Mitsushima D, Marzban F, Luchansky L L, Burich A J, Keen K L, Durning M, Golos T G, Terasawa E
Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, 53715, USA.
J Neurosci. 1996 Apr 15;16(8):2563-73. doi: 10.1523/JNEUROSCI.16-08-02563.1996.
To investigate further the role of GABA in the onset of puberty, this study examines whether glutamic acid decarboxylase (GAD), the catalytic enzyme for GABA synthesis, is involved in the suppression of luteinizing hormone releasing hormone (LHRH) before puberty in rhesus monkeys. First, both GAD67 and GAD65 mRNAs were detectable by reverse transcription-PCR analysis in the preoptic area, medio-basal hypothalamus, posterior hypothalamic area, and hippocampus of the monkey brain. Second, effects of antisense oligodeoxynucleotides (D-oligos) for GAD67 and GAD65 mRNAs on LHRH release were examined in conscious female rhesus monkeys at the prepubertal stage using a push-pull perfusion method. The GAD67 or GAD65 antisense D-oligos or scrambled D-oligos were infused directly into the stalk-median eminence. Both the GAD67 and the GAD65 antisense D-oligos induced a large and prompt increase in LHRH release, whereas the scrambled D-oligos did not induce any significant effect. The results suggest that the removal of GABA inhibition by interfering with GAD synthesis is effective in increasing LHRH release in prepubertal monkeys. Third, the specificity of the antisense D-oligos on GAD levels was examined by incubating basal hypothalami with D-oligos in vitro and subsequent Western blot analysis. The antisense D-oligos consistently decreased the proteins GAD67 and GAD65 compared with respective control D-oligos. We conclude that the decrease of tonic GABAergic inhibition and maturational changes in GAD synthesis may be critical factors for the onset of puberty in nonhuman primates.
为了进一步研究γ-氨基丁酸（GABA）在青春期启动中的作用，本研究探讨了GABA合成的催化酶谷氨酸脱羧酶（GAD）是否参与恒河猴青春期前促黄体生成素释放激素（LHRH）的抑制。首先，通过逆转录-聚合酶链反应（RT-PCR）分析在猴脑的视前区、下丘脑中间基底部、下丘脑后区和海马中均可检测到GAD67和GAD65 mRNA。其次，在青春期前的雌性恒河猴中，使用推挽灌注法研究了针对GAD67和GAD65 mRNA的反义寡脱氧核苷酸（D-oligos）对LHRH释放的影响。将GAD67或GAD65反义D-oligos或乱序D-oligos直接注入垂体柄-正中隆起。GAD67和GAD65反义D-oligos均能迅速大幅增加LHRH的释放，而乱序D-oligos则未产生任何显著影响。结果表明，通过干扰GAD合成去除GABA抑制可有效增加青春期前猴子的LHRH释放。第三，通过体外将下丘脑基部与D-oligos孵育并随后进行蛋白质印迹分析，检测了反义D-oligos对GAD水平的特异性。与各自的对照D-oligos相比，反义D-oligos持续降低了GAD67和GAD65蛋白的水平。我们得出结论，紧张性GABA能抑制的降低和GAD合成的成熟变化可能是非人类灵长类动物青春期启动的关键因素。
