Reward-seeking behavior, crucial for survival, is hijacked by various neuropsychiatric disorders, notably substance use disorder. Prior work implicated reward-seeking with the dorsomedial striatum (DMS), a brain structure expressing the endogenous opioid dynorphin (dyn) in 50% of its neurons. Previous studies also linked dyn, signaling through the kappa opioid receptor (KOR), with escalation and reinstatement in drug-seeking, however, the exact mechanisms of dyn-KOR signaling remain unknown. Recent experiments from the Bruchas lab show that supraphysiological release of dyn in the DMS with excitatory optogenetics elicited reward-seeking behavior, demonstrating that dyn-KOR is sufficient for reward-seeking behavior, but not that it is necessary. To provide evidence of necessity, I plan to inhibit dyn-KOR signaling in the DMS by expressing the novel inhibitory optogenetic tool Platynereis dumerilii (PdCO) that we recently showed to be useful for such studies (Wietek et al., Nat Methods, 2024). PdCO is expressed in a target neuronal population and begins inhibiting under 473 nm light. Hence, in this study, PdCO will be injected into the DMS and expressed selectively in dyn neurons. Following recovery, mice will learn a self-administration procedure to study reward-seeking behavior, where sucrose is delivered when mice poke their nose in the correct 1 of 2 holes, with a 5s light cue before reward delivery. Following learning, I will inhibit dyn release in a counterbalanced fashion by activating PdCO using 20 Hz pulsed 473 nm light. I anticipate that inhibiting dyn release would result in reduced sucrose consumption. Finally, I plan to use an extinction learning procedure, where dyn release is inhibited during perceived reward delivery. Here, I hypothesize that inhibition will accelerate extinction learning. By leveraging the specificity of optogenetics on various procedures, this study begins to isolate the exact mechanisms by which dyn-KOR signaling impacts reward-seeking behavior with potential insights for substance use disorder interventions.

The sensation of acute pain is fundamental to survival, indicating tissue damage that motivates an animal to engage in adaptive protective behaviors. Chronic pain, however, is pain that persists beyond the typical recovery window and serves little adaptive function. Due to the negative affective component inherent in chronic pain, it has been shown to contribute to the development of comorbid psychiatric disorders such as depression, social aggression, and social withdrawal. Our research aims to understand how acute and chronic pain affects social reward, motivation, and behavior. Our experiments used social self-administration (SSA), where pair-housed mice were placed in operant chambers and underwent voluntary lever press trials for the reward of social interaction with their cagemate. Following the acquisition of the SSA task, male and female mice then received a spared nerve injury (SNI) to induce neuropathic pain. Following recovery from the SNI surgery, experimental mice were returned to the lever press trials at different post-operative windows to simulate acute and persistent pain windows. Changes in social behavior were measured via changes in lever press frequency and interactive behavior during trials. Our data shows that male and female mice who underwent SSA trials immediately following nerve ligation maintained normal levels of social interaction. In contrast, female experimental animals that began SSA at a delayed time point, simulating persistent pain, lever pressed significantly less for a social partner compared to their respective control group. Conversely, male experimental animals at the delayed time point displayed high lever pressing behavior for a social partner. This data points towards divergent social adaptations following pain due to the sex of the experimental mice. Future experiments will further delineate these sex-specific adaptations following a traumatic injury. This research can inform social intervention as an adjunct or alternative treatment to pharmacological pain intervention and its comorbidities.

 The locus coeruleus (LC), a nucleus in the pons of the brainstem, is the primary source of norepinephrine (NE) in the brain and is responsible for regulating arousal and stress responses. The LC also has roles in pain, avoidance and reinforcement learning, exploration, and feeding. How LC neurons are mediated to generate these distinct responses has yet to be defined. One area of interest is the peri-LC, which sends local GABAergic projections to the LC and is thought to modulate physiological arousal through direct inhibition of the LC. The peri-LC is composed of various populations of GABAergic neurons that may contribute to the inhibition of the LC. We were interested in exploring the role of neurokinin1/substance P, nociception, and enkephalin GABAergic neurons. Using vGAT-cre, Tac1-cre, Noci-cre, and Penk-cre mice strains, we injected an excitatory DREADD into the peri-LC to be able to activate genetically defined neural subpopulations within the peri-LC. I ran the mice through open field and elevated zero maze testing to assess anxiety levels. The activation of all peri-LC^GABA neurons led to a decrease in anxiety-like behavior, which is consistent with an inhibition of the LC and a decrease in LC^NE release. In contrast, the activation of peri-LC^Tac1 neurons increased anxiety-like behavior, while the activation of peri-LC^Noci and peri-LC^Penk neurons had no behavioral effect in these assays. Together, these results suggest that the different peri-LC^GABA subpopulations likely represent distinct nodes that differentially integrate novel stimuli to influence distinct behaviors. Understanding how the peri-LC regulates LC function can give insight into the neurobiological basis of arousal and stress-related disorders, which informs our understanding of neuropsychiatric pathologies.

