Poster Session

BNST CIRCUITRY IN STRESS-INDUCED CHANGES OF EXPLORATORY BEHAVIOR

A Ly1, ED Prevost1, CJ McNulty1, DJ McGovern1, CP Ford2, DH Root1

1Department of Psychology and Neuroscience, University of Colorado Boulder

2Department of Pharmacology, University of Colorado Anschutz Medical Campus

Stress can induce long-lasting behavioral changes that result in psychiatric illnesses, such as post-traumatic stress disorder (PTSD). Some of these changes include avoidance of future situations that may be stress-inducing or ignoring one’s own basic needs, such as eating. In mice, stress can manifest in similar ways: after a stress-inducing experience, mice will not explore for food or eat in a novel, brightly lit environment in spite of hunger. This phenomenon is known as novelty-suppressed feeding or hyponeophagia. The bed nucleus of the stria terminalis (BNST) is a region of the extended amygdala that is involved in unconditioned and sustained fear responses. The BNST is widely diverse in neural cell-types, and each subclass has yet to be fully characterized. BNST neural activity is also enhanced in response to uncontrollable stress in humans. Using a mouse model of stressor controllability, we sought to explore the behavioral consequences of inescapable stress, particularly regarding approach/avoidance conflict. Our preliminary data establishes that both GABA and glutamate activity within the BNST is increased during uncontrollable stress, extending the previously aforementioned finding in humans to specific BNST neurons. We have also found that BNST neurons form synapses onto the arcuate nucleus (ARC) and paraventricular hypothalamus (PVH), brain regions that play a central role in feeding and stress regulation, respectively. Using RNAscope in situ hybridization, we discovered that the majority of glutamatergic BNST neurons co-express the genetic machinery to vesicularly package both GABA and glutamate. Our preliminary evidence suggests that BNST glutamatergic neurons, defined by VGluT3 expression, functionally release both GABA and glutamate on downstream ARC neurons. Our results may identify novel mechanisms of neurotransmitter co-transmission that may be used to reduce the effects of stress on exploratory behavior.

ESTROUS CYCLE INTERACTS WITH SUBSTANTIA NIGRA-TO-DORSOLATERAL STRIATUM PATHWAY DURING FEAR EXTINCTION TO REDUCE FEAR RELAPSE

AA. Hohorst, MK. Tanner, LM. Alvarez, RA. Abdul, JD. Westerman, R. Han, EC. Loetz, BN. Greenwood

The impaired inhibition of learned fear is a feature of stress-related psychiatric disorders like depression, generalized anxiety disorder and post-traumatic stress disorder. Extinction-based exposure therapy can be an effective treatment for these disorders but has limited efficacy due to the vulnerability of fear memories to relapse. Additionally, women are about twice as likely to experience stress- and trauma-related disorders than men, however, sex differences are not fully considered in neuroscience. Prior data suggests female rats learning extinction during estrous phases with high ovarian hormones (proestrus; Pro & estrus; Est) have enhanced extinction memory and reduced relapse compared to those with low ovarian hormones (metestrus; Met & diestrus; Di). However, how relapse in females learning extinction in Pro/Est compares to males, and mechanisms underlying estrous cycle-modulation of extinction and relapse, are unknown. Increased dopamine (DA) signaling in the striatum can enhance extinction memory, and females in Pro/Est have higher stimulus-evoked striatal DA than males and females in Met. DA neurons originating in the substantia nigra (SN) and terminating in the dorsolateral striatum (DLS) contribute to stimulus-response learning, which can be resistant to memory-disrupting phenomena thought to contribute to relapse. This study aims to characterize effects of sex and estrous cycle during extinction on relapse, and determine the mediating effects of the SN-DLS circuit on Pro/Est. Undergoing extinction during Pro/Est protects females from relapse compared to males and females undergoing extinction during other phases. Inhibiting the SN-DLS circuit during extinction restored relapse in females that learned extinction during Pro/Est. These data suggest estrous cycle interaction with the SN-DLS circuit can render extinction learning resistant to relapse. These results could have clinical implications for the use of exposure therapy in women and men.

