYNTHESIS IN ARCUATE NEUROSECRETORY CELLS

这项建议的广泛目标是澄清 神经分泌细胞合成和释放神经降压素的定位 在大鼠下丘脑弓状核的背内侧 老鼠。该区域NT合成的调节遵循一种模式 暗示NT参与女性特有的、雌激素依赖的分泌 一种或多种腺垂体激素，特别是催乳素和 促性腺激素。为了验证这一假设，将使用NT抗血清来 选择性地干扰内源性释放的NT在意识中、自由地 在发情前期移动雌性动物，以及这种干预对 催乳素、黄体生成素和卵泡的循环水平- 刺激素将会被测定。这项提议的主要目的之一 是在女性体内确定雌激素诱导的程度 大鼠结节漏斗多巴胺能神经元中NT的合成 生理条件。为此，一种原位双标记方法 杂交将被用来确定数量和分布 背内侧弓状神经元共同表达编码NT和 不同培养条件下神经中性素N(NT/N)和酪氨酸羟基酶mRNA的表达 内分泌状况。同样的技术也将用于调查 NT/N m RNA可能与雌激素受体m RNA或 NT受体mRNA在该区域的表达，从而鉴定NT神经元 通过雌激素或NT本身进行直接调节。这些问题的一个基本前提 研究表明，NT/N基因的异质性共表达与某些 背内侧神经元中的其他基因将揭示 这一区域的NT-合成神经元。NT/N基因表达的激素调节 将研究这些神经元的表达情况，以获得更多 关于它们功能的线索。将使用原位杂交来评估 催乳素或孕酮影响NT/N基因表达的可能性 在背内侧弓状核。此外，与An的原位杂交 内含子特异性探针将被用来确定雌激素 通过增加NT/N的转录增加NT/N的mRNA水平 吉恩。雌激素增加NT/N水平的时间进程 初级转录本和成熟的NT/N mRNA将指示雌激素是否- 诱导合成NT可能介导特异性神经内分泌 事件。稳态水平的NT和神经介素N将被测量到 确定雌激素是否改变了它们在正中隆起中的比例 神经中间叶。最后，将使用原位杂交技术来 确定成年男女血浆雌激素水平的差异是否可以解释 NT/N基因在背内侧的性别分化表达 弓形神经元。阐明NT在正常神经内分泌中的作用 功能，这些研究将为今后的研究提供基础 NT参与人类神经内分泌紊乱。

The broad objective of this proposal is to clarify the function of neurotensin (NT) synthesis and-release by neurosecretory cells located in the dorsomedial division of the hypothalamic arcuate nucleus in the rat. Regulation of NT synthesis in this region follows a pattern that implies NT involvement in female-specific, estrogen-dependent secretion of one or more adenohypophysial hormones, particularly prolactin and the gonadotropins. To test this hypothesis, NT antiserum will be used to selectively interfere with endogenously released NT in conscious, freely moving females at proestrus, and the effect of this intervention on circulating levels of prolactin, luteinizing hormone, and follicle- stimulating hormone will be determined. A primary aim of this proposal is to determine in the female the extent to which estrogen-inducible synthesis of NT occurs in tuberoinfundibular dopaminergic neurons under physiological conditions. To that end, a double-label method of in situ hybridization will be used to determine the number and distribution of dorsomedial arcuate neurons that coexpress mRNA encoding NT and neuromedin N (NT/N mRNA) and tyrosine hydroxylase mRNA under different endocrine conditions. The same technique will also be used to investigate likely coexpression of NT/N mRNA with either estrogen receptor mRNA or NT receptor mRNA in this region and thus to identify NT neurons subject to direct regulation by estrogen or NT itself. A basic premise of these studies is that heterogeneous coexpression of the NT/N gene with certain other genes in dorsomedial neurons will reveal functional subgroups of NT-synthesizing neurons in this region. Hormonal regulation of NT/N mRNA expression in these neurons will be investigated to obtain additional clues to their function. In situ hybridization will be used to assess the possibility that prolactin or progesterone influence NT/N mRNA expression in the dorsomedial arcuate nucleus. Also, in situ hybridization with an intron-specific probe will be used to determine whether estrogen increases NT/N mRNA levels through increased transcription of the NT/N gene. The time course over which estrogen increases levels of NT/N primary transcript and mature NT/N mRNA will indicate whether estrogen- induced synthesis of NT could plausibly mediate specific neuroendocrine events. Steady-state levels of NT and neuromedin N will be measured to determine whether estrogen alters their ratio in the median eminence or neurointermediate lobe. Finally, in situ hybridization will be used to determine whether adult sex differences in plasma estrogen levels account for sexually differentiated expression of NT/N mRNA in dorsomedial arcuate neurons. By clarifying the role of NT in normal neuroendocrine function, these studies will provide a basis for future investigation of NT involvement in human neuroendocrine disorders.