The mechanistic target of rapamycin, mTOR, functions in the mTORC1 complex with another protein called raptor as a master regulator of eukaryotic cellular metabolism thereby regulating cell growth including from cancer, cell death including after stroke, inflammation, and aging. In a forward genetic screen for hypoxia resistant mutants, the Crowder lab recently identified a missense mutation in the daf-15 gene, which encodes C. elegans raptor. The mutation produces a heat-sensitive reduction of raptor function, hereafter referred to as daf-15(rf). At 20°C, daf-15(rf) is normally hypoxic sensitive, at 22°C very hypoxia resistant, and at 25°C incapable of normal development. Raptor negatively regulates autophagy, a mechanism for breakdown and recycling of proteins and organelles. Activation of autophagy has been found to promote hypoxic survival in C. elegans and higher organisms. Thus, we hypothesized that activation of autophagy was responsible for the hypoxia resistance of our daf-15(rf) mutant. To test this hypothesis, we first asked whether we could detect increased autophagy using fluorescently-tagged autophagy proteins at 22°C in daf-15(rf) but saw no effect compared to wild type. Next, we asked whether a C. elegans transcription factor, HLH-30, that promotes expression of autophagy proteins was activated by daf-15(rf) and found activation at 25°C but not at 22°C. Finally, we tested whether proteins essential for autophagy were also necessary for the hypoxia resistance of daf-15(rf). By generating double mutant strains, we showed that animals with daf-15(rf) but without essential autophagy proteins were still hypoxia resistant. Thus, we conclude that C. elegans raptor regulates hypoxic sensitivity by an autophagy-independent mechanism. These findings demonstrate that raptor can control hypoxic cellular injury by mechanisms distinct from autophagy. Such mechanisms, if identified, could be targeted for treatment of cancer, stroke, and other diseases where hypoxia plays a role.

mTOR, the mechanistic target of rapamycin, is a serine/threonine kinase that regulates protein synthesis, cell growth, and metabolism in response to nutrients and energy in most eukaryotes. mTOR consists of two distinct complexes, mTORC1 and mTORC2. These complexes can be further divided into three components: mTORC1 consists of mTOR, Raptor, and mLST8, and mTORC2 consists of mTOR, Rictor, and mLST8. mTORC1 is critical in metazoan development and has been implicated in aging, cancer, diabetes, cardiovascular disease, and hypoxia. Previously, the Crowder lab conducted a mutant screen in C. elegans for hypoxia resistant mutations, and identified a missense reduction of function mutation in the daf-15 gene, the C.elegan ortholog of Raptor. A unique feature of this mutation is that the function of Raptor can be turned on and off by varying temperature. It has normal hypoxia resistance at 20 degrees, increased resistance between 21-22, and developmentally arrests at 25 degrees. I and the other authors made use of this conditional developmental arrest phenotype to screen for genetic suppressors. Using genetic mapping, sequencing, and complementation testing, we have identified multiple mutations in three different genes responsible for restoring Raptor function. One of the genes identified in this manner was rnf-126. Results show mutations in rnf-126 suppress the Raptor mutation. A null mutation in rnf-126 similarly suppressed the Raptor mutation. Previous work has implicated mammalian rnf-126 in degradation of the mTORC1 complex in cancer cells, suggesting that reduced levels of daf-15 may produce hypoxia resistance. We tested this hypothesis using auxin-mediated degradation of daf-15, finding that auxin-treated animals are hypoxia resistant. Current work by myself and others will further investigate how rnf-126 controls Raptor function and hypoxia sensitivity. Elaborating the function of this gene will define novel mechanisms whereby Raptor and mTORC1 controls metabolism, hypoxic injury, and development.