ANTEMORTEM DIAGNOSTIC FOR CANINE COGNITIVE DYSFUNCTION USING PLASMA BIOMARKERS

AD Hines1, S MgGrath2, NL Trinh1, NZ Madrid1, O Yanouri1, A Alsulami1, A Latham1, AZ Burynska3, L Mulligan2, B Kusick2, JA Moreno1

Canine Cognitive Dysfunction (CCD) is a well-recognized neurodegenerative disease, affecting up to 35% over the age of 8. As canines age, they may experience a build-up of misfolded proteins, causing decline of cognition with similarities to Alzheimer’s disease (AD). Canines provide an excellent translational model for human dementias due to their natural development of clinical signs. Currently, antemortem diagnostic testing for CCD is limited to owner questionnaires and magnetic resonance imaging to rule out other causes of cognitive decline. The objective of this project is to design non-invasive antemortem diagnostic methods able to detect biomarkers of cognitive decline in plasma. Immunohistochemical (IHC) staining of canine brain tissue from both young and aged canines has shown presence of Aβ and hyperphosphorylation of tau, as well as increased glial cell inflammation, marked by GFAP, Iba1 and S100β. The biomarkers of CCD found by IHC staining are also present within extracellular vesicles, specifically exosomes, in both plasma and cerebrospinal fluid (CSF). Concentration of extracted exosomes can then be run on a western blot and probed for the same biomarkers of CCD as found in IHC. To date, we have observed neurofibrillary light chain (NfL) to be present in both the plasma and in the CSF at significantly higher levels in canines with CCD compared to young or aged canines without clinical signs of CCD. Detection of NfL in plasma and CSF? supports our hypothesis that other biomarkers of neurodegeneration will be present in the plasma and CSF of CCD patients. Development of an antemortem plasma and CSF based assay would be instrumental in helping veterinarians to diagnose CCD, and due to the similarities between CCD and AD, these diagnostics have the potential for strong translation to human medicine.

NEURONAL ACTIVATION IN FEAR, MEMORY, AND MESOLIMBIC STRUCTURES FOLLOWING MODEL PREDATOR EXPOSURE IN THE TRINIDADIAN GUPPY

AK Alayoubi1, LR Stein2, KL Hoke1.

1Department of Biology, Colorado State University

2Department of Biology, University of Oklahoma

Anti-predator behaviors such as hiding, freezing, and fleeing vary across taxa, though the neural circuitry mediating these behaviors have been studied in relatively few taxa. The Trinidadian guppy displays unusual anti-predator behaviors (e.g., approaching predators for inspection), offering an opportunity to investigate the neural underpinnings of unique anti-predator behaviors. Here, we identify four brain regions in the Trinidadian guppy that are differentially activated after exposure to predatory stimuli. Guppies were third-generation lab-reared fish derived from natural populations with evolutionary history of high- and low-predation. We exposed guppies to either a model predator replica or control condition. Subsequently, we immunohistochemically labelled their brains for a marker of neuronal activation (phosphorylated ribosomal protein S6; PS6). Optimizing a novel, semi-automated cell quantification technique using the open-source software FIJI allowed us to accurately and efficiently quantify PS6 positive cells in three dimensions. We found little evidence for an interaction of brain region, predator exposure, and source population on neuronal activation. However, we did find four regions with moderate to strong evidence of differential neuronal activation. Regions include the central, ventral, dorsal, and lateral areas of the telencephalon, presumptive homologs of the basolateral amygdala, nucleus accumbens, and hippocampus of mammals, respectively. Altogether, our findings suggest neuronal activation in regions that innervate reward, memory, and fear circuits may mediate anti-predator behaviors.

WITHIN-PERSON CHANGES IN THE AGING WHITE MATTER: A META-ANALYSIS AND SYSTEMATIC REVIEW OF LONGITUDINAL DIFFUSION TENSOR IMAGING STUDIES

Andrea Mendez Colmenares1,3, Ben Prytherch2, Michael Thomas1, Agnieszka Burzynska3

1Department of Psychology/Molecular, Cellular and Integrative Neurosciences,

2Statistics,

3Department of Human Development and Family Studies/Molecular, Cellular and Integrative Neurosciences, Colorado State University

Objective:

This combined meta-analysis and systematic review aimed to synthesize the evidence from longitudinal MRI studies on the magnitude, direction, spatial patterns, and possible modifiers of naturally occurring within-person changes in adult white matter (WM). Background: Age-related WM deterioration leads to cognitive impairments in older age even in the absence of dementia. Despite the emerging evidence from animal studies showing experience-induced changes in both myelin and axons, adult human WM is often perceived as static and not involved in neuroplasticity. However, because the evidence of experience-induced changes in the adult human WM microstructure is scarce, WM is rarely considered the primary target for treatments and interventions against cognitive decline. Design/Methods: We focused on the most widely used WM technique, diffusion tensor imaging (DTI). Results: Of 28 studies included, 22 (79%) reported negative changes in DTI fractional anisotropy (FA). The meta-analytic pooled effect among 2906 participants (66.4 ± 8.9 years) showed a decline in the whole WM FA (d= -0.12, 95% CI:-0.21 to -0.03, p= 0.008, average follow-up = 26 months), and this decline was greater with longer follow-up time. Similarly, we found a negative change of FA in the anterior corpus callosum (d= -0.14, 95% CI: -0.22 to -0.06, p =0.003). Additionally, we found that the magnitude of change in FA was greater with advancing age, and greater in women than in men. In our qualitative review, we found that older age was associated with greater longitudinal change in FA in late-myelinating regions (e.g., anterior corpus callosum) than in early myelinating regions (e.g., superior corona radiata). Conclusions: Our results demonstrate that WM microstructure undergoes significant within-person changes in older age as measured with DTI. Understanding the naturally occurring within-person changes in adult WM will lay the foundation for studying the plastic and regenerative potential of WM in future clinical trials.

