5-HT1A RECEPTOR/G PROTEIN COUPLING AND ADAPTATION

许多证据表明血清素5-HT1A受体与焦虑和抑郁有关。5-HT1A受体激动剂表现出抗焦虑/抗抑郁作用，5-HT1A受体功能在抗焦虑和抗抑郁药物反复治疗后发生改变。虽然已经积累了很多关于5- HT1A受体信号转导在基因工程细胞中表达受体的数据，但对受体在其天然环境中的情况知之甚少。我们之前的研究表明，受体/效应物偶联效率存在区域差异，最近的证据表明，抗焦虑/抗抑郁药物的慢性治疗引发了区域特异性的5-HT1A受体适应，从而强调了5-HT1A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-HT1A受体信号转导，特别是在不同类型抗焦虑/抗抑郁药物反复治疗后，受体/G蛋白偶联、G蛋白特异性和5-HT1A受体适应的区域差异。提出以下具体目的：1)确定在受体/G蛋白偶联水平上，先前描述的受体/效应器偶联效率的区域差异是否可以证明。在部分不可逆受体阻断前后，将生成激动剂刺激的[35S]GTPgammaS结合（在膜和脑部分）的剂量-反应曲线，以验证5-HT1A受体激活G蛋白偶联在体树突背中缝（DR）自身受体上的效率高于海马-campus突触后受体的效率。还将确定外侧隔（LS）和内侧前额叶皮层（mPFC）突触后受体的耦合效率；2)确定5-HT1A受体在不同脑区是否优先与特定的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 ODNs）选择性敲除百日咳毒素（PTX）敏感的G蛋白α亚基，将验证5- HT1A受体与G蛋白的偶联是区域特异性和激动剂特异性的假设。我们预测，在DR中，5-HT1A受体将优先与Galphai3偶联，介导激动剂诱导的5-HT合成抑制；在海马中，5-HT1A受体会优先与Galphai2偶联，介导福斯克林刺激的腺苷酸环化酶的抑制。我们还将验证一些激动剂可能优先偶联不同的Galphai/ Galphai亚基来介导这些作用的假设；3)确定不同类别抗焦虑/抗抑郁药物的重复治疗是否会导致区域特异性的5-HT1A受体适应和受体/G蛋白偶联的变化。我们将评估激动剂对前脑区域5-羟色胺合成的抑制作用，以验证反复使用伊沙匹龙、帕罗西汀或克洛昔林（但不包括丙咪嗪）治疗（21天）会使体突5-HT1A自身受体脱敏，并减少dr中激动剂刺激的[35S]GTPgammaS结合。我们还将验证在海马中，激动剂刺激的[35S]GTPgammaS结合会通过丙咪嗪反复治疗而增加的假设。克劳格林降低，但伊萨匹龙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.