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

描述(申请人提供)：中枢神经系统中的大麻素CB1受体介导大麻的精神活动效应，是内源性大麻素系统的关键组成部分，该系统涉及动机和奖励、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在很大程度上被认为是由纹状体/基底节(纹状体/BG)中的CB1受体介导的。在这个系统中，CB1受体被发现与多巴胺能神经传递相互作用，并与D2受体共存于中等刺状GABA能神经元的亚群。CB1和D2受体都是G蛋白偶联受体(GPCRs)，通过Gi/o激活抑制腺酰环化酶(AC)的活性，在某些情况下可以通过Gs/olf激活来激活腺酰环化酶。与其他脑区相比，G-蛋白γ亚基7(Gng7)在纹状体/BG高表达，在Gng7基因敲除(KO)小鼠中，Gng7在D1R对腺苷酸环化酶的刺激和调节G-α-olf的表达中发挥作用。然而，Gng7在GPCRs对AC的抑制调节中的潜在作用还没有被研究过。我们的初步发现表明，在Gng7基因敲除小鼠中，纹状体CB1和D2受体对AC的抑制作用减弱，这表明该G-伽马亚单位在抑制Gi/o信号转导中发挥作用。或者，Gng7 KO可以通过伴随G-olf的减少而改变可被Gi/o抑制的纹状体AC活性的比例。建议的R03项目将检验这一假设，即在Gng7基因敲除的小鼠中，CB1和D2介导的AC抑制被减弱，因为G-Gamma-7在抑制Gi/o信号中发挥作用。目的1测定Gng7基因敲除小鼠纹状体/BG亚区，包括尾壳核、伏隔核和苍白球，以及其他含有CB1和D2的区域，如前额叶皮质和海马区，CB1和D2受体对腺苷环化酶抑制的丧失程度。表达CB1受体但不表达Gng7的小脑将作为阴性控制区。目的1还将确定Gng7的缺失对AC抑制的影响是否仅限于CB1和D2受体，或与其他纹状体GPCR(如阿片类药物)有关。目的2将通过激动剂刺激的[35S]GTPGammaS结合来确定在Gng7KO小鼠中AC抑制减弱的区域，CB1或D2刺激的GI/O蛋白活性是否降低。G蛋白活性的任何降低是否由于受体表达的丧失将通过CB1和D2受体的免疫印迹来测试。目的2还将通过检测杂合子G-α-OLF KO小鼠中G蛋白的激活和CB1和D2受体对AC的抑制，来验证另一种假说，即减弱AC抑制是由于Gng7 KO小鼠中G-OLF伴随减少所致。这些研究将作为未来R01应用的基础，该应用将更广泛地研究CB1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的分子机制，如药物滥用和精神分裂症，以及运动障碍。与公共健康相关：大麻(大麻)和其他滥用药物通过调节大脑纹状体区域的生化信号通路产生奖励效应。该区域还参与控制自主运动和习惯性行为，并受一种名为多巴胺的化学信使的控制。纹状体中这些信号通路中的一个关键蛋白是G蛋白伽马7型亚单位。该项目将通过研究基因改变而不表达这种蛋白的小鼠，来确定这种蛋白在大麻素和多巴胺受体激活的生化效应中的作用。这些研究将提供信息，有助于我们理解药物成瘾的神经机制，以及可能的神经精神和运动障碍，如精神分裂症和帕金森氏症。

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.