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)

Various fatal and debilitating genetic diseases are caused by mitochondrial dysfunction. To study mitochondrial diseases in humans, the Kaeberlein Lab uses NDUFS4-KO mice as a model of the human mitochondrial disease Leigh Syndrome. These mice have a non-functioning protein in their energy-producing oxidative phosphorylation process. We previously observed an increase in total iron in livers of NDUFS4-KO mice, which can damage cells by producing reactive oxygen species. Thus, we hypothesized a low-iron diet may alleviate the effects of the mitochondrial disease. My project aimed to study the molecular consequences of a low-iron diet in NDUFS4-KO mice by quantifying mRNA transcript levels and protein expression of genes involved in iron uptake, storage, or efflux, and comparing these levels between wild-type (WT) and NDUFS4-KO mice fed a normal or low-iron diet. First, I purified mRNA through cell lysis from 35-day old mice liver samples and used the mRNA to perform a one-step Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Next, to quantify protein levels, I performed western blot analysis in collected protein extracts. I observed an increase protein expression and mRNA transcript levels of iron-uptake proteins such as TfR1 in both WT and NDUFS4-KO low-iron diet mice compared both control groups. This suggests iron levels were reduced to safer levels, supporting my hypothesis. Furthermore, when analyzing the expression of genes which respond to iron overload, such as ferritin, I observed a decrease in the low-iron diet NDUFS4-KO mice compared to control diet NDUFS4-KO mice. This observation shows that low iron diet contributes to a reduction in excess iron. My data may illuminate the involvement of iron in mitochondrial diseases as well as support further research into the consumption of low-iron containing foods, and eventually lead to the development of therapeutic drugs to treat humans who suffer from such ailments.

 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.