The effects of GABA on embryonic gonadotropin-releasing hormone neurons in rat hypothalamic primary culture
The effects of GABA on embryonic gonadotropin-releasing hormone neurons in rat hypothalamic primary culture
复制标题
DOI：
10.1262/jrd.18103
复制
发表时间：
2007-04-01
影响因子：
1.8
通讯作者：
Nishihara, Masugi
中科院分区：
文献类型：
--
作者：
Fujioka, Hitomi;Yamanouchi, Keitaro;Nishihara, Masugi
在胚胎发育过程中，促性腺激素释放激素(GnRH)神经元出现在嗅觉胎盘，迁移到视前区(POA)，然后将轴突延伸到正中隆起。在妊娠晚期，当GnRH神经元到达POA并形成神经元网络时，关于GnRH神经元的特性的信息很少，尽管许多研究已经在早期发育阶段或出生后对这种特性进行了研究。本研究利用GnRH启动子控制下表达增强型绿色荧光蛋白(EGFP)的转基因大鼠，研究了调节GnRH神经活性的主要神经递质--γ-氨基丁酸(GABA)在胚胎18.5天(E18.5)对GnRH基因表达的调控作用。首先，采用RT-PCR方法检测了E18.5胚胎POA组织中谷氨酸脱羧酶(GAD)两种亚型GAD65和GAD67的mRNA表达。免疫组织化学显示GAD67阳性细胞位于GnRH阳性细胞附近，GnRH神经元内GABA-A和GABA-B受体亚单位均呈免疫反应阳性。接下来，取E18.5胚胎下丘脑前部组织进行原代培养，以EGFP表达为指标评价GABA及其激动剂对GnRH启动子活性的影响。GABA和GABA-A受体激动剂Muscimol，而不是GABA-B受体激动剂巴氯芬显著增加EGFP阳性/GnRH阳性细胞比率。这些结果提示，在妊娠晚期，GABA可能通过GABA-A受体刺激胚胎GnRH神经元GnRH基因的表达。
Gonadotropin-releasing hormone (GnRH) neurons arise in the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence during embryogenesis. Little information is available concerning the properties of GnRH neurons during the late gestational period when GnRH neurons reach the POA and form neuronal networks, although many studies have examined such properties during earlier developmental stages or the postnatal period. The present study was performed to elucidate the involvement of gamma-aminobutyric acid (GABA), one of the major neurotransmitters modifying GnRH neural activity, in regulation of GnRH gene expression on embryonic day 18.5 (E18.5) using transgenic rats expressing enhanced green fluorescence protein (EGFP) under the control of GnRH promoter. First, using RT-PCR, the mRNA of two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), GAD65 and GAD67 was detected in E18.5 embryonic POA-containing tissues. GAD67-positive cells were also demonstrated in close vicinity to GnRH-positive cells by immunohistochemistry, and immunoreactivity for both the GABA-A and GABA-B receptor subunits was detected in GnRH neurons. Next, primary cultures derived from anterior hypothalamic tissue of E18.5 embryos were prepared, and the effects of GABA and its agonists on GnRH promoter activity were evaluated using EGFP expression as a marker. GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, significantly increased the EGFP-positive/GnRH-positive cell ratio. These results suggest that GABA plays a role in stimulating GnRH gene expression through GABA-A receptors in embryonic GnRH neurons in late gestational stages.