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

促性腺激素释放激素（GnRH）神经元在生殖神经内分泌学中起着重要作用。然而，这些神经元的功能分析是有限的，尽管通过生产转基因小鼠，可以用增强的绿色荧光蛋白（EGFP）特异性地标记GnRH神经元。在本研究中，我们培育了转基因大鼠，用EGFP标记GnRH神经元。在乙醚麻醉下切除1-7日龄幼鼠（新生儿）或35-40日龄大鼠（青春期）的大脑。切开布洛卡斜带和内侧视前区，酶解分散。神经元在neurobase - a培养基中培养1 d。将穿孔膜片钳应用于鉴定的GnRH神经元，分析电压门控Ca^<2+>电流。在新生儿GnRH神经元中，可以清楚地观察到高压激活的Ca^<2+>电流，但低压激活的Ca^<2+>电流可以忽略不计。尼莫地平（l型通道阻滞剂）和ω- concontoxin GVIA （n型通道阻滞剂）各使电流衰减约20%。r型通道阻滞剂SNX-482将电流衰减约55%。P/ q型通道阻滞剂ω-agatoxin对IVA的抑制作用较小。然而，在青春期GnRH神经元中，可以观察到高电压和低电压激活的Ca^<2+>电流。硝苯地平、ω-螺毒素GVIA和SNX-482对GnRH神经元的抑制作用与新生儿神经元相似，而o-agatoxin IVA对GnRH神经元的抑制作用在40-61%的检测中明显可见。电压门控Ca^<2+>电流的表达谱无性别差异。这些结果表明，GnRH神经元在功能上表达L-、N-、P/Q-、R-和t型通道。P/Q-和t型通道的表达受发育调控。

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.