Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive compound found in Cannabis sativa. The psychoactive and cannabimimetic behaviors associated with THC have been well described as being dependent on the partial agonist activity of THC at the endogenous cannabinoid 1 receptor (CB₁R). We are investigating the direct action of THC on the medial prefrontal cortex (mPFC, a brain region primarily responsible for executive function), and the effects of adolescent THC exposure on µ-opioid receptor (MOR) expression in adult periaqueductal grey (PAG, a brain region involved in opioid-mediated pain inhibition). To increase our understanding of the cannabimimetic behavioral effects of THC, and its direct pharmacological action in the brain, it is important to map the neuro-anatomical expression of target proteins. We examined expression patterns of CB₁R and MOR in the mPFC and the PAG, respectively. To do this, we utilized a form of in situ hybridization, RNAscope. We leveraged RNAscope by preparing tissue samples from brain regions of interest for treatment with mRNA-specific probes, allowing us to target CB₁R and MOR mRNA. After a series of washes and incubations, these fluorescent probes hybridize to our target mRNAs and allow us to visualize their expression under a confocal microscope. Analysis of mRNA expression informs us on the localization of the CB1R/MOR and known neuron types within our brain regions of interest. After imaging, we are able to utilize HALO software to analyze the levels of expression and co-localization of CB₁R/MORs with neuronal markers for glutamatergic and GABAergic neuron types. By creating and optimizing a workflow for extraction, preparation, hybridization, and analysis, we determined CB₁R mRNA is primarily co-localized with glutamatergic neurons in the mPFC. Moving forward, we are utilizing this RNAscope technique to investigate differential CB₁R expression GABA interneuron subpopulations in the mPFC. (Funded by DA051558)

 Cannabis sativa is one of the most widely used drugs in the world. In humans, Cannabis sativa is commonly used to alleviate anxiety and pain, among other things, in medical and recreational contexts. In mice, intraperitoneal (i.p.) injections of its primary psychoactive compound, Δ9-tetrahydrocannabinol (THC) produce a characteristic triad of behavioral responses consisting of hypolocomotion, hypothermia, and analgesia. However, injections of THC do not accurately represent how humans typically administer THC, which primarily consists of inhalation and oral consumption. To better model a typical route of administration used by humans, we developed a voluntary oral consumption paradigm in mice whereby THC is formulated in gelatin. Following habituation, mice were given ad libitum access to THC gelatin for 2 hours. We measured the triad behaviors immediately following consumption to determine whether voluntary oral consumption of THC-gelatin using this paradigm induces acute cannabimimetic behaviors. Due to the slow pharmacokinetic activity of orally consumed THC, we measured triad responses immediately, 1 hour, and 2 hours after consumption. To compare our relative THC-gelatin-induced cannabimimetic behaviors to published data, we replicated the triad experiment and demonstrated our ability to obtain dose-dependent triad responses by using i.p. injections. At high concentrations (4mg/15mL) of THC-gelatin, cannabimimetic behavioral responses matched those of mice treated with low-dose (3 mg/kg) of THC i.p. injections. From these initial studies we conclude that development of THC-gelatin formulation triggers characteristic cannabimimetic behavioral effects in mice. These results suggests that classical THC and cannabinoid-dependent behaviors in mice can feasibly be studied with a more translational model (Funded by DA051558). Optimizing an oral administration model of cannabinoids in mice will enable future research on the pharmacology of oral cannabinoid therapeutics.