mTOR, the mechanistic target of rapamycin, is a serine/threonine kinase which is an enzyme that phosphorylates the hydroxyl group on a serine or threonine side chain. mTOR kinase 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, a state in which tissues are deprived of oxygen and can not carry out normal metabolic activity. Previously, the Crowder lab conducted a mutant screen in C. elegans, for hypoxia resistant mutations, and has recently identified a missense (changes one amino acid to another) 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, mutations that suppressed developmental arrest. Using genetic mapping, sequencing, and complementation testing, we have identified multiple mutations in three different genes responsible for restoring Raptor function. Preliminary results show mutations in the gene rnf-126 (ring finger protein) suppress the Raptor mutation. Current work by myself and others is designed to answer how these genes control Raptor function and hypoxia sensitivity. Elaborating the function of these genes will define novel mechanisms whereby Raptor and mTORC1 controls metabolism, hypoxic injury, and development.

Chronic pain affects 30% of children and adolescents, putting them at risk for physical and psychological impairments. Given poor access to pediatric pain care, psychological treatments such as cognitive behavioral therapy are more accessible through digital health interventions. One intervention our lab created is Web-based Management of Adolescent Pain (WebMAP). In this mobile app, youth with chronic pain develop pain management strategies by progressing through interactive modules. Although digital health interventions like WebMAP are transforming accessibility to chronic pain care, limited information is available to understand how best to enhance youth engagement in a mobile app or whether there are differences in engagement among youth with different background characteristics. Our project aimed to 1) identify barriers to engagement with WebMAP among subgroups defined by user demographics and 2) develop recommendations for enhancing engagement with WebMAP and extending its reach to target underserved populations. In a previous cluster randomized controlled trial, participants assigned to the WebMAP intervention were 73 youth aged 10 to 17 (84.3% female) with various chronic pain conditions. We analyzed the app metadata to assess module completion rates across various subgroups, including groups defined by age, race, sex, and annual household income. We also coded interview and questionnaire responses from users to identify app preferences and areas of improvement. Results indicated that although users liked WebMAP’s design and content, they suggested changes to its display and features. By viewing our findings on usage and perception through a health equity lens, we summarized the accessibility of WebMAP and outlined recommendations to enhance its cultural relevance to different groups. Ultimately, our research identified factors that impacted engagement with WebMAP and offered valuable insight into optimizing digital health psychological interventions for chronic pain management. Moving forward, we hope to apply these findings as we develop the next release of WebMAP.

The lateral hypothalamus (LHA) is an important brain region for motivated behaviors including feeding. The LHA contains GABAergic (inhibitory), Glutamatergic (excitatory), and other neuropeptide neuron populations. Previous research demonstrated that optogenetic stimulation of LHA GABA neurons increases food consumption while stimulation of glutamate neurons decreases food consumption, but both populations increase in activity during consumption. The caveats to these previous research experiments are that they do not isolate consummatory behaviors from appetitive behaviors, and they only focus on the role of neuronal stimulation on caloric consumption, not on non-caloric rewards or aversive tastants. In our experiments, we use a multi-spout head-fixed mouse behavioral system to isolate consumption from other behavioral variables, and measure consumption over a range of different concentrations of either rewarding or aversive taste solutions. Using fiber photometry, I recorded calcium dynamics from both neuronal cell types of interest simultaneously, and we found that GABA neuron activity scales with increased lick rate regardless of the solution, while glutamate neuron activity scales with aversive but not rewarding solutions. When I stimulate LHA GABA neurons during consumption using the red-shifted excitatory opsin, Chrimson, we see an increase in licking regardless of solution. Stimulation of LHA vglut2 neurons reduced licking regardless of solution. We also ran inhibition experiments using the red-shifted inhibitory opsin, JAWS, of the two populations and saw that GABA inhibition reduces consumption, while glutamate inhibition increases consumption. Our research has shown how both populations work to drive consummatory behaviors and how their activity level influences consumption. This research is important because it uncovered the function of LHA GABA and Glut neurons in bidirectionally mediating consummatory behaviors for both rewarding and aversive solutions and will contribute to understanding of health issues related to consumption such as obesity.

