It is established that stress can promote addictive drug use and relapse in humans with substance use disorders, thus understanding the stress mechanisms responsible is likely important in developing effective treatments for drug addiction. One effect of stress is the stress-induced release of endogenous dynorphin neuropeptide systems in the brain which activate kappa opioid receptors, that in turn stimulate p38 mitogen-activated protein kinase pathway (MAPK). P38 MAPK activation results in the dysphoria experienced during a stress response. We want to learn more about how p38 affects mood and develop efficient CRISPR techniques to manipulate p38 activation. Other techniques of silencing p38 have been used but come with disadvantages. With a CRISPR approach, virus expressing excision sequences for gene editing can be injected into transgenic mice encoding Cre recombinase in a cell-type specific manner. In this study, I tested a CRISPR/Cas9 virus (AAV1-Flex-SaCas9-sqMapk14), designed to excise the p38 gene. As expected, wild type male mice developed significant conditioned place aversion to the KOR agonist, U50,488. In contrast, mice injected with virus bilaterally in the VTA did not acquire aversion. This finding suggested that successful excision of p38 in VTA had occurred. To confirm with immunohistochemistry, I stained CRISPR injected and U50,488 activated brain slices with p38 and phospho-p38 selective antibodies. We expect slices from CRISPR treated mice to show fewer positive cells in the VTA as compared to controls. My characterization of these antibodies is on-going, but preliminary results suggest differences in CRISPR injected and wild type mice. With the development of a technology allowing for efficient manipulation of p38 MAPK within cell site and brain regional specificity, we hope to provide further insight to the stress response and to better understand its role in addiction.

Peripherally restricted drugs are extremely appealing to drug developers because they remain effective while minimizing adverse side effects such as addiction. This is possible because the drugs stay out of the central nervous system, which allows them to have an impact on the body without affecting the brain. I examined Klight 1.2A transfected Neuro 2a cells under a fluorescent microscope when treated with three different drugs that are thought to be peripherally restricted. Klight is a Kappa opioid receptor agonist sensor that makes the cells fluoresce when they are activated. Neuro 2a cells are a neuroblastoma mouse cell line that is used to study signaling pathways. When I imaged these cells under different drug treatments I quantified the fluorescence. I then compared that quantity to the fluorescence of the cells when treated with U50, a full agonist, at maximal activation. I repeated this procedure for both a high and low drug concentration for each drug. The first two drugs this study examined are Difelikefalin and TP-2021 which are anti-itch drugs from Titan Pharmaceuticals. The third is Asimadonline which plays a role in hot flashes during menopause. My results of these experiments showed significant dose-dependent activation of KLight for all three drugs. These in vitro experiments on plated cells will provide a baseline which can then be compared to the in vivo fluorescence in the brain. In future experiments, when we compare the in vitro and in vivo levels of fluorescence, we can determine if the drugs are peripherally restricted or not.

Alcohol withdrawal exists as a significant barrier to care for those who struggle with alcohol use disorder. Furthermore, there is evidence of worsening withdrawal symptoms after repetitive cycles of alcohol use and abstinence. Our preliminary research points to neuroinflammation primarily regulated by microglia, the immune cells in the brain, as a potential causal component in the escalation of recurring alcohol withdrawal symptoms. Previous work in our lab has shown that mice subjected to chronic intermittent exposure (CIE) to ethanol vapor showed elevated markers of neuroinflammation, as evidenced by transcriptome analysis in microglia. We found that the “unfolded protein response (UPR)” was activated; it plays a critical role in the development of neuroinflammation, and the UPR is mediated significantly by the C/EBP homologous protein (CHOP) an apoptotic transcription factor. Therefore, we hypothesize that CHOP in microglia is necessary for promoting alcohol withdrawal symptoms. We expect to observe measurable reductions in the withdrawal symptoms experienced by transgenic male and female mice with and without CHOP following CIE exposure. In our pilot study, transgenic male and female mice with and without CHOP were subjected to five weeks of CIE to ethanol vapor and then tested for withdrawal phenotypes. These tests include body temperature, locomotion, marble-burying behavior, novelty suppressed feeding, and sucrose preference. We have also characterized microglia morphology and CHOP expression in pilot mice brains. Having measured changes in these mice's neuroinflammatory mechanisms and subsequent behaviors, our results will determine the efficacy of decreasing CHOP gene expression to ameliorate the kindling effect in alcoholics. Therefore, our work provides a promising direction in tackling the inescapable cycle of worsening alcohol withdrawal that limits the road to recovery for alcoholics.

