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。伏隔核（NAc）和纹状体多巴胺释放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。刺激黑质致密部（SNc）和腹侧被叶区（VTA）多巴胺能神经元的胆碱能传入可以分别增加NAc和纹状体的多巴胺释放，这种作用对mAChR拮抗剂敏感。在这些中脑多巴胺能神经元上唯一可检测到的mAChR是M5品种，对M5敲除小鼠的研究支持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。由于在所有五种毒毒碱受体亚型（M1-M5）中乙酰胆碱（ACh）结合位点的高度保守性，M5受体的选择性正构激动剂的开发相对不成功。然而，通过靶向从正构口袋中移除的变构位点，Conn实验室成功地开发了M5受体的正变构调节剂和负变构调节剂（分别为VU0238429和VU0419959）。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，我计划对大脑切片进行电生理学研究，以验证碳水化合物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来确定这些调节剂输注到SNc和VTA是否可以改变清醒，自由活动的大鼠的细胞外多巴胺水平。我假设M5正调节性会增加多巴胺神经元向内电流并增加多巴胺释放，而负调节性会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调节在多种中枢神经系统疾病（如帕金森病、多动症和药物成瘾）中的治疗潜力具有重要意义。

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.