In recent years, the United States has seen an increasing number of opioid overdoses, causing many research studies to focus on opioid drugs of abuse. However, we still lack a fundamental understanding of the opioid receptor that these drugs bind to. The mu-opioid receptor (MOR) is involved in both the pain-relieving and euphoric effects of opioid drugs. The Ventral Tegmental Area (VTA), known to be a major source of dopamine, contains a significant amount of inhibitory gamma-aminobutyric acid (GABA) neurons that express MORs. Previous research has shown that activity at the MOR in the VTA is rewarding and that dopamine projections from the VTA to the Nucleus Accumbens (NAc) are activated to reinforce behavior. However, whether the expression of MORs on GABA neurons in the VTA is important for influencing VTA to NAc dopamine activity during reward seeking behavior is unclear. To investigate the role of the MORs in reward seeking behaviors, I knocked out MORs in the VTA and used dLight, a fluorescent dopamine sensor, to measure dopaminergic release from synapses in the NAc during Operant and Pavlovian conditioning tasks in the absence of these receptors. Pavlovian conditioning utilizes chambers in which a house light turns on and a sucrose pellet is delivered via food hopper to food-restricted mice. In the operant conditioning task, mice must learn to complete a nose poke in order to receive the sucrose pellet reward. These tasks allow me to gain a more complete understanding of how the absence of MORs in the VTA changes behavior and dopamine activity during reward-seeking. This research furthers our understanding of how the MOR affects natural reward and motivated behaviors and is crucial in helping us understand how opioids of abuse alter existing brain circuitry to cause opioid use disorder.

The use of opioid drugs for pain management in postoperative settings has been widespread since the 1860s. However, the risk factor for developing an opioid use disorder (OUD) has increased substantially with its continued use, with addiction rates of more than 10% for those taking opioids. An obstacle to abstinence in opioid addiction are the adverse side effects that occur during cessation of drug use once dependence has formed, including nausea, anxiety, vomiting, and depression. The Nucleus Accumbens (NAc) is part of the mesocorticolimbic reward pathway. Decades of pharmacological studies demonstrate that nearly all abused drugs evoke dopamine release within the NAc, thus altering innate systems for how reward is processed. The activity of NAc neurons is strongly regulated by efferent excitatory input from numerous brain regions. The paraventricular thalamus (PVT) a relatively understudied brain region, regulates behavioral responses to reward and aversive stimuli as well as to drugs of abuse such as morphine. Our preliminary data demonstrate that the activity of these projections is highly regulated by the Cannabinoid 1 Receptor (CB1), which mediates the primary psychoactive effect of cannabis. This is particularly relevant on account of recent clinical findings demonstrating that activation of CB1 can ameliorate the aversive effects of opiate withdrawal. Using fiber photometry (which uses fluorescence emission of the calcium sensitive fluorophore, GCaMP, as a proxy measurement for neural activity), I have shown that this circuit is activated by aversive stimuli and inhibited by rewarding stimuli. Furthermore, treatment with morphine can attenuate the pain-induced activation of this circuit. However, whether cannabinoids can influence this circuit's activity to reduce withdrawal symptoms remains untested. Our research will contribute to our understanding of the neurophysiological basis for opiate withdrawal and how cannabinoids could represent a novel class of therapeutics for the treatment of opiate use disorder.

Mitochondria are the main oxygen consumers in eukaryotic cells and as such are the primary organelles affected by oxygen deprivation, hypoxia. Hypoxia alters the size and shape of mitochondria, called the mitochondrial dynamics, but their role in hypoxic cell death is unknown. The Crowder lab has recently discovered that a mutation in the Mechanistic Target of Rapamycin Complex One (mTORC1) protein Raptor confers hypoxia resistance in the nematode C. elegans. mTORC1 is a master regulator of metabolism and is known to affect certain aspects of mitochondrial biology. Given these two facts, we tested the hypothesis that the hypoxia resistance of the C. elegans Raptor mutant is from alterations of mitochondrial dynamics. First, I showed that hypoxia induces small, rounded mitochondria in C. elegans caused from mitochondrial fission. Second consistent with the hypothesis, I showed that the mitochondria appear to have more normal morphology before and after hypoxia in the Raptor mutant. However, not consistent with the hypothesis, a C. elegans mutant with excess mitochondrial fission was not hypersensitive to hypoxia. Then combining the hyper fission mutant with the Raptor mutant did not diminish the hypoxia resistance produced by reduced Raptor function. Thus, our data demonstrates abrogating mitochondrial fission is not necessary for the hypoxia resistance produced by the Raptor mutant and leads us to reject our hypothesis. By exploring the interaction of mitochondrial fusion and fission with Raptor, we are beginning to understand how these important organelle and metabolic regulators combine to control hypoxic cell death.