PRELIMINARY ANALYSIS OF GENETIC MARKERS FOR FUNCTIONAL ETHANOL TOLERANCE IN HONEY BEES (APIS MELLIFERA)

B Brown1, C Peterson1, PC Ferlin1, J Steenblik1, N Boehm1, KN Stauch2, TE Black1.

1Department of Psychological Sciences, Weber State University

2Department of Psychology, Oklahoma State University

Over the past several decades, honey bees have become a sound model for examining the behavioral effects of ethanol consumption and tolerance. Analyses of ethanol tolerance in Drosophila, have led to promising candidate genes within the species that are conserved to both insect and mammalian systems. These genes include Heat Shock Protein 70 (HSP70), a chaperone protein and measure of cellular oxidative stress, and the “slowpoke” gene (slo), which codes for several isoforms of sodium ion channels necessary for axonal transmission. The current work attempts to compare expression of these genes via RT-qPCR, along with performance in a free foraging task, in order to determine their role in development of functional ethanol tolerance.

SEX DIFFERENCES OF DEPRESSIVE-LIKE BEHAVIORS INDUCED BY VICARIOUS CHRONIC SOCIAL DEFEAT STRESS

Branden Cahill1, Madeline Morgan1, Alisandra Marvel1, Yun Li1.*

1Department of Zoology and Physiology, University of Wyoming, 1000 E University Avenue, Laramie, WY 82071, USA

Major depressive disorder is a sexually dimorphic disorder yet, sex differences are prudent in divergence in symptomology. A behavioral paradigm, Chronic Social Defeat Stress (CSDS) model, has been well established to produce depressive-like behaviors in male rodents. Unfortunately, female rodents, for various reasons cannot be used successfully in this paradigm. The vicarious chronic social defeat stress (VCSDS) paradigm was created to overcome these issues. In the present study, we employed vicarious chronic social defeat stress paradigm to examine sex and age dependent behavior changes in response to chronic emotional stress in wild type mice. Our preliminary analysis demonstrated that a proportionally more female mice displayed depressive-like behavior compared to male mice exposed to VCSDS. Our data support that VCSDS model is a suitable model to study biological basis of sex dimorphism in stress induced depression.

UNDERSTANDING THE RELATIONSHIP BETWEEN EXPOSURE TO VIOLENCE DURING CHILDHOOD AND/OR ADULTHOOD AND GROSS COGNITIVE FUNCTION AND PSYCHOSOCIAL VULNERABILITIES AMONG JUSTICE-INVOLVED ADULTS WITH BRAIN INJURIES

CF Marchi1, KA Gorgens1, JA Gallagher2

1Graduate School of Professional Psychology, University of Denver, 2University of Colorado

According to the most recent estimates, nearly 2.5 million Americans have been the victim of violence (DOJ, 2022). After someone has been exposed to violence, their risk of poorer short and long term cognitive and psychological consequences increases. People in the criminal justice system are more likely than their peers outside of the system to have been the victim of violence. People in the criminal justice system also report significantly higher rates of traumatic brain injury (TBI) than the general population. Both TBI and exposure to violence are related to significant changes in cognitive and psychological functioning; however, the literature investigating how exposure to violence across the lifespan compounds these vulnerabilities is sparse. The current study investigates the unique cognitive and psychosocial vulnerabilities of individuals in the criminal justice system who have a significant reported history of TBI and were exposed to violence as children and/or as an adult. Data from 586 justice-involved individuals in the Colorado criminal justice system were utilized for the analysis. Results suggest that people who were exposed to violence as a child and also as an adult performed significantly poorer on measures of learning, attention/processing speed, working memory, and delayed memory. People who were ever exposed to violence reported significantly higher rates of mental illness than people who had never been exposed to violence. The findings of this study highlight the relationship between violence and mental illness. They also highlight the importance of secondary violence prevention efforts for persons with TBI who have been the victim of violence during their childhood, since their cognitive function is worse when they also experience violence as an adult. For example, efforts to promote self-advocacy for children in the child welfare system may reduce their victimization risk as an adult and thus lower their risk of poorer outcomes.