Drug addiction and abuse exacerbate many health issues globally. The nucleus accumbens (NAc), a region within the brain, contains dopamine receptor D1 and D2 medium spiny neurons (MSNs) with relevance to addiction-related behaviors. I investigated the effect of D2 MSNs on behavioral economics of cocaine-seeking through chemogenetic activation of Designer Receptors Exclusively Activated by Designer Drugs (DREADD). I used a transgenic line of rats specifically expressing the Cre-recombinase enzyme in D2 MSNs. This enzyme is responsible for selective expression of otherwise-inert viral vectors by reversing its sequence, allowing for neuron-specific genetic manipulation. I bilaterally injected either a Cre-dependent AAV containing the DREADD hM3Dq or a fluorescent marker as control into the NAc of male rats; additionally, I catheterized the rats to allow for cocaine self-administration. Activation of hM3Dq required binding to clozapine-N-oxide (CNO)—the “designer drug”—and because only D2 MSNs expressed this DREADD, they were the only neurons to activate upon administration of CNO. After recovery, rats were trained to press a lever to receive cocaine infusions; and once this self-administration task was learned, the animals were introduced to reinforcement schedules with blocks of trials with differing unit doses of cocaine. Once this phase was learned, the rats were tested in sessions that were preceded either with an injection of saline or CNO, to test how chemogenetic activation of D2 MSNs affects the cocaine demand curve. Based on studies discussing the roles of D2 MSNs, I predict that chemogenetic activation of these neurons will correlate with decreased demand for cocaine concentrations compared to those injected with vehicle or green fluorescent protein alone. This study can produce valuable insight into the role this pathway plays in the development of drug-seeking behavior, ultimately leading to a deeper understanding of the system and possible avenues for treating addiction.

Morphine and other mu-opioid receptor (MOR) ligands are commonly used treatments for pain management. Long-term administration of opiates results in receptor desensitization and tolerance, inhibiting treatment efficacy and contributing to the current opioid epidemic. Following opioid administration, peroxiredoxin 6 (PRDX6) is recruited to the opioid receptor complex through JNK (cJun N-terminal Kinase)-dependent signaling. PRDX6 activation generates reactive oxygen species (ROS), resulting in opioid receptor desensitization and one form of opioid tolerance. Recently, cannabidiol (CBD) has been implicated in decreasing acute morphine tolerance. The present study will establish a connection between morphine and CBD cotreatment with the hypothesis that cotreatment will decrease ROS production. To measure ROS, I will employ the genetically encoded ROS sensor HRM63, which fluoresces proportionally to ROS production. Because ROS is a product of JNK/PRDX6 signaling, measuring the strength of ROS-dependent fluorescence is a simple way to visualize my hypothesis using in vitro cell models. In each experiment, treatments of morphine, CBD, or combined morphine and CBD will be delivered to plated HEK293 cells stably expressing both MOR and HRM63. I will then image the cells using fluorescence microscopy to quantify ROS response to treatment. I predict that cotreatment of CBD with morphine will result in lower ROS production compared to morphine treatment alone. These results will be crucial in the ongoing characterization of CBD's role in opioid tolerance.

Organelles - membrane bound structures within eukaryotic cells - allow compartmentalization and simultaneous execution of thousands of biochemical reactions within one cell. Rather than serving just one purpose, they are constantly interacting with outside molecules and each other to complete processes. This function is described as being signaling platforms, surfaces that can facilitate interactions between proteins. Mitochondria house signaling proteins that are important for innate immunity and mitochondrial quality control on their surfaces and has been shown to have signaling complexes that associate with the outer mitochondrial membrane to coordinate local protein synthesis. Based on these findings, we hypothesized that mitochondria function as signaling platforms for protein interactions. We chose to focus on two signaling cascades, insulin and calcium, due to their significance in metabolic processes and clinical relevance. To investigate, we used proximity labeling technology, in which a molecular tag is attached to proteins within a certain cellular location. We labeled proteins that alter their mitochondrial association in response to these two stimuli. After cells were treated with stimuli, the lysed and tagged proteins were visualized through Western blots. This process also enables their identification through mass spectrometry. Our goal is to have a detailed characterization of mitochondria associated proteins after insulin and calcium stimulation. Such data will create openings for further research into the mitochondria as a signaling platform and potential for implications in the medical world.

Decades of research have demonstrated that RNA molecules can serve as architectural scaffolds, templating the assembly of subcellular compartments in all kingdoms of life. In mammals, architectural RNAs scaffold an array of subnuclear structures that are essential to cellular function including metabolism, DNA repair, and epigenetic programming. Many of these architectural RNAs are causally dysregulated in diseases, including cancer and neurodegenerative disorders. However, the molecular mechanisms of these architectural RNAs remain poorly understood, partially because technologies for identifying the molecules (proteins, DNAs, other RNAs), with which RNAs interact are lacking. To address this challenge, the Shechner Lab has developed a technology termed Oligonucleotide-Directed Biotinylation (ODB), a universal method for elucidating RNA subcellular interactions. ODB applies a powerful method called proximity-biotinylation to individual RNAs. In proximity-biotinylation, a promiscuous biotinylating enzyme (e.g. Horseradish Peroxidase, HRP) is targeted to a subcellular compartment of interest. This enzyme then tags nearby (~10 nm) molecules with biotin, enabling their straightforward isolation and analysis. ODB advances this technology by using RNA-in situ Hybridization (RNA¬–FISH) methods to precisely deploy HRP to individual RNAs. Ongoing work, using a series of model RNA targets, has demonstrated that ODB can reveal the proteins, RNAs, and genomic loci near a target RNA at exceptional depth and precision. My goal is to generalize this ODB protocol, developing methods that can be applied to any RNA target. To examine how RNA abundance influences ODB experimental design, I use pulse-chase, RNA decay experiments to manipulate the expression of the architectural RNA NEAT1, and examine how to adjust parameters of the ODB protocol to compensate for this altered expression. Likewise, I am using imaging-based assays to investigate how ODB's biotinylation radius varies with labeling conditions, increasing the range of ODB’s target radii. Collectively, this establishes general guidelines for adapting ODB to novel RNA targets.

