Dissecting PFC Endocannabinoid-THC -regulated circuits in movement behavior

摘要：美国的大麻使用量从2016年的8.9%急剧增加到2019年的17.5%， 美国公民（12岁以上）在过去一年中使用过大麻。随着大麻使用的增加，我们必须 了解有害效应的生物活性，例如抑制患者 活动和功能正常。四氢大麻酚（THC）是THC中的主要精神活性化合物， 作为内源性大麻素（eCB）受体CB 1 R的部分激动剂，在小鼠中诱导运动障碍， 运动功能减退和僵住症。除此之外，对THC的低动性反应的神经回路基础， 其他CB 1 R激动剂，仍然在很大程度上未开发。使用新型eCB传感器GRAB-eCB 2.0，在前边缘区， 皮质（PrL），我们观察到自发运动事件和eCB活性瞬变之间的相关性。 这些eCB瞬变和复制的GCaMP 6 f钙瞬变与运动的开始时间锁定 并且在THC处理的小鼠中显著大于载体处理的小鼠。的中心假设 这一建议是THC激活选择的PrL GABA能中间神经元亚群上的CB 1 R， 解除抑制PrL内的多巴胺能活性以调节自发运动。目标1将决定是否 选择GABA能亚群调节THC依赖性PrL介导的自发运动。我会利用 原位杂交以探测GABA能中间神经元（GABA-IN）的差异表达模式。结合 GABA-IN-Cre系与病毒技术和光遗传电生理学，我将研究生理 THC引起的变化。我们将测量IPSC中THC依赖的变化，并使用尖端的 探测大麻素依赖性兴奋/抑制去极化抑制（DSE/DSI）的技术 2/3层至5/6层。目的2将利用GABA-IN-Cre和VGLUT-Cre的光纤光度法 表达cre依赖性GCaMP 6 f或eCB 2.0的动物，以记录特定神经元活性和eCB水平 在用THC和/或一种 一系列的药物。我们还将病毒表达CRISPR-CB 1 R构建体，以消除CB 1 R。 GABA-IN和多巴胺能神经元，以确定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的选定亚群介导了THC依赖性的PrL增加 与自发运动有关的活动。为了确定THC处理的小鼠中PrL活性的充分性， GABA-IN和谷氨酸能神经元将被光遗传学刺激或抑制以介导运动。的 这个建议的目的是训练我成为一个独立的研究者，专注于神经药理学 神经调节，利用新方法的培训来破译THC的机制， 在运动过程中调节前边缘皮层活动，同时诱导强烈的运动不足。

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.