CONTROL OF CELLS OF THE REPRODUCTIVE AXIS

我们的目标是填补我们在电生理方面的空白 了解促性腺激素释放激素神经元和促性腺激素是如何 被刺激分泌，如果它们有内在的机制 解释分泌物的节律性和脉搏形状。三种细胞类型 最初将用于：一种神经样促性腺激素释放激素分泌细胞系 来自转基因小鼠肿瘤；性腺激素样αT-3细胞系 来自转基因小鼠脑垂体瘤；以及幼年和成年 促性腺激素在酶促性腺激素的作用下从鲑鱼前脑垂体中分离出来。 响应将主要通过膜生物物理技术进行研究。 包括：全细胞和膜片钳表征离子的方法 这些细胞的通道及其对促性腺激素释放激素的反应；膜的变化 电容作为分泌囊泡吐出作用的指标； Indo-1荧光染料使细胞内钙离子发生变化。具体的 目标是：1)清点电压门控离子通道并 确定它们可以支持的电活动。2)清点 快速配基门控通道。3)确定促性腺激素释放激素对 通道、细胞内钙和分泌物。4)确定调节性 通过G蛋白作用的其他多肽和神经递质的作用 偶联受体。5)探索所使用的细胞内信号通路 用于对GnRH和其他调制发射机的响应。6)测试 关于兴奋的电生理学基础的假设， 抑制性和极端的节奏性。

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.