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Physiological analysis of medial preoptic GnRH neurons - basis for the mechanism of onset of puberty

内侧视前 GnRH 神经元的生理分析 - 青春期开始机制的基础

基本信息

批准号：
13680883
负责人：
KATO Masakatsu
金额：
$ 2.11万
依托单位：
Nippon Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680883/
关键词：
GnRH neuron EGFP transgenic calcium channel R-type calcium channel T-type calcium channel patch clamp

项目摘要

Gonadotropin releasing hormone (GnRH) neurons play an important role in reproductive neuroendocrinology. Functional analysis of these neurons is, however, limited although it has been facilitated by producing transgenic mice to specifically label GnRH neurons with enhanced green fluorescence protein (EGFP). In the present study we produced transgenic rats to label GnRH neurons with EGFP. The brains were excised from 1-7 day-old pups (neonate) or 35-40 day-old rats (puberty) under ether anesthesia. A diagonal band of Broca and the medial preoptic area were cut and were enzymatically dispersed. The neurons were incubated in Neurobasal-A medium for 1 das. A perforated patch clamp was applied to the identified GnRH neurons to analyze the voltage-gated Ca^<2+> currents. In neonatal GnRH neurons, high voltage-activated Ca^<2+> currents were clearly observed but low voltage-activated Ca^<2+> currents were negligible. Nimodipine (L-type channel blocker) and ω-conotoxin GVIA (N-type channel blocker) each attenuated the current by 〜20%. The R-type channel blocker SNX-482 attenuated the current by 〜55%. Inhibition by P/Q-type channel blocker ω-agatoxin IVA was small. In pubertal GnRH neurons, however, both high and low voltage-activated Ca^<2+> currents were observed. Inhibitions by nifedipine, ω-conotoxin GVIA and SNX-482 were similar to that in neonatal neurons, whereas the inhibition by oo-agatoxin IVA was clearly seen in 40-61% of GnRH neurons examined. There was no sex difference in the profile of expression of the voltage-gated Ca^<2+> currents. These results indicate that GnRH neurons functionally express L-, N-, P/Q-, R- and T-type channels. Expressions of P/Q- and T-type channels are develonmentally regulated.

促性腺激素释放激素（GnRH）神经元在生殖神经内分泌中起重要作用。然而，这些神经元的功能分析是有限的，虽然它已经通过生产转基因小鼠，以特异性标记GnRH神经元与增强的绿色荧光蛋白（EGFP）促进。在本研究中，我们生产了转基因大鼠标记的GnRH神经元与绿色荧光蛋白。在乙醚麻醉下，从1-7日龄幼仔（新生儿）或35-40日龄大鼠（青春期）切除脑。切下Broca斜角带和内侧视前区，并酶促分散。将神经元在Neurobasal-A培养基中孵育1天。将穿孔膜片钳应用于所鉴定的GnRH神经元以分析电压门控性Ca^<2+>电流。在新生的GnRH神经元中，可以清楚地观察到高电压激活的Ca^<2+>电流，但低电压激活的Ca^<2+>电流可以忽略不计。尼莫地平（L型通道阻断剂）和ω-芋螺毒素GVIA（N型通道阻断剂）均使电流衰减约20%。R型通道阻断剂SNX-482使电流衰减约55%。P/Q型通道阻断剂ω-agatoxin IVA的抑制作用较小。然而，在青春期GnRH神经元中，观察到了高电压和低电压激活的Ca^2+电流。硝苯地平、ω-芋螺毒素GVIA和SNX-482的抑制作用与新生神经元相似，而卵圆蝮蛇毒素IVA的抑制作用在40-61%的GnRH神经元中明显可见。电压门控性Ca^<2+>电流的表达没有性别差异。这些结果表明，GnRH神经元功能表达L-，N-，P/Q-，R-和T-型通道。P/Q-和T-型通道的表达是发育调节的。