DISTINCT VTA GLUTAMATERGIC POPULATIONS DIFFERENTIALLY SIGNAL REWARD VALUE AND ECONOMIC DECISION MAKING

DJ McGovern1, K Siletti1, A Ly, E Prevost1, D Root1

1Department of Psychology and Neuroscience, University of CO Boulder

The Ventral Tegmental Area (VTA) is a cellularly heterogeneous midbrain region that contributes to drug-seeking, reinforcement learning, stress, and motivated behavior. VTA dopamine neurons, defined by the expression of tyrosine hydroxylase, are recruited for consummatory reward behaviors and economic decision making in mice. Recent data implicate glutamate neurons, defined by the expression of vesicular glutamate transporter 2 (VGluT2), in reward behavior. Several subtypes of VGluT2 neurons intermingle within the VTA.. In these projects we used a combination of transgenic mice and intersectional or subtractive viral monitoring and manipulation strategies to characterize the contribution of 1) VTA VGluT2+VGaT+ neurons 2) VTA VGluT2+VGaT- neurons 3) and VTA VGluT2+TH+ neurons to consummatory reward value and motivated behavior. Mice were injected with GCaMP6m respective to the cell-type of interest and were recorded using fiber photometry during a two-bottle choice consummatory reward task (sucrose, fat, saccharine). Escalating concentrations of sucrose were used to assess neuronal responses to changes in reward value. VTA glutamate cell-types scaled calcium signaling. The VGluT2+VGaT+ population signaled more for sucrose than fat while the VGluT2+VGaT- population signaled more for fat than sucrose, which suggests that subjective reward value may be more salient for this population of neurons. We adapted a behavioral economic paradigm to assess the causal role of VGluT2+VGaT+ co-expressing neurons in sucrose reward valuation. We artificially manipulated reward price by reducing sucrose delivery. Optical stimulation of VTA VGluT2+VGaT+ neurons increased responding for reward at higher price. These results suggest VTA VGluT2+VGaT+ neurons can amplify sweet reward value. Further, this project provides novel insights into the functional contributions of genetically-distinct VTA glutamatergic cell-types in reward processing.

PRESEASON PERFORMANCE DIFFERENCES BETWEEN YOUTH CONCUSSION HISTORY ON COMPUTERIZED NEUROPSYCHOLOGICAL ASSESSMENT MEASURE

E Osherow1, N Golub2, K Little1, B Davidson3 & K Gorgens1

1Graduate School of Professional Psychology, University of Denver

2School of Education, University of Indiana-Bloomington

3Ritchie School of Engineering & Computer Science, University of Denver

Baseline neurocognitive testing for athletes in pre-season can inform individualized recovery and may reduce negative outcomes post-concussion. In 2019, about 15% of all U.S. high school students self-reported one or more sports or recreation-related concussions within the preceding 12 months (Center for Disease Control [CDC], 2022). Concussion symptoms commonly include memory loss, fatigue, mood dysregulation, issues with balance and vision, headaches, and delayed reaction time (CDC, 2019; Tommerdahl, 2020). While most athletes recover in a few weeks, they are at risk for future concussions and worsening symptoms (McCrea et al., 2020). High school athletes with multiple concussions report longer recovery times as well as delayed return to play. This study aims to examine the relationship between reported concussion history and performance on preseason baseline computerized cognitive tests from 71 high school athletes. We compared throughput scores on the five subtests from Automated Neuropsychological Assessment Metrics (ANAM) between athletes with (n=25) and without (n=46) a concussion history. Groups were compared using a t-test and Cohen’s d effect size. Results indicate that athletes with one or more concussions prior to baseline measurement performed statistically worse on the repeated simple reaction time test. Throughput scores suggest that athletes with a concussion history had fewer average correct responses per minute than athletes without concussion history (p=0.04, d=0.51). This moderate effect size is larger than the effect size reported by similar studies, which strengthens the need to reinvestigate more sensitive methodology through repeat subtests to evaluate longer-term deficits (Cook et al., 2022). Given that the ANAM test is unique in youth sports on this repeated measure, the data shows that repeat testing might start to unveil cognitive fatigue and compromised endurance in athletes with what first appears to be invisible at baseline testing.

SENSITIVITY AND TOLERANCE TO ALCOHOL IN GCKR HUMAN SNP RS1260326 (P446L) IN A MOUSE MODEL OF ALCOHOL BEHAVIORS AND METABOLISM

EA Mehrhoff1,2, M Bower1,2, N Fowler1, E Yang1, G Verner1, C Aki1, L Hendricks3, H Lee1, A Funke1, M Branney1, MA Ehringer1,2.

