5-HT1A RECEPTOR/G PROTEIN COUPLING AND ADAPTATION

许多证据表明，5-羟色胺5-HT 1A受体参与焦虑和抑郁。 5-HT 1A受体激动剂显示抗焦虑/抗抑郁作用，并且5-HT 1A受体功能在用抗焦虑和抗抑郁药物重复治疗后改变。 虽然已经积累了大量关于5-HT 1A受体在基因工程细胞中表达受体的信号转导的数据，但对受体在其天然环境中的了解要少得多。 我们以前的研究表明受体/效应器偶联效率的区域差异，最近的证据表明抗焦虑/抗抑郁药物的长期治疗会引起区域特异性5-HT 1A受体适应，这强调了5-HT 1A受体功能区域差异的潜在重要性。 因此，本项目建议更好地了解5-HT 1A受体信号转导，特别是在受体/G蛋白偶联、G蛋白特异性和5-HT 1A受体适应性方面的区域差异。 提出了以下具体目的：1）确定先前描述的受体/效应物偶联效率的区域差异是否在受体/G蛋白偶联水平上是可证明的。 将生成部分不可逆受体阻断前后激动剂刺激的[35 S] GTP γ S结合（在膜和脑切片中）的剂量-反应曲线，以检验以下假设：5-HT 1A受体激活G蛋白偶联在体树突中缝背核（DR）自身受体上的效率比在突触后受体上的效率更高。 还将确定外侧隔（LS）和内侧前额叶皮质（mPFC）中突触后受体的偶联效率; 2）确定5-HT 1A受体是否优先与不同脑区域中的特定G蛋白偶联。 通过脑室内或组织内输注反义寡脱氧核苷酸（AS ODN）选择性敲低百日咳毒素（PTX）敏感性G蛋白α亚基将检验5-HT 1A受体偶联G蛋白将是区域特异性和激动剂特异性的假设。 我们预测，在DR中，5-HT 1A受体将优先耦合到Galphai 3介导激动剂诱导的5-HT合成抑制;在海马中，5-HT 1A受体将优先耦合到Galphai 2介导抑制毛喉素刺激的腺苷酸环化酶。 我们还将检验这样的假设，即一些激动剂可以优先偶联到不同的Galphai/Galphao亚基以介导这些作用; 3）确定用不同类别的抗焦虑/抗抑郁药物重复治疗是否导致区域特异性5-HT 1A受体适应和受体/G蛋白偶联的变化。 将评估前脑区域中5-HT合成的激动剂抑制，以检验用伊沙匹隆、帕罗西汀或氯吉林重复治疗（21天）（但不是丙咪嗪）将使体树突5-HT 1A自身受体脱敏并减少DR中激动剂刺激的[35 S] GTP γ S结合。我们还将检验在海马中，激动剂刺激的[35 S] GTP γ S结合将通过丙咪嗪的重复治疗而增加，通过氯吉兰而减少，但在伊沙匹隆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.