Substance use disorder (SUD) can be defined as the misuse of pharmaceuticals, illegal drugs, and alcohol, and suggested to be due to the co-opting of existing pathways in the brain controlling natural reward. The endogenous opioid dynorphin (dyn), signaling via the kappa opioid receptor (KOR), has shown great promise in being targeted as an effective treatment strategy for SUDs. However, the role for dyn-KOR signaling in natural reward seeking and specifically, the location of its action in the brain are unknown; information that is critical in informing intervention times for treatment. The Dorsomedial Striatum (DMS) is an area of the brain where dynorphin is ample, and it’s crucial for reward-seeking behavior. However, what dyn is doing to influence these behaviors is unknown. To study natural reward-seeking, we set up an operant behavior task where the mice learn to nosepoke (seeking) into an “active” port for sucrose (reward), whereas nosepoking into an “inactive” port yields nothing. While wild-type mice learn this behavior, we observe that mice lacking DMS dyn are slower and do not seek as many rewards upon learning. This suggests that DMS dyn is necessary for reward-seeking behavior. To determine if the converse is true, we artificially boosted DMS dyn release using in vivo optogenetics. We show that when we stimulated dyn release during reward delivery, mice enhance their seeking behavior. Hence, we hypothesized that dyn may help shape the value of a reward, thereby impacting seeking. We designed a reward devaluation task, where animals are given free access to the reward prior to a session of reward-seeking, to decrease/devalue their seeking. We observe that wild-type animals decrease their responding, but animals lacking DMS dyn don’t devalue their seeking. Altogether, we reveal that dyn transmission in the DMS during reward shapes seeking, thereby enabling value-guided flexibility of reward-seeking behavior.

FKBP51 is a protein that acts as a co-chaperone for glucocorticoid receptors and is active during the stress response. FKBP51 blunts glucocorticoid receptor signaling and can interfere with feedback inhibition of stress responses. Overall, increased levels of FKBP51 and its gene FKBP5 positively correlates with an increased risk of stress-related neuropsychiatric disorders. FKBP5 is expressed in serotonin neurons of the dorsal raphe nucleus (DRN), a brain region and system important to stress and anxiety responses. In order to investigate the function of FKBP5 in the DRN, new plasmids were generated to knock down or overexpress FKBP5, thereby changing FKBP51 expression in serotonin neurons. My first goal was to validate these plasmids using Neuro2A cells which endogenously express FKBP5. In order to do this, I cultured Neuro2A cells so that we could transfect the cells with either a CRISPR plasmid to decrease FKBP5 expression or an overexpression plasmid to increase FKBP5 expression. I used western blots to test for changes in FKBP51 protein, and that data was analyzed using integrated density in ImageJ. I found that the CRISPR knockdown plasmid successfully decreased expression of FKBP51 in cells and that the overexpression virus upregulated FKBP5. My second goal was to validate the CRISPR FKBP5 knockdown in vivo. Using Pet1-CRE mice that express Cre recombinase in serotonin neurons, we injected the CRISPR virus for a control virus into the DRN. I then used fluorescent in situ hybridization to look for changes in FKBP5 mRNA levels. I found that CRISPR successfully reduced FKBP5 relative to controls, indicating this virus is a viable way to reduce FKBP5 expression in vivo. This research is a clear step to better understanding stress-related neuropsychiatric disorders such as depression, anxiety, and PTSD.

