Elucidation of M5 Muscarinic Receptor Neurophysiology using Allosteric Modulators

描述（由申请人提供）：拟议研究的长期目标是利用M5毒蕈碱受体（mAChR）的新型变构调节剂来阐明中脑多巴胺能神经元中M5受体激活的生理后果。多巴胺在中脑核（NAc）和纹状体中释放的失调被认为是多种中枢神经系统（CNS）疾病的基础，包括帕金森病、注意力缺陷障碍（ADHD）和药物成瘾。刺激黑质腹侧被盖区（VTA）和黑质背侧部（SNc）的多巴胺能神经元的胆碱能传入可分别增加NAc和纹状体中的多巴胺释放，这种效应对mAChR拮抗剂敏感。在这些中脑多巴胺能神经元上唯一可检测的mAChR是M5种类的，并且在M5敲除小鼠中的研究支持M5激活导致这些多巴胺能神经元兴奋的假设。然而，M5的治疗潜力的验证还有待于M5选择性调节剂的开发。M5受体的选择性正构激动剂的开发相对不成功，这是由于在所有五种毒蕈碱受体亚型（M1-M5）中正构乙酰胆碱（ACh）结合位点的高度保守性。然而，通过靶向从正构口袋中去除的变构位点，Conn实验室成功开发了M5受体的正变构和负变构调节剂（分别为VU 0238429和VU 0419959）。在这些研究中，我建议使用分子药理学技术来充分表征这些M5选择性调节剂改变乙酰胆碱介导的信号传导的机制。然后，我计划在脑切片中进行电生理学研究，以验证卡巴胆碱诱导的多巴胺神经元内向电流是由M5受体激活介导的这一假设。最后，我的目标是使用微透析，以确定是否这些调制器到SNc和VTA的输液可以改变清醒，自由活动的大鼠细胞外多巴胺水平。我假设，M5正调制器将增加多巴胺神经元内向电流和增加多巴胺释放，而负调制器将降低兴奋性和细胞外多巴胺水平。这些研究对于阐明M5的生理作用以及验证M5调节在多种CNS疾病（如帕金森病、ADHD和药物成瘾）中的治疗潜力都很重要。 公共卫生相关性：中脑多巴胺能神经元活性的改变与几种中枢神经系统疾病有关，包括帕金森病、注意力缺陷障碍和成瘾。阐明M5和其他毒蕈碱受体亚型在通过亚型特异性变构调节剂调节多巴胺能神经元中的生理作用将对开发治疗这些疾病的新方法具有重要意义。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to utilize novel allosteric modulators of the M5 muscarinic receptor (mAChR) to elucidate the physiological consequence of M5 receptor activation in midbrain dopaminergic neurons. Dysregulation of dopamine release in the nucleus accumbens (NAc) and striatum is thought to underlie multiple central nervous system (CNS) disorders including Parkinson's disease, attention deficit disorder (ADHD), and drug addiction. Stimulation of cholinergic afferents to dopaminergic neurons in the substantia nigra pars compacta (SNc) and ventral tegemental area (VTA) can increase dopamine release in the NAc and striatum respectively, an effect that is sensitive to mAChR antagonists. The only detectable mAChR on these midbrain dopaminergic neurons is of the M5 variety and studies in M5 knock- out mice support the hypothesis that M5 activation leads to excitation of these dopaminergic neurons. However, validation of the therapeutic potential of M5 has awaited the development of an M5-selective modulator. The development of selective orthosteric agonists for the M5 receptor has been relatively unsuccessful due to the high conservation of the orthosteric, acetylcholine (ACh) binding site across all five muscarinic receptor subtypes (M1-M5). However, by targeting allosteric sites that are removed from the orthosteric pocket the Conn lab has successfully developed both a positive and negative allosteric modulator of the M5 receptor (VU0238429 and VU0419959 respectively). In these studies I propose to use molecular pharmacological techniques to fully characterize the mechanism whereby these M5-selective modulators alter ACh-mediated signaling. I then plan to perform electrophysiology studies in brain slices to test the hypothesis that carbachol-induced inward currents in dopamine neurons are mediated by M5 receptor activation. Finally, I aim to use to use microdialysis to determine if infusion of these modulators into the SNc and VTA can alter extracellular dopamine levels in awake, freely moving rats. I hypothesize that the M5 positive modulator will increase dopamine neuron inward currents and increase dopamine release while the negative modulator will decrease both excitability and extracellular dopamine levels. These studies are important to both elucidate the physiological role of M5 as well as to validate the therapeutic potential of M5 modulation in multiple CNS diseases such as Parkinson's Disease, ADHD, and drug addiction. PUBLIC HEALTH RELEVANCE: Alterations in midbrain dopaminergic neuron activity have been implicated in several central nervous systems disorders including Parkinson's disease, attention deficit disorder, and addiction. Eludicating the physiological role of M5 and other muscarinic receptor subtypes in regulating dopaminergic neurons via subtype-specific allosteric modulators will have important implications for the development of novel approaches to treat these diseases.