Role of CB1R expressed in the prefrontal cortex in the control of locomotion

摘要：近年来，美国的大麻使用量惊人地翻了两番：从2016年的8.9%增加到2017年的17.5%。 2019年，12岁以上的人在过去一年中使用大麻。因此，我们必须更好地了解 大麻中的主要致醉化合物β 9-四氢大麻酚（THC）的生物活性。THC激活 大麻素受体1（CB 1 R），并损害几种行为，包括自发运动。虽然 已知四氢大麻酚减少小鼠的自发运动，神经回路基础和参与 内源性大麻素（eCB），2-花生四烯酸甘油（2-AG），这种反应仍然在很大程度上未被探索。使用 一种新的2-AG传感器（eCB2.0）表达在前边缘皮层（PrL）神经元，我们发现了一个紧密的相关性 在2-AG水平增加、钙瞬变和自发运动开始之间。值得注意的是， 与载体相比，THC处理的小鼠中2-AG和GCaMP 6 f钙瞬变显著更大。 治疗的小鼠，和启动的运动事件的数量大大减少，作为hypolocomotion的函数 反应我们的假设是2-AG激活选择的PrL GABA能中间神经元亚群上的CB 1 R， 其解除抑制PrL内的神经递质能活性以控制自发运动的起始，和 THC加强了这种机制。 目的1将确定THC如何优先调节选择的GABA能神经递质的生理活性。 亚群和多巴胺能神经元。我们将使用RNAscope原位杂交技术来定位CB 1 R PrL中GABA能中间神经元（GABA-IN）的表达。将GABA-IN-Cre系与病毒组合 技术和通道视紫红质辅助电路映射与切片电生理学，我们将确定如何 2-AG对PFC中CB 1-R的作用改变了突触传递以及THC如何影响突触传递。 目标2将利用GABA-IN-Cre和VGLUT-Cre小鼠的体内纤维光度测定和光遗传学操作 在小鼠PrL中表达cre依赖性GCaMP 6 f或eCB 2.0，以记录神经元活性和2-AG 递增剂量给药小鼠自发运动过程中抑制性和兴奋性神经元的水平 的THC。我们将确定具体的2-AG信号成分参与这一反应，使用选择性 药理学抑制剂。我们还将病毒表达CRISPR-CB 1 R构建体以从GABA中消除CB 1 R。 IN和多巴胺能神经元来确定该靶点的参与。基于这个前提，我们将测试 假设GABA-IN的选择亚群介导与增加相关的PrL活性的变化， 2-AG水平和自发运动的控制。确定PrL活动的必要性和充分性 在THC处理的小鼠中，我们将光遗传学刺激或抑制GABA-IN和谷氨酸能神经元， 测量载体和THC处理的小鼠中自发运动的变化。 这一探索性的R21拨款提案为旨在 他们致力于破译eCB调节PrL活性的机制，以及THC如何损害运动行为。

SUMMARY: Cannabis use in the US has alarmingly quadrupled in recent years: from 8.9% in 2016 to 17.5% in 2019 in individuals of age 12+ that used Cannabis in the last year. Thus, it is critical that we better understand the bioactivity of ∆9-tetrahydrocannabinol (THC), the primary intoxicating compound in Cannabis. THC activates cannabinoid receptors 1 (CB1R) and impairs several behaviors, including spontaneous locomotion. While it is known that THC reduces spontaneous locomotion in mice, the neural-circuit basis and involvement of the endocannabinoid (eCB), 2-arachidonoyl glycerol (2-AG), of this response remains largely unexplored. Using a novel 2-AG sensor (eCB2.0) expressed in the prelimbic cortex (PrL) neurons, we discovered a tight correlation between increase in 2-AG levels, calcium transients and initiation of spontaneous locomotion. Remarkably, both 2-AG and GCaMP6f calcium transients were significantly greater in THC-treated mice compared to vehicle- treated mice, and the number of initiated locomotion events greatly reduced as a function of the hypolocomotion response. Our hypothesis is that 2-AG activates CB1R’s on select PrL GABAergic interneuron subpopulations, which disinhibits the glutamatergic activity within the PrL to control the initiation of spontaneous locomotion, and THC potentiates this mechanism. ■ Aim 1 will determine how THC preferentially modulates the physiological activity of select GABAergic subpopulations and glutamatergic neurons in the PrL. We will use RNAscope in situ hybridization to map CB1R expression in GABAergic interneurons (GABA-IN’s) in the PrL. Combining GABA-IN-Cre lines with viral techniques and channel-rhodopsin assisted circuit mapping with slice electrophysiology, we will determine how 2-AG’s action at CB1-R in PFC changes synaptic transmission and how this is impacted by THC. ■ Aim 2 will utilize in vivo fiber photometry and optogenetic manipulation of GABA-IN-Cre and VGLUT-Cre mice expressing cre-dependent GCaMP6f or eCB2.0 in mouse PrL to record changes in neuronal activity and 2-AG levels of inhibitory and excitatory neurons during spontaneous locomotion of mice treated with increasing doses of THC. We will determine the specific 2-AG signaling components involved in this response using selective pharmacological inhibitors. We will also virally express a CRISPR-CB1R construct to eliminate CB1R from GABA- IN’s and glutamatergic neurons to establish the involvement of this target. Based on this premise, we will test the hypothesis that select subpopulations of GABA-IN’s mediate changes in PrL activity associated with increase in 2-AG levels and control of spontaneous locomotion. To determine the necessity and sufficiency of PrL activity in THC treated mice, we will optogenetically stimulated or inhibited GABA-IN’s and glutamatergic neurons and measured changes in spontaneous locomotion in vehicle and THC-treated mice. This exploratory R21 grant proposal provides an ideal mechanisms and scientific foundation for studies aimed at deciphering the mechanisms by which eCBs modulates PrL activity and how THC impairs locomotor behavior.