Research demonstrates that acute and chronic stress can both reduce as well as potentiate an animal's drive to seek reward. In humans, anxiety disorders are also highly comorbid with substance-use disorders. A better understanding of the underlying circuitry connecting these behaviors is necessary for developing better treatment strategies. Neuropeptide S (NPS) acts to reduce anxiety-like behavior and drives drug-seeking through activation of its cognate Gq-coupled protein receptor, NPSR1. The peri locus coeruleus (periLC) produces NPS and sends projections to the orbitofrontal cortex (OFC), a region dense with expression of NPSR1. Although we know the NPS population is involved in cue-reward processing, the elucidation of specific NPS/NPSR1 neuronal circuits correlated to observed behaviors have yet to be documented. In preliminary studies, my mentor, Dr. Kasey Girven, successfully identified a projection from NPS neurons in the periLC to the OFCNPSR1 neuron population. Currently I am investigating the role of the OFCNPSR1 population in drug-seeking behaviors. In my preliminary work I utilized NPSR1-cre mice that expressed a cre-inducibleGCaMP6s in the OFC and a photometry fiber implant above the expressing population to examine the OFCNPSR1 activity during a fixed ratio one task constructed to incentivize an active nose poke with 10 seconds of access to a 10ug/ml fentanyl solution. Using this paradigm combined with fiber photometry, I was able to test the effects of oral fentanyl self-administration on OFCNPSR1 neurons and I uncovered a bidirectional response to delivery of the conditioned stimulus (enhanced activity), and fentanyl reward (quiescence). This experimentation seeks to further our findings on the role of NPS transmission in the OFC and its involvement in drug-seeking behaviors.

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.

The increasing availability of both prescription and illicit opioids has caused substance use disorders to skyrocket. Treatment options such as therapeutics that can inactivate Kappa opioid receptors (KOR) have been shown to reduce drug-seeking behavior through the modulation of intracellular signaling pathways. Downstream KOR activation, G-protein coupled receptor kinase 3 (GRK3)/arrestin-dependent pathway leads to activation of p38 mitogen-activated protein kinase (p38 MAPK) and feelings of dysphoria. In another pathway downstream of KOR receptor activation, a G-protein mediated response and activation of cJun kinase (JNK) leads to the generation of reactive oxygen species (ROS). Selective activation by biased ligands of the JNK mediated pathway result in the release of ROS, which leads to the eventual depalmitoylation of the G-αi/o subunit of the KOR. This results in the long-term inactivation of KOR, which is predicted to improve stress resilience and to prevent drug-seeking behavior. Drugs such as Nalfurafine and Nalmefene can selectively activate KOR such that ROS is produced. Using 2-photon microscopy to detect fluorescence that indicates the release of ROS by Nalmefene and Nalfurafine into the ventral tegmental area of transgenic mice, I can better understand the potential of these drugs for long-term inactivation of KOR. I have observed under a light microscope that when Nalfurafine was washed onto slice, an increase in ROS was observed. Nalmefene showed a similar trend to Nalfurafine but increased ROS to a lesser extent. Additionally, when either Nalfurafine or Nalmefene were added to a solution of naloxone, there was no significant increase in ROS. Using the data collected from slice and behavioral assays such as the tail-flick test, we can illustrate the positive therapeutic effects that KOR inactivators can have on substance use disorders in the long term.

Leigh Syndrome (LS) is the most common form of mitochondrial disease in children. It affects 1 in every 40,000 births and is characterized by ataxia, seizures, failure to thrive and premature death. There are more than 75 gene mutations that have been associated with LS. Among them is NDUFS4, the gene that codes for a subunit of the protein complex I of the mitochondria. Mice carrying a whole-body knockout (KO) of this gene greatly model this illness; they recapitulate multiple phenotypes of LS in patients. Prior studies in the lab have shown that the KO of Ndufs4 in GABAergic neurons, not in excitatory neurons, across all brain regions, reproduce the epilepsy phenotype seen in the global KO mice. Moreover, GABAergic neurons in a specific brain region such as the brainstem are sufficient to lead to epilepsy in mice. Mice with Ndufs4 KO in brainstem and cerebellum interneurons, mediated by GlycineCre, have epilepsy. However, it is still unclear as to what brain regions housed neurons involved in seizure activity in these mice. In this study, brain regions experiencing neuronal hyperactivity and hypersynchrony during seizures in this new model of LS were examined. A thermal seizure was induced in the Ndufs4 GlycineCre KO mice. Forty-five minutes after the seizures, the mice were anaesthetized, the brains were fixed, and harvested. Brain slices were prepared and stained with a c-Fos antibody and finally imaged on the confocal microscope. Surprisingly, high c-Fos immunoactivity was observed in the cerebellum alone and not in other brain regions generally known to be involved in seizure generation. These findings indicate the participation of the cerebellum in seizure generation in Leigh Syndrome epilepsy. In future studies, we will repeat this experiment to increase the sample size and confirm these findings.