From previous clinical cases, doctors found that children with Leigh syndrome are sensitive to volatile anesthetics. Leigh syndrome can be caused by defective Ndufs4, a subunit in complex I of the electron transportation chain. Previously, we used the Ndufs4 knockout C57Bl/6 mice to model Leigh syndrome and noticed that individuals with Ndufs4-knockout astrocytes go under anesthesia at a normal concentration, but require a lower concentration to emerge. We also found that isoflurane inhibits the effect of norepinephrine in astrocytes. Since norepinephrine is associated with individual emergence from an anesthetized state, this might help us to understand the mechanism behind our observations of the astrocyte specific Ndufs4 knockout. We furthered our study by assessing the role of gliotransmitters in the mechanism of mitochondrial disease and volatile anesthesia. Gliotransmitters are chemicals released by glial cells and used by astrocytes to modulate neuronal information processing. A pilot study was conducted in vitro in the wildtype mice assessing the release of different gliotransmitters, including cyclic adenosine monophosphate, adenosine, adenosine triphosphate, gamma-aminobutyric acid, serine, glutamine, and glutamic acid. Previously we found that the level of cAMP increased significantly after the norepinephrine treatment. Thus, we expect to see a larger acceleration in cAMP concenration in knockout mice, compared to wildtype. I am continuing this project studying the effect of the gliotransmitters in astrocyte cell cultures from the Ndufs4-knockout mice, to compare to the wildtype counterparts. Each astrocyte culture will be randomly assigned to a treatment: with or without norepinephrine. The amount of each gliotransmitter released per unit of protein is measured. I will analyze the data generated and compare to the wildtype data. Future study includes the assessment of the relationship between the metabolites within astrocytes, and the response to isoflurane and norepinephrine. This study would accelerate our understanding of the mechanism of volatile anesthesia, as well as the mitochondrial disease, which in turn will benefit numerous patients with safer and wiser treatment plans.

One third of patients in America diagnosed with depression or anxiety are resistant to treatment, creating an urgent need to develop improved therapeutics. In such disorders, motivation to seek rewarding outcomes (‘approach’ behavior) is commonly reduced while apathy (‘avoid’ behavior) is often increased, suggesting that circuitry regulating approach-avoidance (Ap-Av) behaviors may become disrupted in these disorders. Previous research in our lab showed that neurons that release the endogenous neuropeptide nociceptin in the paranigral ventral tegmental area (pnVTA) become highly activated when a large amount of effort is required to obtain a reward, and that activation of these neurons drives avoidance behavior. To investigate pnVTA nociceptin circuitry in directing approach and avoidance behavior simultaneously, I am using NOPLight, a nociceptin biosensor, in an Ap-Av task to record nociceptin release in vivo during Ap-Av decision making in mice. The Ap-Av behavioral assay I am using utilizes a head-fixed apparatus with an aversive LED light positioned at the mouse’s eye level and a sucrose sipper positioned at their mouth. At variable time intervals, either a tone indicating a sucrose reward outcome or a tone indicating an aversive light outcome will play. After the tone plays, the mice will have a short response period where the number of times they lick the sucrose sipper determines the magnitude of the outcome (more licks = more sucrose reward/aversive light, less licks = less sucrose/light). Thus, this task includes simultaneous approach and avoidance components that collectively influence decision-making, and it gives us insight into the role of nociceptin in regulating these behaviors. This research is clinically significant as it can help us understand possible mechanisms underlying the expression of symptoms related to motivation in psychiatric disorders and identify new therapeutic targets to treat them.