1Department of Integrative Physiology, University of Colorado Boulder

2Institute for Behavioral Genetics, University of Colorado Boulder

3Department of Psychology and Neuroscience, University of Colorado Boulder

Multiple large Genome Wide Association Studies of alcohol behaviors found a single nucleotide polymorphism (SNP) in the human gene for the glucokinase regulatory protein (GCKR). SNP rs1260326 corresponds to an amino acid change at position 446 from Proline to Leucine (P446L), where the P allele is associated with increased levels of consumption (“risk” allele). GCKR is a protein that binds glucokinase and inhibits it, with high levels of both mRNA and protein expression in the liver. Gckr mRNA is also expressed in the brain, however the importance of the role it plays in the brain is unknown. An initial characterization of Gckr 446P and 446L mice previously done in the lab showed that female PP and PL mice voluntarily consume and prefer alcohol compared to the female LL mice in two-bottle choice. More recent work found no differences in baseline metabolism of alcohol across two hours between genotypes, in either sex. Alcohol sensitivity and tolerance behaviors are currently being tested within these Gckr 446P and 446L mice, utilizing the stationary dowel test, modified loss of righting reflex, and balance beam, across two days of testing. There is strong evidence that a low response to alcohol (initial sensitivity) is a strong predictor of future problematic alcohol use, and this phenotype is highly heritable. Acute functional tolerance is often combined with measures of initial sensitivity and assesses tolerance development during the first exposure to alcohol. Rapid tolerance, however, is a reduction in the effect of alcohol that occurs within 8-24 hours following the first exposure to alcohol. The relationship between initial sensitivity to alcohol, acute functional tolerance, rapid tolerance, and chronic tolerance is complex and remains poorly understood. This relationship is being explored in the Gckr 446P and 446L mice to understand the effect this variant has on these alcohol related behaviors.

BRAINWIDE TRACING OF MONOSYNAPTIC INPUTS TO VENTRAL TEGMENTAL AREA GLUTAMATE-GABA CO-TRANSMITTING NEURONS

ED Prévost1, AG Phillips1, K Lauridsen1, DJ McGovern1, C McNulty1, YS Kim2, L Fenno2, C Ramakrishnan2, K Deisseroth2, DH Root1.

1Department of Psychology and Neuroscience, University of Colorado Boulder

2Stanford University

The ventral tegmental area (VTA) is a heterogenous midbrain structure involved in the processing of motivated behaviors. With the advent of INTRSECT viral vectors allowing specific targeting of neurons defined by multiple genetic characteristics, the roles of molecularly diverse VTA cell-types have begun to be dissected. The recently discovered glutamate and GABA co-transmitting VTA neurons signal rewarding and aversive outcomes. However, the pattern of neuronal integration onto this unique population of cells is heretofore unknown. Using a genetically modified mouse line that expresses Cre recombinase in cells expressing vesicular glutamate transporter type 2 (VGluT2) and Flp recombinase in cells expressing vesicular GABA transporter (VGaT), we have mapped brainwide synaptic inputs to glutamate-GABA co-transmitting neurons in the VTA. We infused eight VGluT2::Cre/VGaT::FlpO double transgenic mice with Cre- and Flp-dependent helper viruses (AAV8-nEF-Con/Fon TVA-mCherry and AAV8-Ef1a-Con/Fon oG) followed by a monosynaptic retrograde rabies virus (EnvA-ΔG rabies-GFP). Via high-throughput imaging and a novel tool for semi-automated brain registration (SHARCQ), we quantified the presynaptic input neurons by brain region according to a mouse brain atlas. Glutamate-GABA VTA neurons received the most inputs from the lateral hypothalamus, superior colliculus, periaqueductal gray, lateral habenula, VTA, and dorsal raphe. Cell-type identification and functional assessments of presynaptic neurons are ongoing and will be discussed at the meeting.

PREFRONTAL DOPAMINE IN FEMALES DISRUPTS THE PROTECTIVE EFFECTS OF BEHAVIORAL CONTROL

Gianni N. Bonnici1, Connor J. McNulty1, J Amat1, RJ Sanchez1, DH Root1, MV Baratta1

1Department of Neuroscience, University of Colorado Boulder

Control over stress mitigates many of the neurochemical and behavioral outcomes of a physically identical, uncontrollable stressor. Current evidence suggests that the recruitment of a prefrontal cortex (PFC) dependent circuit drives the stress-buffering effects of behavior control in male rats. Surprisingly, although females perform the controlling response as effectively as males, the protective effects of behavioral control are absent in females, and they do not engage in the PFC dependent circuitry used by males. Consequently, high catecholamine levels, such as those evoked during stress, can impair PFC function. Here, we argue that the ventral tegmental area (VTA), the primary dopaminergic input to the PFC, could be responsible for the circuit difference observed between males and females in control over stress. In vivo microdialysis of the PFC during control revealed a prolonged increase of dopamine in female, but not male rats (EXP1). Intra-PFC infusions of the D1 antagonist SCH-23390 (1 μg/hemisphere) led control to be protective in females (EXP2). Because membrane bound proteins are difficult to target with immunohistochemistry, we ran an in-situ hybridization on vesicular glutamate transporter and tyrosine hydroxylase mRNAs in VTA cells projecting to PFC. We found that in both cases, VTA projections had increased cfos mRNA in female behavioral control rats compared to males (EXP3). These findings suggest that the VTA potently mediates the sex-specific disparity in the stress-buffering effects of behavioral control. Furthermore, reduced benefit from a resilience factor may present a novel approach to understanding sex differences in stress-linked psychiatric disorders.

