CONTROL OF CELLS OF THE REPRODUCTIVE AXIS

我们的目标是填补我们的电生理学的空白， 了解GnRH神经元和促性腺激素，以了解它们如何 被刺激分泌，如果他们有内在的机制， 解释分泌的节律性和脉冲形状 三种细胞类型 最初将使用：神经元样GnRH分泌细胞系， 来自转基因小鼠肿瘤;性腺激素样α T-3细胞系 来自转基因小鼠垂体瘤;和青少年和成年人 促性腺激素从鲑鱼前垂体酶解分离。 反应将主要通过膜生物物理技术进行研究 包括：全细胞和膜片钳方法来表征离子 通道及其对GnRH的反应;膜的变化 电容作为分泌囊泡的胞吐作用的指数;以及 胞内钙随indo 1荧光染料变化。 具体 目的是：1）清点电压门控离子通道， 确定它们能够支持的电活动。 2)盘点 快速配体门控通道 3)为了确定GnRH对 通道、细胞内钙和分泌。 4)为了确定调节 其他肽和神经递质通过G蛋白发挥作用， 偶联受体 5)探索细胞内信号通路 对GnRH和其他调节性递质的反应。 6)测试 关于兴奋的电生理学基础的假设， 抑制和超昼夜节律。

The goal is to fill in gaps concerning our electrophysiological understanding of GnRH neurons and gonadotropes in order to learn how they are stimulated to secrete and if they have intrinsic mechanisms that would explain the rhythmicity and pulse shape of secretion. Three cell types will be used initially: a neuron-like GnRH-secreting cell line derived from a transgenic mouse tumor; a gonado trope-like alphaT-3 cell line derived from a transgenic mouse pituitary tumor; and juvenile and adult gonadotropes enzymatically dissociated from salmon anterior pituitaries. Responses will be studied primarily by membrane biophysical techniques including: whole-cell and patch clamp methods to characterize the ionic channels and their response to GnRH in these cells; changes of membrane capacitance as an index of exocytosis of secretory vesicles; and intracellular calcium changes with indo 1 fluorescent dye. The specific aims are: 1) To inventory the voltage-gated ionic channels and to determine the electrical activity they can support. 2) To inventory the fast ligand-gated channels. 3) To determine the actions of GnRH on channels, intracellular calcium, and secretion. 4) To determine modulatory actions of other peptides and neurotransmitters acting through G-protein- coupled receptors. 5) To explore the intracellular signaling pathways used for responses to GnRH and other modulatory transmitters. 6) To test hypotheses regarding the electrophysiological basis of excitation, inhibition, and ultradian rhythmicity.