Light sheet imaging differs from other forms of microscopy by illuminating the entire plane of an ex vivo extracted brain sample and computationally compiling these planes post-hoc into a 3-dimensional image. This technique allows users to preserve the structural integrity and map biological units such as cells, axons, and genes onto a standardized brain atlas. Using the SHIELD clearing, preservation, and staining protocol, we prepared fluorescently labelled ex vivo whole, intact brains for light sheet imaging and used the ClearMap open source pipeline on the Hyak cluster operated by University of Washington-IT to overcome computational limitations. While ClearMap registers images and identifies cell counts per brain region, it lacks quality assurance metrics as well as figures for visual confirmation of preprocessing performance. To overcome these deficits, we have built a quality assurance package for ClearMap that includes registration validation and group-level quantification figures. In testing this package we analyzed multiple mouse brain samples exhibiting Fos expression patterns under varied conditions. Using this dataset we found that registration performance between expected and actual slices measured in F1 score averaged 0.7 in medial sections of the brain and we were able to visually inspect the alignment of subregions identified in a standardized brain atlas. Additionally, we created an interactive script to visualize heatmaps that indicate expression patterns for user-defined brain regions on an animal level. This work will enable rapid validation of collected data and third-party package performance while streamlining the analysis, interpretation, and visualization of results. In the future we intend to include useful statistics (one-way ANOVA, classification models, etc.) to provide additional functionality and further increase the speed and quality of data analysis.

An important part of addiction recovery is degrading high value associations between drug cues and the drugs themselves. Dopamine plays a crucial role in learning, and is specifically implicated in the prefrontal cortex (PFC) and reversal learning - learning to update and change behavior when it is no longer being rewarded. Past studies have reported elevations in dopamine during contingency reversal, but the timescale of how activity of PFC dopamine neurons maps to reversal learning remains unclear. Here we investigated the activity of PFC dopamine during reversal learning in a longitudinal fiber photometry study, recording dopamine signal on a timescale of seconds. Mice were trained on a reversal learning task where they initially learned that two of four presented odors precipitated a sucrose reward in 85% of the trials while the remaining two odors precipitated the reward for only 15% of the trials. Once the learning was stable, reward probability flipped for two odors (one 85% odor and one 15% odor) and the mice had to update their behavior to the new odor/reward structure. Fiber photometry recordings were conducted during pre-reversal, reversal, and post-reversal stages of the study. Our data replicate findings demonstrating elevated dopamine release during the reversal period, centered around the 15-85 cue. Analysis of the relationship between the dopamine signal and behavior also revealed significant cue, reward prediction error, and 15-85 reversal coding in the majority of animals, suggesting a multi-faceted role for dopamine in the PFC. Given this, dopamine in the PFC may play an important mediating role in the enhancement of associations between drugs and drug cues, but does not play a clear role in contingency degradation.

The World Health Organization reports that drowning is the third leading cause of unintentional injury deaths worldwide. Intervention efforts are crucial to prevent drownings from occurring and maximize survival in people who do experience drowning. While multiple factors contribute to the risk of drowning, age affects drowning settings, patterns, and rates. Therefore, drowning prevention efforts should be tailored based on age. However, most drowning prevention interventions focus on pediatric populations, which fails to address the increased injury burden in older adults that disproportionately affects aging societies. To address this knowledge gap, our research team conducted a formal rapid review of published drowning prevention efforts in adults aged 50 years and older. We will analyze our results using Haddon's Matrix, a systemic tool that provides a comprehensive view of different types of prevention acting on discrete injury components. Our search strategy yielded 1914 titles and abstracts, of which we eliminated 1850 studies as irrelevant to our research question. We then conducted full-text screening on 64 studies, retaining a total of 7 studies for data extraction, which is currently ongoing. Preliminary results show discrepancies in the age definitions of older adults and a failure to address equity issues related to drowning prevention in this population. Many studies included older adults as part of the study population but did not stratify outcomes by age. Hence, no determination could be made regarding the intervention’s efficacy in older adults. Finally, most older adult drowning prevention efforts targeted a behavior change in the host rather than modifying the environment, which is often a more efficient method of injury prevention. Our preliminary results illustrate the need for a global collective effort to diversify drowning prevention and research efforts to alleviate the drowning injury burden in older adults.

