McCarthy M M, Masters D B, Rimvall K, Schwartz-Giblin S, Pfaff D W
Rockefeller University, Laboratory of Neurobiology and Behavior, New York, NY 10028.
Brain Res. 1994 Feb 14;636(2):209-20. doi: 10.1016/0006-8993(94)91019-7.
Increased GABA activity in the medial hypothalamus (HYP) and midbrain central gray (MCG), but not the preoptic area (POA), facilitates sexual receptivity in the female rat [40]. In the current experiments, ovariectomized females were chronically treated with estrogen (via silastic capsules) to maintain a continuously high level of lordosis response. Administration of crystalline antisense oligodeoxynucleotide to the GABA synthetic enzyme, GAD67, into the HYP and MCG significantly and reversibly reduced lordosis response for 1-2 days, but did not inhibit lordosis when administered into the POA. Administration of a control oligonucleotide, consisting of the same nucleotide bases but in a scrambled sequence, did not significantly modulate behavior when infused into any brain areas. When oligodeoxynucleotide antisense to GAD67 was suspended in oil and then infused into the HYP or MCG it was more effective and resulted in less inter-animal variability. Subsequent experiments involving infusions into the MCG compared the effectiveness of antisense oligonucleotides to the two different forms of GAD, known as GAD65 and GAD67. Oligodeoxynucleotides antisense to the mRNA for either gene were effective at reducing lordosis behavior but with a different time course. Oligonucleotide antisense to GAD67 significantly reduced behavior within 24 h of infusion and there was full recovery by 4 days post-infusion. GAD65 antisense oligonucleotide did not significantly reduce behavior until 48 h post infusion and animals did not fully recover to pretest levels of lordosis until 5 days post-infusion. When antisense oligonucleotide for the two genes was administered simultaneously, the inhibition of lordosis was maximal at 24 h and stayed depressed for 4 days. There did not appear to be an additive effect of the two different antisense oligonucleotides when administered together. Tissue GABA levels in HYP and MCG of individual rats assayed by HPLC were no longer correlated with lordosis score after antisense oligonucleotide infusion but were after infusions of scrambled control oligos. Immunoblotting for the two forms of GAD revealed that GAD67 antisense oligonucleotide infusion led to significant decreases in both GAD67 and GAD65 protein levels as compared to infusions of scrambled control oligo. In addition, the levels of a neuronal marker, neuron-specific enolase, also decreased (although nonsignificantly) suggesting either a temporary shutdown of protein synthesis or a degeneration of GABAergic neurons after GAD67 antisense oligonucleotide infusion.
内侧下丘脑（HYP）和中脑中央灰质（MCG）而非视前区（POA）中γ-氨基丁酸（GABA）活性的增强，可促进雌性大鼠的性接受能力[40]。在当前实验中，对卵巢切除的雌性大鼠长期给予雌激素（通过硅橡胶胶囊），以维持持续高水平的脊柱前凸反应。向HYP和MCG注射针对GABA合成酶GAD67的结晶反义寡脱氧核苷酸，可显著且可逆地降低脊柱前凸反应1 - 2天，但注射到POA中时并不抑制脊柱前凸。注射由相同核苷酸碱基但序列混乱组成的对照寡核苷酸，注入任何脑区时均未显著调节行为。当将针对GAD67的反义寡脱氧核苷酸悬浮于油中然后注入HYP或MCG时，效果更佳且动物间变异性更小。随后涉及向MCG注射的实验比较了针对两种不同形式GAD（即GAD65和GAD67）的反义寡核苷酸的效果。针对任一基因mRNA的反义寡脱氧核苷酸均可有效降低脊柱前凸行为，但时间进程不同。针对GAD67的寡核苷酸在注射后24小时内显著降低行为，注射后4天完全恢复。针对GAD65的反义寡核苷酸直到注射后48小时才显著降低行为，动物直到注射后5天才完全恢复到测试前的脊柱前凸水平。当同时给予针对这两个基因的反义寡核苷酸时，脊柱前凸的抑制在24小时时最大，并持续抑制4天。两种不同的反义寡核苷酸一起给药时似乎没有相加作用。通过高效液相色谱法测定的个体大鼠HYP和MCG中的组织GABA水平，在注射反义寡核苷酸后不再与脊柱前凸评分相关，但注射混乱对照寡核苷酸后相关。对两种形式GAD的免疫印迹显示，与注射混乱对照寡核苷酸相比，注射针对GAD67的反义寡核苷酸导致GAD67和GAD65蛋白水平均显著降低。此外，一种神经元标志物神经元特异性烯醇化酶的水平也降低（尽管不显著），这表明在注射针对GAD67的反义寡核苷酸后，要么是蛋白质合成暂时停止，要么是GABA能神经元发生退化。