Proteins often interact with other proteins to relay signals or to cause physiological effects. Knowing the components of cell signaling pathways is important for understanding diseases and developing treatments. However, it can be difficult to isolate and detect protein complexes because some experimental conditions are too harsh on the weak inter-protein interactions. We therefore questioned whether we could use cross-linking to maintain native protein complexes for rapid isolation and detection. I used formaldehyde (FA), a protein cross-linker, in conjunction with a 100k molecular weight cut-off (MWCO) spin filter to isolate protein complexes above 100k molecular weight (MW). FA treatment for a few minutes should only cross-link proteins that are near each other. Protein complexes formed through cross linkage should be massive (>100k MW) compared to most unlinked proteins. Therefore, complexes of closely-associated proteins can be rapidly purified from a 100k MWCO filter. I specifically studied the proteins associated with epidermal growth factor receptor (EGFR) in response to extracellular epidermal growth factor (EGF) treatment. EGFR itself is above 100k MW, but the proteins that associate with it when EGF is bound to it, such as MAPK and GRB2, are below 100k MW. The presence of the smaller proteins in filtered cell lysate samples prepared with EGF and FA treatment would show that the described experimental setup can be used to isolate smaller proteins in complexes above 100k MW. This can be further applied in structural and mechanistic studies of protein complexes involved in normal and pathological physiological processes, in determining novel complexes after further purification and identification steps, and in time course experiments—where different proteins may arrive in a complex at different times post treatment. The aforementioned applications can help pave the way for novel and more effective disease treatments and provide a better understanding of the inner workings of cells.

Binge eating disorder is a debilitating disease which can arise from many kinds of traumas, pains, and stresses of life. Previous characterization of a binge-eating model developed by our lab shows that mice will consume greater quantities of high palatable diet (HPD) following exposure to specific types of psychological stressors including forced swim and foot shock compared to mice exposed to such psychological stressors. It was found that the claustrum of the brain had increased neural activity following bouts of binge-eating. One aspect of my research required me to quantify the density of neural activation in the claustrum from its most rostral to caudal area. We found that stressed mice displayed significantly higher levels of claustrum neural activation compared to controls. For the behavioral pattern we wanted to rule out influence of energy expenditure in the stress paradigm. Mice were given access to running wheels for an hour and then received access to HPD. Mice who displayed high levels of running had similar food intake to that of mice who did not display running activity. This suggests that psychological stress is an underlying component in this model for stress eating. As an ongoing project we are utilizing 1-photon imaging in the claustrum to monitor single cell activity across no stress vs. stress sessions and subsequent feeding behavior. We have thus far found increased neural activity in response to onset of a feeding bout in no stress conditions and we are investigating how stress modulates the effect of neurons tracked across time. This research potentially has great impact on the scientific community’s knowledge behind why psychological stressors contribute to binge-eating behaviors and could one day have astounding translational benefits for treating humans with binge-eating disorder.

The nucleus accumbens core (NAc), among other brain regions, plays a key role in drug seeking behavior and relapse, particularly in determining incentive value as cocaine consumption escalates. Neurons in this region have two genetically-distinct output projections which form the direct and indirect pathways, D1 and D2 respectively. My experiment is based on the Incubation of Craving model, in which animals show increased drug seeking after a period of abstinence from self-administered cocaine. The behavioral side of this project seeks to better understand how these two pathways contribute to drug seeking behavior following a period of forced abstinence, and I am also investigating how RNA translation changes in and between the direct and indirect pathways following the escalation of cocaine taking and incubation of craving by forced abstinence. I used a transgenic line of rats expressing the Cre-Recombinase enzyme in the D1 and D2 medium spiny neurons (MSNs) to selectively express and manipulate MSNs in the direct and indirect pathways. I bilaterally injected either DIO-hM4Di RiboTag, DIO-hM3Dq RiboTag or a fluorescent control virus into the NAc, and the rats were catheterized during this time to allow for cocaine self-administration. The rats underwent the Incubation of Craving experiment where they experienced an acquisition and abstinence period. Once the Incubation of Craving was complete, I collected and homogenized the NAc from each subject and performed RNA purification. Then, I performed qPCR and RNA sequencing to investigate, and validate any pathway-specific changes in mRNA expression following these behaviors with the goal to discover new therapeutic targets. My hypothesis is that ribosome-associated mRNA in synaptosomes differs from the cell bodies, with enrichment of RNAs known to be trafficked to dendrites. Additionally, I predict that incubation of craving will induce distinct patterns of RNA changes in neurons of the direct and indirect pathway.