EFFECTS OF MYCOBACTERIUM VACCAE ATCC 15483, A BACTERIUM WITH ANTI-INFLAMMATORY AND IMMUNOREGULATORY PROPERTIES, ON ANXIETY-LIKE DEFENSIVE BEHAVIORS FOLLOWING A "TWO-HIT" STRESSOR MODEL OF CHORNIC DISRUPTION OF RHYTHMS AND A HIGH-FAT DIET IN MALE AND FEMALE C57BL/6N MICE

Hunter EAH1, Dawud LM1, Marquart BM1, Zhang H1, Concha AE1, Zabat RN1, Gebert MJ2,3, Frank MG4, 5, Lowry CA1, 3, 4, 5, 6, 7

1Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO, 80309, USA

2Department of Ecology and Evolutionary Biology, Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado Boulder, Boulder, CO 80309, USA

3Center for Microbial Exploration, University of Colorado Boulder, Boulder, CO, 80309, USA

4Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, 80309, USA

5Center for Neuroscience, University of Colorado Boulder, Boulder, CO, 80309, USA

6Department of Physical Medicine and Rehabilitation and Center for Neuroscience, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA

7Veterans Health Administration, Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Rocky Mountain Regional Veterans Affairs Medical Center (RMRVAMC), Aurora, CO 80045, USA

Circadian disruption and consumption of a Western high-sugar/high-fat diet, particularly common in persons engaged in shift work, have been linked with increased inflammation, which is considered a risk factor for development of stress-related psychiatric disorders, including anxiety disorders, mood disorders, and trauma- and stressor-related disorders. Currently, the therapy that exists for those suffering from the effects of shift work is timed restrictive eating which many individuals find difficult to continue with in the long term. Due to limited availability of effective interventions, novel prevention and therapeutic strategies are needed. The “Old Friends” hypothesis proposes that one mechanism underlying the increase in inflammatory disease, as well as stress-related psychiatric disorders, in modern urban societies is reduced exposure to microorganisms with which humans coevolved, in particular those microorganisms that served to limit inappropriate inflammation. One of these “Old Friends” is Mycobacterium vaccae ATCC 15483, a bacterium previously shown to have anti-inflammatory, immunoregulatory and stress resilience properties. We tested the hypothesis that immunization with a whole-cell, heat-killed preparation of M. vaccae ATCC 15483 prevents the negative impacts of chronic disruption of rhythms and a high-fat “Western style” diet on stress-induced changes in physiology and behavior in mice. To determine if immunization with M. vaccae ATCC 15483 prior to and during a “two-hit” stressor model consisting of consumption of a high-fat diet (HFD) with chronic disruption of rhythms (CDR) prevents HFD/CDR-induced anxiety-like defense behavioral responses, male and female C57BL/6N mice were assessed in the open-field and elevated plus-maze tests. Briefly adult male and female mice received a subcutaneous injection of a whole-cell, heat-killed preparation of M. vaccae ATCC 15483 (0.1 mg; 1 x 108 bacteria) or vehicle (sterile borate-buffered saline) on day –21 and once weekly for the following 10 weeks. On day 0, mice were assigned to either a continuous control diet (CD) or HFD and either a normal light: dark condition (NLD) or CDR condition for eight weeks. Mice were then tested in the open-field test test on day 55 of the protocol and the elevated plus-maze test on day 57 of the protocol. Analysis of results is ongoing and we predict that M. vaccae ATCC 15483 will prevent HFD/CDR-induced anxiety-like defense behavioral responses inC57BL/6N mice.

