Dissecting PFC Endocannabinoid-THC -regulated circuits in movement behavior

摘要：美国的大麻使用率从2016年的8.9%大幅上升到2019年的17.5%， 去年吸食过大麻的美国公民(12岁以上)。随着大麻使用量的增加，我们必须 了解有害影响的生物学活性，例如抑制患者运动能力的低血压 活动和功能正常。∆9-四氢大麻酚(THC)是四氢大麻酚中的主要精神活性化合物，ACTs 作为内源性大麻素(ECB)受体CB1R的部分激动剂，可诱导小鼠的运动损伤，如 运动迟缓和下丘脑瘫。除此之外，低速运动对THC反应的神经电路基础，以及 其他CB1R激动剂在很大程度上仍未被开发。使用一种新型的ECB传感器Grab-eCB2.0，在初步的 在大脑皮质(PRL)，我们观察到自发运动事件和ECB活动瞬变之间的相关性。 这些ECB瞬变和复制的GCaMP6f钙瞬变与运动的开始是时间锁定的 与赋形剂治疗的小鼠相比，THC治疗的小鼠显著更大。的中心假说 这一建议是THC在选定的PRL GABA能中间神经元亚群上激活CB1R，这是 抑制催乳素体内的谷氨酸能活动，以调节自发运动。目标1将决定是否 部分GABA能亚群调节依赖的PRL介导的自发运动。我会利用 原位杂交检测GABA能中间神经元(GABA-IN‘s)的差异表达模式组合 用病毒技术和光遗传电生理学技术研究GABA-IN-CRE系的生理 THC引起的变化。我们将测量IPSC中依赖THC的变化，并使用尖端技术 大麻素依赖的兴奋/抑制去极化抑制(DSE/DSI)的探测技术 第2/3层到第5/6层相反。AIM 2将使用GABA-In-Cre和VGLUT-Cre的光纤光度法 表达cre依赖的GCaMP6f或eCB2.0的动物记录特定的神经元活动和ECB水平 THC和/或一种药物对小鼠自发运动过程中抑制性和兴奋性神经元的影响 一系列药理制剂。我们还将病毒表达CRISPR-CB1R结构，以消除CB1R GABA-IN和谷氨酸能神经元确定THC是否能够在神经元中产生相同的变化 活动。我们假设GABA-IN的特定亚群介导了THC依赖性的PRL增加 与自发运动相关的活动。为了确定THC处理的小鼠PRL活性的充分性， GABA-IN和谷氨酸能神经元将受到光遗传刺激或抑制来调节运动。这个 这项提议的目标是培养我成为一名专注于神经药理学的独立研究员 神经调节的研究，利用新方法的培训来破译THC 在运动中调节大脑皮层的活动，同时诱导强健的低速运动。

SUMMARY: Cannabis use in the U.S. has been dramatically increasing from 8.9% in 2016, to 17.5% in 2019, of U.S. citizens (age 12+) having used Cannabis in the last year. As Cannabis use increases, it is critical that we understand the biological activity of unwanted effects such as hypolocomotion that inhibit a patient’s ability to move and function normally. ∆9-Tetrahydrocannabinol (THC), the primary psychoactive compound in THC, acts as a partial agonist at the endocannabinoid (eCB) receptor, CB1R, to induce motor impairments in mice such as hypolocomotion and catalepsy. Beyond this, the neural-circuit basis of hypolocomotive responses to THC, and other CB1R agonists, remains largely unexplored. Using a novel eCB sensor GRAB-eCB2.0, in the prelimbic cortex (PrL), we observed a correlation between spontaneous movement events and eCB activity transients. These eCB transients, and replicated GCaMP6f calcium transients, are time-locked to the initiation of movement and were significantly greater in THC-treated mice compared to vehicle-treated mice. The central hypothesis of this proposal is that THC activates CB1R’s on select PrL GABAergic interneuron subpopulations, which disinhibits the glutamatergic activity within the PrL to modulate spontaneous movement. Aim 1 will determine if select GABAergic subpopulations modulate THC dependent PrL-mediated spontaneous movement. I will utilize in situ hybridization to probe differential expression patterns of GABAergic interneurons (GABA-IN’s). Combining GABA-IN-Cre lines with viral techniques and optogenetic electrophysiology, I will investigate the physiological changes induced by THC. We will measure THC-dependent changes in IPSCs as well as use cutting-edge techniques to probe the cannabinoid-dependent depolarized suppression of excitation/inhibition (DSE/DSI) in layers 2/3 to layers 5/6 contralaterally. Aim 2 will utilize fiber photometry of GABA-IN-Cre and VGLUT-Cre animals expressing cre-dependent GCaMP6f or eCB2.0 to record the specific neuronal activity and eCB levels of inhibitory and excitatory neurons during spontaneous movement of mice treated with THC and/or one of a series of pharmacological agents. We will also virally express a CRISPR-CB1R construct to eliminate CB1R from GABA-IN’s and glutamatergic neurons to determine if THC is able to produce the same changes in neuronal activity. We hypothesize that select subpopulations of GABA-IN’s mediate the THC-dependent increase in PrL activity associated with spontaneous movement. To determine the sufficiency of PrL activity in THC treated mice, GABA-IN’s and glutamatergic neurons will be optogenetically stimulated or inhibited to mediate movement. The goal of this proposal is to train me to become an independent investigator focused on the neuropharmacology of neuromodulation, leveraging training in novel approaches to decipher the mechanisms by which THC modulates prelimbic cortical activity during movement while inducing robust hypolocomotion.