Mitochondrial calcium is essential for energy metabolism and cell survival. Deranged mitochondrial calcium leads to pathological remodeling of the heart. Little is known regarding the regulation and roles of mitochondrial calcium in cardiomyocyte growth. Mitochondrial calcium uniporter (MCU) is a major channel for mitochondrial calcium uptake. Germline knockout of MCU on the inbred C57BL/6 background is lethal. However, αMHC-Cre-driven MCU deletion in the heart just before birth yields viable offspring with normal heart function. In this study, we will use human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) to study the regulation and roles of MCU in cardiomyocyte growth. First, the expression of MCU in iPSCs and iPSC-CMs at different stages of their differentiation and maturation process will be determined at mRNA and protein levels. Then, we will delete MCU gene in undifferentiated iPSCs and follow a protocol to differentiate them into beating cardiomyocytes. The iPSC-CMs will be monitored for their morphological changes, cardiac troponin T expression, and electric pacing-induced calcium transients and cell contraction. The proliferation of iPSC-CMs will also be evaluated by using BrdU staining and molecular markers. This study will demonstrate how MCU expression changes during the differentiation and maturation of iPSC-CMs and whether it plays a role in cardiomyocyte growth.

A key neuromodulatory system involved in anxiety disorders is the locus coeruleus noradrenergic system (LC-NE), which projects broadly throughout the central nervous system. The LC is stress responsive and tonic activation of the LC and its projections to the BLA is anxiogenic. Previously, the Bruchas Lab has used two-photon calcium imaging to show that a powerful stressor (predator odor) increased synchronous activity of LC neurons. They also found that mimicking this predator odor evoked activity with optogenetics altered the activity of individual neurons downstream in the BLA in a β-adrenergic receptor (β-AR) dependent manner. Although these data support the LC's involvement in promoting aversion and increasing anxiety-like behavior, the specific neurotransmitter, neuronal cell types, and receptors responsible for these effects remain unidentified. Therefore in hopes of identifying these specific signaling molecules and neuronal cell types and receptors, I first used fiber photometry and a novel biosensor (GRABNE2m) to detect norepinephrine (NE) release in the BLA while mice were exposed to a predator odor. I found that predator odor produced robust increases in NE release in the BLA compared to control odor (n=5, 3 male, 2 female) Further, we found that optogenetic activation of terminals from the LC to the BLA produced very similar levels of NE release compared to what was evoked by predator odor. To determine the cell type and receptor that is sensing this stress-induced NE release, I used a CRISPR/SaCas9 virus, developed in collaboration with Dr. Larry Zweifel’s lab, to knock-down β2-adrenergic receptors (β2-ARs) in glutamatergic BLA neurons to test their causal role in stress-induced anxiety-like behavior. CRISPR knockdown of β2-ARs in the BLA blocked several stress-induced anxiety-like behaviors (n=4, 4 female). By understanding the circuit-based mechanisms of how stress-induced anxiety is regulated, researchers could identify potential targets for therapeutic treatments of anxiety disorders.

Falls are a leading cause of injury, disability, and death in older adults. One common but understudied risk factor for falls includes knee instability (KI), defined as the loss of postural support across the knee during weight bearing, which is more common in older adults with knee osteoarthritis (KOA). The goals of our project are to (1) evaluate the feasibility of using a new thigh worn accelerometer device called Move4 to measure movement-evoked pain and (2) explore the association between movement-evoked pain and KI in older adults with KOA. Move4 uses sensing technology to trigger a brief survey (called an Ecological Momentary Assessment [EMA]) on a participant’s study phone asking them to rate their pain after a bout of physical activity (e.g., 15 minutes of walking) or inactivity (e.g., 30 minutes of sitting). EMAs are also collected at random times in the day (morning, early and late afternoon, and evening) over a 1-week period to assess pain level, mood, and incidence of KI. Feasibility is established if >80% of participants wear the Move4 device for 6-7 days and complete >70% of the triggered EMAs over a 1-week period. Multilevel statistical modeling is used to examine movement-evoked pain and KI occurrence and examine physical activity patterns before and after KI. The findings of this study may establish a novel method to measure movement-evoked pain in daily life that can overcome challenges of recall bias and improve estimates of KI incidence through real-time data capture. This research has the potential to improve our understanding of a prevalent symptom that increases risk of falling and limits physical functioning in a rapidly growing population of older adults with KOA.