COGNITIVE DECLINE AND NEURONAL DEGENERATION IN GUINEA PIGS WITH SYSTEMATIC MYCOBACTERIUM TUBERCULOSIS INFECTION

Isla Anderson1, Amanda Latham2, Charlize Geer1, David F Ackart1, Amelia Day Hines1, Randall J Basaraba1, Julie A Moreno2

1Department of Microbiology, Colorado State University

2Department of Environmental and Radiological Health Sciences, Colorado State University

Tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb), infects approximately ten million people each year. Primarily a disease of the lungs, TB induces a robust peripheral immune response of cytokine producing macrophages and T cells. Cross-sectional studies have found associations between TB and an increased risk for neurodegenerative diseases, including Parkinson’s Disease (PD) and dementia. TB patients co-infected with HIV also experience accelerated cognitive decline compared to HIV infection alone. These correlations exist without diagnoses of central nervous system (CNS) Mtb infection or tuberculosis meningitis (TBM), where infection progresses to the brain causing inflamed meninges.To better understand the CNS effects of peripheral TB disease, we used guinea pigs, a pathologically relevant model, infected with Mtb by aerosol exposure. Through use of behavior testing and immunohistochemical staining, animals with systemic Mtb infection show impaired cognitive functioning and markers of neurodegeneration compared to uninfected controls. Guinea pigs 60- and 90-days post infection demonstrate non-spatial memory loss and anxiety-like behaviors indicative of declined cognition. Neuropathological analysis, including quantification of neurons and H&E staining, also shows death and degradation of neurons in the hippocampus. Interestingly, these results occur without any detectable bacteria in the brains of these animals. This helps us establish a correlation between peripheral Mtb infection and damage to the CNS. These findings will enhance our ability to prevent human patients with TB from experiencing permanent deficiencies, as well as deepen our understanding of how the peripheral immune response affects the brain and contributes to neurological disorders.

DIFFUSION TENSOR IMAGING ANALYSIS OF DIFFERENCES IN WHITE MATTER MICROSTRUCTURE BETWEEN DOGS WITH AND WITHOUT CANINE COGNITIVE DYSFUNCTION

JE Henry1, S McGrath2 , M Ukai2, M Antonakakis2, JA Moreno3, AZ Burzynska4

1 Department of Biomedical Sciences, Colorado State University

2 Department of Clinical Sciences, Colorado State University

3 Department of Environmental and Radiological Health Sciences, Colorado State University

4 Department of Human Development and Family Studies, Colorado State University

With neurodegenerative diseases becoming increasingly prevalent in our society, the need for an effective animal model continues to grow. Dogs with Canine Cognitive dysfunction (CCD) can serve as a reliable model for studying the pathological process, magnetic resonance imaging (MRI) changes and novel treatments for Alzheimer’s disease. Studies have shown that a decline in volume and microstructural integrity of corpus collosum (CC) has been associated with cognitive decline in humans. Furthermore, Diffusion Tensor imaging (DTI) confirmed the vulnerability of CC fibers to aging. In this study, Diffusion Tensor modeling was used to extract mean fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD) in the corpus callosum of dogs with and without CCD. The aim of this study was to examine whether DTI modeling parameters can identify changes in CC microstructure in dogs with and without CCD and determine whether DTI can be used as a reliable diagnostic imaging modality in dogs with CCD. To the authors’ knowledge there have been no studies examining white matter degradation in dogs with CCD using DTI. This study revealed an overall decrease in mean FA, increase in mean MD, increase in AD, and slight increase in mean RD in the CC in dogs with CCD compared to dogs without CCD. There was no statistical significance between aged dogs with and without CCD; however, this could be attributed to the low resolution and distortion of images and small subject groups. Future investigations will include acquisition of opposing phase-encoding directions and development of an enhanced preprocessing guideline, including topup to account for anterior to posterior distortions and eddy to correct for eddy movement distortions. Additionally, analysis of other structures, including white matter in the frontal lobe and fornix may illuminate more distinct changes in myelin microstructure secondary to CCD.

HOW THE RELATIVE TIMING OF PROSECTION AND DISSECTION INFLUENCES PERFORMANCE: ACCOUNTING FOR APTITUDE, INSTRUCTIONAL TIME, AND EXPERIENCES

JF Martin1, A Linton1,2, AC Garrett1,2, GR Cahill2, G Gloeckner3, C Magee1,2.

1Department of Biomedical Sciences, Colorado State University

2Virtual Veterinary Educational Tools, Colorado State University 3School of Education, Colorado State University

