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

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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.