Role of G-gamma-7 in striatal cannabinoid and D2 dopamine

描述（由申请人提供）：中枢神经系统中的大麻素CB 1受体介导大麻的精神活性作用，是内源性大麻素系统的关键组成部分，参与动机和奖励、运动控制、食欲、学习和记忆以及疼痛和体温调节。内源性大麻素系统对运动和动机行为的控制被认为主要由纹状体/基底神经节（纹状体/BG）中的CB 1受体介导。在该系统中，已发现CB 1受体与多巴胺能神经传递相互作用，并与D2受体共定位于中等棘状GABA能神经元的亚群中。CB 1和D2受体都是G蛋白偶联受体（GPCR），其通过Gi/o激活抑制腺苷酸环化酶（AC）活性，并且在某些情况下可以通过Gs/olf激活刺激腺苷酸环化酶。相对于其他脑区，G蛋白γ亚基7型（Gng 7）在纹状体/BG中高度表达，并且已显示在腺苷酸环化酶的D1受体刺激中起作用，并调节G-alpha-olf表达，如在Gng 7敲除（KO）小鼠中所确定的。然而，Gng 7在GPCR对AC的抑制性调节中的潜在参与尚未被研究。我们的初步研究结果表明，AC抑制纹状体CB 1和D2受体减少Gng 7基因敲除小鼠，这表明该G-γ亚基的抑制性Gi/o信号的作用。或者，Gng 7 KO可以通过伴随的G-olf减少来改变可以被Gi/o抑制的纹状体AC活性的比例。拟议的R 03项目将检验以下假设：由于G-γ-7在抑制性Gi/o信号传导中起作用，因此在Gng 7敲除小鼠中CB 1和D2介导的AC抑制作用减弱。目的1将确定Gng 7基因敲除小鼠纹状体/BG亚区中CB 1和D2受体对腺苷酸环化酶抑制作用的丧失程度，包括尾壳核、丘脑核和苍白球，以及其他含有CB 1和D2的区域，如前额皮质和海马。表达CB 1受体但不表达Gng 7的小脑将作为阴性对照区。目的1还将确定Gng 7缺失对AC抑制的影响是否仅限于CB 1和D2受体，或在其他纹状体GPCR（例如阿片类药物）中观察到。目的2将确定在Gng 7 KO小鼠中AC抑制减少的区域中，CB 1或D2刺激的Gi/o蛋白活性是否减少，如通过激动剂刺激的[35 S] GTP γ S结合所确定的。将通过CB 1和D2受体的免疫印迹检测G蛋白活化的任何降低是否是由于受体表达的丧失。目的2还将通过测定杂合子G-α-olf KO小鼠中CB 1和D2受体的G蛋白激活和AC抑制，检验另一假设，即减弱的AC抑制是由于Gng 7 KO小鼠中G-olf的伴随减少。这些研究将作为未来R 01应用的基础，该应用将更广泛地研究CB 1和D2受体在纹状体功能调节中的关系，以及这种调节对运动和动机行为控制的贡献。这些研究将有助于确定导致神经精神障碍（如药物滥用和精神分裂症）以及运动障碍的分子机制。公共卫生相关性：大麻和其他滥用药物通过调节大脑中被称为纹状体的生化信号通路产生奖励效应。这个区域也参与控制随意运动和习惯行为，并且也受称为多巴胺的化学信使的控制。纹状体中这些信号通路中的关键蛋白是G蛋白γ 7型亚基。该项目将确定这种蛋白质在大麻素和多巴胺受体激活的生化效应中的作用，方法是研究经过基因改造而不表达这种蛋白质的小鼠。这些研究将提供有助于我们理解药物成瘾和可能的神经精神和运动障碍，如精神分裂症和帕金森氏症的神经机制的信息。

DESCRIPTION (provided by applicant): Cannabinoid CB1 receptors in the CNS mediate psychoactive effects of marijuana and are a critical component of the endogenous cannabinoid system, which is involved in motivation and reward, motor control, appetite, learning and memory, and pain and thermoregulation. Control of motor and motivational behaviors by the endocannabinoid system is thought to be largely mediated by CB1 receptors in the striatum/basal ganglia (striatum/BG). Within this system, CB1 receptors have been found to interact with dopaminergic neurotransmission, and are colocalized with D2 receptors in subpopulations of medium spiny GABAergic neurons. Both CB1 and D2 receptors are G-protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase (AC) activity via Gi/o activation, and under certain circumstances can stimulate adenylyl cyclase through Gs/olf activation. The G-protein gamma subunit type 7 (Gng7) is highly expressed in striatum/BG relative to other brain regions, and has been shown to play a role in D1 receptor stimulation of adenylyl cyclase and to regulate G-alpha-olf expression as determined in Gng7 knockout (KO) mice. However, potential involvement of Gng7 in inhibitory regulation of AC by GPCRs has not been examined. Our preliminary findings indicate that AC inhibition by striatal CB1 and D2 receptors is diminished in Gng7 knockout mice, suggesting a role for this G-gamma subunit in inhibitory Gi/o signaling. Alternatively, Gng7 KO could, by concomitant reduction in G-olf, alter the proportion of striatal AC activity that can be inhibited by Gi/o. The proposed R03 project will test the hypothesis that CB1 and D2-mediated inhibition of AC is attenuated in Gng7 knockout mice because G-gamma-7 plays a role in inhibitory Gi/o signaling. Aim 1 will determine the extent of loss of adenylyl cyclase inhibition by CB1 and D2 receptors in subregions of the striatum/BG in Gng7 knockout mice, including caudate-putamen, nucleus accumbens and globus pallidus, as well as other CB1 and D2 containing regions such as prefrontal cortex and hippocampus. Cerebellum, which expressed CB1 receptors but does not express Gng7, will serve as negative control region. Aim 1 will also determine whether the effect of loss of Gng7 on AC inhibition is limited to CB1 and D2 receptors or is seen with other striatal GPCRs (e.g. opioid). Aim 2 will determine whether CB1- or D2-stimulated Gi/o-protein activity is diminished in regions in which AC inhibition is diminished in Gng7 KO mice, as determined by agonist-stimulated [35S]GTPgammaS binding. Whether any reduction in G-protein activation is due to a loss in receptor expression will be tested by immunoblotting of CB1 and D2 receptors. Aim 2 will also test the alternative hypothesis that attenuated AC inhibition is due to concomitant reduction in G-olf in Gng7 KO mice, by determining G-protein activation and AC inhibition by CB1 and D2 receptors in heterozygous G-alpha-olf KO mice. These studies will serve as a basis for a future a R01 application that will more extensively investigate the relationship between CB1 and D2 receptors in the regulation of striatal function, and the contribution of this regulation to control of motor and motivational behavior. These studies will help to determine molecular mechanisms contributing to neuropsychiatric disorders such as substance abuse and schizophrenia, as well as motor disorders. PUBLIC HEALTH RELEVANCE: Marijuana (cannabis) and other abused drugs produce their rewarding effects through the regulation biochemical signaling pathways in a brain region known as the striatum. This region is also involved in control of voluntary movement and habitual behavior, and is also under the control of a chemical messenger known as dopamine. A key protein in these signaling pathways in striatum is the G-protein gamma type 7 subunit. This project will determine the role of this protein in the biochemical effects of cannabinoid and dopamine receptor activation by studying mice that have been genetically altered to not express this protein. These studies will provide information that contributes to our understanding of the neural mechanisms of drug addiction and possibly neuropsychiatric and movement disorders, such as schizophrenia and Parkinsonism.