Human history is marked by intergroup and interpersonal conflict. Over time we have begun to understand that humans, and other animal species, are imperfect decision-makers influenced by learned social biases. Specifically, we are interested in understanding in-group bias: the tendency to favor those of one’s own group over those in other groups. In order to investigate this behavior at the neural circuit and cellular level we developed a social behavior paradigm using male mice. Our paradigm utilizes the resident-intruder assay to determine which social behaviors the resident mouse (C57BL/6J male, n = 16) displays in response to a novel ‘in-group’ (C57BL/6J male) or ‘out-group’ (C57BL/6J albino male) intruder placed in their home cage. We focused on investigative and aggressive behaviors and found that a little over 50% of our resident male mice displayed an out-group aggression bias. This bias was eliminated after early life exposure to a C57BL/6J albino, supporting the hypothesis that this behavior is learned. To better understand the development of in-group bias, our future experiments aim to recapitulate out-group aggression bias without the use of a genetic variant by artificially creating groups with neutral odors. We plan to group-house half of the mice in neutral odor A and the other half in neutral odor B to fabricate an in-group and out-group, and determine whether this model produces out-group aggression bias. These insights will help us to interrogate the neurobiology of aggressive behavior and provide insight on out-group aggression and potential ways to reduce this bias.

Sickle cell disease (SCD), the most common genetic blood disease in North America, is characterized by recurrent episodes of acute severe pain due to blockages of red blood cells. In past studies, digital cognitive-behavioral interventions have been shown to be beneficial in other chronic pain conditions through teaching pain-management skills. Our research investigates the effectiveness of iCanCope SCD (iCC-SCD), a web and mobile-based pain-management program for SCD pain in youth ages 12-18 years. The final enrolled sample for the study was 137 participants, of which 26 participants were excluded because they did not complete pre-treatment assessments. Thus, the final sample consisted of 111 adolescents (107 caregivers), 54 randomized to Education control, and 57 randomized to iCC-SCD. The iCC-SCD program includes modules teaching coping strategies, symptom and goal-tracking, and peer-based social support, while the attentional-control contains static education about SCD. The efficacy of the program is determined through self-report scales at pre-treatment, post-treatment (2 months), and follow-up (6-months) periods, targeting the primary outcomes of adaptive coding, pain reduction, and pain-related disability. A statistically significant effect of treatment group (iCC-SCD vs. Education) on change over time in average pain intensity from baseline to 6-month follow-up was found. While most youth engaged with the program (40/57, 70%), the overall usage was highly variable. Therefore, I will explore the differences between participant website and app engagement from this study, feedback on why participants may or may not have been able to complete the iCanCope program and determine areas to enhance engagement. The information collected in this analysis can help to improve web- and mobile-based interventions for not only youth coping with SCD pain but also those with other pain-related conditions, given the flexibility and universality of cognitive-behavioral frameworks.

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.

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 (so called mitochondrial dynamics) but the responsible mechanisms and 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. mTORC1 is a master regulator of metabolism and is known to affect certain aspects of mitochondrial biology. In this work I show that disrupting mitochondrial dynamics with mutants in mitochondrial fission produce hypoxia resistance but that mutants with altered fusion have normal hypoxic sensitivity. I have built compound mutants containing both fission and fusion machinery mutants together with the hypoxia resistant Raptor mutant. Using these mutants, I am testing how Raptor controls fission and fusion and whether either is required for its hypoxia resistance. Our preliminary findings indicate that the hypoxia resistance of the Raptor mutant does not require FZO-1-mediated mitochondrial fusion. 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.

Two-photon microscopy enables single-cell resolution recording of neural activity via the expression of proteins that change fluorescence brightness levels based on neural activity. This technology can be used in conjunction with behavioral tests in animal models to investigate the neural mechanisms underlying cognition, sensory processing, and internal states. Here we present findings to improve 2-photon data quality through a denoising algorithm, which removes random non-neural noise from data, and subsequently extract neural-behavioral relationships through deep-learning classification. In this project, I wrote custom python scripts to perform these complex analyses using open-source packages on the UW high performance computing cluster. Two-photon in vivo images of fluorescent indicators can be contaminated by varying levels of noise, related to the recording device or the environment. Such noise is prohibitive for detecting neural structures. Here, I apply a convolutional neural network (CNN)-based denoising algorithm, DeepInterpolation, to mitigate the noise present in neural activity recordings. We hypothesize that denoising will achieve a significantly higher single-pixel signal-to-noise ratio (SNR) compared to the raw data, and enable significantly more neural structures to be detected by segmentation algorithms. Deep learning techniques have shown promising results in improving the classification of video data. Data acquired from two-photon microscopy are sequences of images across time, yet most analyses focus on pixel-averaged time-series extracted from individual neurons. The relational information between space and time that may inform of underlying neural mechanisms is therefore lost in these approaches. Here, we propose the application of Deep 3-dimensional convolutional networks (3D ConvNets) to learn spatiotemporal features of two-photon imaging data and to classify local circuit interactions related to animal behavior.As a whole, the goal of this work is to provide an open-source working example for the classification and feature extraction of two-photon imaging neural activity recordings. This pipeline can be used to gain insight into spatiotemporal dynamics related to event-related behaviors in two-photon imaging datasets.

