Cannabis use has dramatically increased in response to legalization in the U.S., with total sales in the U.S. jumping 46% from 2019 to 2020. ᐃ9-tetrahydrocannabinol (THC) is the primary psychotomimetic compound in Cannabis and has been shown to modify memory and motivation, processes mediated by the prefrontal cortex (PFC) brain region. I sought to test the effects of THC on PFC activity during appetitive Pavlovian conditioning in mice- a behavior in which a subject learns to associate a non-rewarding stimuli to a reward. THC acts on the endocannabinoid (eCB) CB1 receptor (CB1R), a presynaptic signaling protein responsible for modulating neural activity throughout the brain, with robust expression in the PFC. To monitor neural activity during behavioral trials, we implanted optic fibers into the PFC and virally expressed biological sensors: GCaMP6f to track Calcium activity, and the novel GRABeCB2.0 to measure eCB activity. VGAT-Cre and VGLUT1-Cre animals were presented with a house light prior to a sucrose reward to observe the neuronal GABAergic and glutamatergic activity during the conditioning, respectively. After 5 days of conditioning, I administered vehicle or THC (i.p., 5 mg/kg) to observe behavioral and neural effects of THC. We observed neural activity that transferred from the sucrose reward to the house light cue suggesting these neurons encode for this learning. Endocannabinoid activity also transitioned from sucrose reward to the house light cue suggesting cannabinoid involvement in regulating this association. THC pre-treatment reduced licking and motivation for sucrose while modifying neural activity without eliminating it. This provided much needed insight into the formation of memory during learning and reward motivation under the effect of THC.

Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive compound found in Cannabis sativa and acts on the cannabinoid-1 receptor (CB₁R). Given its well-documented analgesic effects, THC’s therapeutic value in treating pain such as those associated with motor neuron disease states, muscle spasticity-related pain, chronic pain, and muscular sclerosis has gained traction. THC’s psychotomimetic locomotor impairing effects causes patients to cease treatment. However, this relationship between THC and locomotor control is poorly understood. To address this, we are investigating THC’s effects on Pre-Frontal Cortex (PFC) neural activity during natural, unprompted movement behavior in mice. The PFC historically is known for its role in executive function but is also a target for THC’s psychotomimetic effects. We expressed GRAB_eCB2.0, an endocannabinoid biosensor, or GCaMP6f, a Ca²⁺ biosensor, in the PFC and recorded neural activity through fiber photometry during uninhibited movement behavior. We found a novel, THC- and locomotion-dependent transient of Ca²⁺ and endocannabinoid activity in the PFC at the initiation of movement. I investigated the activity of glutamatergic and GABAergic neuron activity in the PFC by utilizing genetic mouse lines and found the Ca²⁺ activity transients were primarily driven by the GABAergic interneurons that constitute 20% of the anatomical population. I hypothesized that this is due to THC-dependent activation of the CB1R on distinct GABAergic interneuron subpopulations in the PFC, which would disinhibit glutamatergic activity and in turn promote spontaneous movement. I utilized in situ hybridization to examine colocalization of CB₁R with distinct GABAergic interneuron subpopulations. We found that while CB₁R does, in fact, colocalize with GABAergic interneurons, there was no differential localization between subpopulations. Overall, this project furthers our understanding of the ways in which THC modulates neuronal activity and locomotive behaviors.

Melanoma is the leading, most deadly, cause of skin cancer. The survival rate of melanoma was observed from 2011-2017 to drop to 68% for patients with regional spread, and less than 30% for distant, metastatic tumours. Treatment is possible, and therapies such as immune checkpoint blockade (ICB) have been developed, increasing 5-year survival rates to over 50%. Current research has just begun to understand the importance of the tumour microenvironment (TME) for cancer survival. To address the hypothesis that specific gene expression within the TME influences the depth and quality of T-cell infiltration, and thus is a tumour-intrinsic property, I will be investigating infiltration across different subtypes of melanoma to assess whether T-cell exclusion can be predicted by transcriptional signatures. Tumours can be classified as immune-inflamed, (a result of CD8 T-cell infiltration), immune-excluded (CD8 T-cells localized around the border), or as an immune desert, entirely void of CD8 T-cells. A better understanding of these phenotypical and genotypical distinctions, propensities, and consequences is integral to healthcare. By implementing clinically relevant in vivo models, I am able to rigorously investigate TME effects on immunotherapies. The development of innovative humanized mouse models of melanoma, using genetically engineered ‘MISTRG’ recipient mice, has allowed us to mimic an entirely functional human immune system to respond to human melanoma tumours in vivo. Results from my lab suggest that the positioning of T cells in the tumour microenvironment is a tumour-intrinsic property, and modelling of the difference between infiltrated (hot) and excluded (cold) tumours is made possible with extensive modelling software, flow cytometry, epigenetics, and data analytics. The data that TME is intrinsic to patient success are thrilling, and future developments within the immunological field are sure to increase patient success even further.