5-HT1A RECEPTOR/G PROTEIN COUPLING AND ADAPTATION

许多证据表明，5-HT1a受体参与了焦虑和抑郁。5-HT1a受体激动剂表现出抗焦虑/抗抑郁作用，经抗焦虑和抗抑郁药物反复治疗后，5-HT1a受体功能发生改变。虽然已经积累了许多关于5-HT1A受体在基因工程细胞中表达该受体的信号转导的数据，但对该受体在其自然环境中的信息转导知之甚少。我们之前的研究表明，受体/效应偶联效率的地区差异，以及最近的证据表明，抗焦虑/抗抑郁药物的慢性治疗会引起地区特异性的5-HT1a受体适应，这突显了5-HT1A受体功能的地区差异的潜在重要性。因此，本项目建议更好地了解5-HT1a受体的信号转导，特别是在受体/G蛋白偶联、G蛋白特异性和5-HT1a受体经不同类别的抗焦虑/抗抑郁药物反复治疗后的区域差异。提出了以下具体目标：1)确定先前所描述的受体/效应器偶联效率的区域差异是否在受体/G蛋白偶联水平上表现出来。在部分不可逆受体阻断前后，将产生激动剂刺激结合[35S]GTP-GammaS的剂量-反应曲线，以检验5-HT1a受体激活G蛋白偶联在体树突背中缝(DR)自身受体上比在突触后受体上表现出更高的效率的假设。还将测定外侧隔区(LS)和内侧前额叶皮质(MPFC)突触后受体的偶联效率；2)确定5-HT1A受体是否优先与不同脑区的特定G蛋白偶联。通过脑室或组织内注射反义寡核苷酸(AS ODN)选择性地敲除百日咳毒素(PTX)敏感的G蛋白α亚单位，将检验5-HT1A受体与G蛋白偶联将是区域和激动剂特异性的假设。我们预测，在DR，5-HT1a受体将优先与Galphai3偶联，介导激动剂对5-HT合成的抑制；在海马区，5-HT1a受体将优先与Galphai2偶联，介导对Forsklin刺激的腺苷环化酶的抑制。我们还将检验假设，即一些激动剂可能优先偶联到不同的Galphai/Galpao亚基来介导这些效应；3)确定不同类别的抗焦虑/抗抑郁药物重复治疗是否导致区域特异性的5-HT1A受体适应和受体/G蛋白偶联的变化。将评估激动剂对前脑区5-羟色胺合成的抑制作用，以检验以下假设：重复使用伊帕米松、帕罗西汀或氯丙吉林(但不包括丙咪嗪)可使躯体树突状细胞5-HT1A自身受体脱敏，并减少激动剂刺激的[35S]GTP-Gamma在博士体内的结合。我们还将测试一种假设，即在海马区，激动剂刺激的[35S]GTP GammaS结合将因重复使用丙咪嗪而增加，被clorgyline减少，但在伊帕西酮或帕罗西汀后保持不变。

Much evidence implicates the involvement of the serotonin 5-HT1A receptor in anxiety and depression. 5-HT1A receptor agonists display anxiolytic/antidepressant effects, and 5-HT1A receptor function is altered after repeated treatment with anxiolytic and antidepressant drugs. While much data has accumulated about 5- HT1A receptor signal transduction in cells genetically engineered to express the receptor, much less is known about the receptor in its native environment. The potential importance of regional differences in 5-HT1A receptor function is underscored by our previous studies demonstrating regional variations in receptor/effector coupling efficiency and recent evidence that chronic treatment with anxiolytic/antidepressant drugs elicits regionally specific 5-HT1A receptor adaptation. This project therefore proposes to gain a better understanding of 5-HT1A receptor signal transduction, especially with regard to regional differences in receptor/G protein coupling, G protein specificity, and 5-HT1A receptor adaptation after repeated treatment with various classes of anxiolytic/antidepressant drugs. The following Specific Aims are proposed: 1) To determine if previously described regional differences in receptor/effector coupling efficiency are demonstrable at the level of receptor/G protein coupling. Dose-response curves for agonist-stimulated binding of [35S]GTPgammaS (in membranes and brain sections) before and after partial irreversible receptor blockade will be generated to test the hypothesis that 5-HT1A receptor activation of G protein coupling will exhibit greater efficiency at somatodendritic dorsal raphe (DR) autoreceptors than at postsynaptic receptors in hippo-campus. Coupling efficiency at postsynaptic receptors in the lateral septum (LS) and medial prefrontal cortex (mPFC) will also be determined; 2) To determine if 5-HT1A receptors couple preferentially to specific G proteins in different brain regions. Selective knockdown of pertussis toxin (PTX)-sensitive G protein alpha subunits by intracerebroventricular or intra-tissue infusion of antisense oligodeoxynucleotides (AS ODNs) will test the hypothesis that 5- HT1A receptor coupling to G proteins will be region- and agonist- specific. We predict that in the DR, 5-HT1A receptors will couple preferentially to Galphai3 to mediate agonist-induced inhibition of 5-HT synthesis; in the hippocampus, 5-HT1A receptors will couple preferentially to Galphai2 to mediate inhibition of forskolin-stimulated adenylyl cyclase. We will also test the hypothesis that some agonists may preferentially couple to different Galphai/Galphao subunits to mediate these effects; 3) To determine if repeated treatment with different classes of anxiolytic/antidepressant drugs results in regionally- specific 5-HT1A receptor adaptation and changes in receptor/G protein coupling. Agonist inhibition of 5-HT synthesis in forebrain regions will be assessed to test the hypothesis that repeated treatment (21 days) with ipsapirone, paroxetine or clorgyline (but not imipramine) will desensitize somatodendritic 5-HT1A autoreceptors and reduce agonist-stimulated binding of [35S]GTPgammaS in the DR. We will also test the hypothesis that in the hippocampus, agonist-stimulated binding of [35S]GTPgammaS will be increased by repeated treatment with imipramine, decreased by clorgyline, but remain unchanged after ipsapirone or paroxetine.