Spatial transcriptomics is a set of neuroscience methods that enables the visualization of gene expression patterns across the brain. Using Expansion-Assisted Iterative Fluorescence in situ hybridization (EASI-FISH) protocols, a cutting-edge spatial transcriptomic approach, we can label unique RNA segments with multiple colored fluorescent probes in neurons throughout an extracted ex vivo brain section. Brain sections are then volumetrically imaged using a confocal microscope and collected images undergo analyses to align images and segment cells and fluorescence expression. Current limitations of these analysis techniques include computing resource usage and the lack of parallel dataset analysis which we aim to address by adapting open source software to be compatible with cloud-based computing. Here we utilize the Hyak cluster operated by University of Washington-IT to overcome memory, CPU, and GPU limitations of desktop computers. To benchmark the analysis package, we analyzed 3D volumetric tissue from the medial preoptic area (MPOA) of the mouse brain labeled for the genes Vgat and Esr1, markers for inhibitory neurotransmitter and estrogen receptor expression respectively. Our scientific goal is to understand the enrichment of such genes during female mating behaviors. From previous transcriptomic data, we expect that around 70% of Esr1 positive cells in the MPOA will be Vgat positive and will be located in the nucleus part of the MPOA. These neurons will tend to be activated after mating in female mice. However, how these double positive neurons locate in high resolution 3D space is not known and will provide novel insights. The future goal is to apply the analysis package to whole-brain light sheet microscopy data. The broader impact of this work is the development of a high-throughput open-source analysis pipeline for the quantification of multiplexed gene expression patterns across multiple spatial scales.

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.

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.

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.

Trauma is the 6th leading cause of mortality among Americans aging 65-79 years and outcomes after traumatic injury are worse among older patients. However, identifying which older adults will have worse outcomes after trauma is difficult. Frailty, an aging-related syndrome of physiological decline, has been associated with worse outcomes post traumatic injury. Most tools used to identify frailty rely on subjective data, which is often unattainable if patients are unconscious, delirious, or suffering from mental health illnesses. The aim of this study is to determine the utility of ultrasound and CT measurements of the masseter muscle (jaw muscle) in quantifying frailty by diagnosing sarcopenia, analyzing its correlation with clinical measures of frailty, and its association with geriatric patient outcomes after traumatic injury. Following IRB approval, I screen trauma patients for eligibility. Patients must be adults, have had a head CT taken in the last 24 hours, have no injury to the masseter muscle, and are able to answer questions about daily life activities. After obtaining consent, I interview to assess nutritional status and overall health using frailty questionnaires. Then, I perform a bedside ultrasonographic exam of the patients’ masseter muscle on both left and right sides. Lastly, I analyze the masseter muscle on the head CT scan. I measure the masseter’s width, depth, and cross-sectional area (CSA) using the Centricity Software. Our preliminary data shows that changes in masseter muscle size are detectible in radiological studies. I found that a representative patient’s masseter muscle width on ultrasound decreased by 29% eighteen days post trauma. Additionally, I collected four CT measurements on the same patient during their hospital stay: masseter muscle CSA decreased by 28% over 12 days and by 41% over 29 days. Results from this study regarding the association of masseter muscle size and frailty are still being analyzed. If significant, these measurements will allow for quick detection of frailty, allow physician to take appropriate treatment decisions, and improve patient outcomes.