Gross anatomy is a time-consuming and expensive foundational course in biomedical science. Modern anatomy instruction places increased focus on problem-solving skills to encourage the transfer of anatomical knowledge to novel experiences. The promotion of problem-based learning and integrative objectives has occurred in parallel with calls to reduce reliance on cadavers and a reduction in instructional time. Prior research has reported that the adoption of integrative instruction is not directly responsible for decreases in student contact hours. We hypothesize that when accounting for aptitude, instructional duration, and experiences, the relative timing of prosection and dissection instruction plays a role in anatomy exam performance and student perceptions. To test this hypothesis, a 5-year (2018-2022) mixed-method retrospective study compared exam performance (N = 437) in three populations: Prosection-Only (PO), Consecutive Enrollment (CE), and Simultaneous Enrollment (SE). First, average dissection exam scores (N = 110; Mdn[IQR], .86[.12]) were lower than prosection exams (.87[.16]), p = .014, r = -0.20. Second, SE had higher mean ranks than PO (317.58 v 177.61) and CE (64.32 v 41.76) on prosection exams, U = 3378, p < .001, r = -.45, and U = 849.5, p < .001, r = -.35, respectively. Third, the average dissection exam score had a higher mean rank for CE (63.76) than SE (50.20), U = 1085.5, p = .030, r = -.21. Although, only dissection exams one (63.76 v 50.20) and two (48.72 v 66.06) contribute to this difference, U = 964, p = .003, r = -.28 and U = 986.5, p = .005, r = -.27, respectively. Fourth, survey data suggests CE students were more critical of changes that disrupt established study strategies. This study identified increased anatomy performance when provided instruction occurred at two separate periods compared to an equivalent condensed instruction. However, that benefit may come at the cost of increased sensitivity to changes in learning ecology.

CORRELATES OF CHILDHOOD TRAUMA, EMOTION REGULATION, AND FAMILY HISTORY OF HARMFUL ALCOHOL USE IN HEALTHY YOUNG ADULTS

K Andereas1, KT Kirk-Provencher1, JL Gowin1

1Department of Radiology, School of Medicine, University of Colorado Anschutz Medical Campus

Introduction Childhood trauma and emotion regulation deficits are associated with developing substance use disorders and with family history of harmful substance use. We expect that participants with family history of harmful alcohol use (FH+) compared to those without family history of harmful alcohol use (FH-) will report more childhood trauma experiences, less use of cognitive reappraisal, and will differ in neural activation in response to viewing negative images in emotion processing regions of the brain (i.e., amygdala).

Methods Participants were 75 healthy young adults (ages 18-23, 44% male, 76% white) screened for family history of harmful alcohol use ( FH+ = 31, FH- = 44). Participants completed the Childhood Trauma Questionnaire and Emotion Regulation Questionnaire. During an fMRI scan, participants completed an emotion regulation task where they were passively viewed negative images and asked to rate how the images made them feel.

Results FH+ participants (M = 45.1, SD = 18.8) had more childhood trauma experiences than FH- (M =35.6, SD =10.4; W = 481, p = 0.03, d = 0.65). The groups did not differ on self-reported use of cognitive reappraisal (t[73] = 0.69, p = 0.49, d = 0.16). Regression analyses revealed no significant main effects of family history or childhood trauma, or a significant family history-by- trauma interaction on neural activation in the right (F[3,71] = 0.69, p = .56) or left (F[3,71] = 1.14, p = .34) amygdalae. Childhood trauma was moderately associated with self-reported cognitive reappraisal for FH+ participants (r = -.34), but not for FH- participants (r = -.15).

Conclusion Results suggest that differences in experiences of childhood trauma are not associated with neural activation in the amygdala in response to negative images, but may be related to self-reported emotion regulation, in healthy young adults with a history of harmful alcohol use.

CORRELATES OF CHILDHOOD TRAUMA, EMOTION REGULATION, AND FAMILY HISTORY OF HARMFUL ALCOHOL USE IN HEALTHY YOUNG ADULTS

K Andereas1, KT Kirk-Provencher1, JL Gowin1.

1Department of Radiology, School of Medicine, University of Colorado Anschutz Medical Campus

Childhood trauma and emotion regulation deficits are associated with developing substance use disorders and with family history of harmful substance use. We expect that participants with family history of harmful alcohol use (FH+) compared to those without family history of harmful alcohol use (FH-) will report more childhood trauma experiences, less use of cognitive reappraisal, and will differ in neural activation in response to viewing negative images in emotion processing regions of the brain (i.e., amygdala).

Methods Participants were 75 healthy young adults (ages 18-23, 44% male, 76% white) screened for family history of harmful alcohol use ( FH+ = 31, FH- = 44). Participants completed the Childhood Trauma Questionnaire and Emotion Regulation Questionnaire. During an fMRI scan, participants completed an emotion regulation task where they were passively viewed negative images and asked to rate how the images made them feel.

Results FH+ participants (M = 45.1, SD = 18.8) had more childhood trauma experiences than FH- (M =35.6, SD =10.4; W = 481, p = 0.03, d = 0.65). The groups did not differ on self-reported use of cognitive reappraisal (t[73] = 0.69, p = 0.49, d = 0.16). Regression analyses revealed no significant main effects of family history or childhood trauma, or a significant family history-by- trauma interaction on neural activation in the right (F[3,71] = 0.69, p = .56) or left (F[3,71] = 1.14, p = .34) amygdalae. Childhood trauma was not associated with self-reported cognitive reappraisal for FH+ participants (r = -.34, p = .06) or FH- participants (r = -.15, p = .34).

Conclusion Results suggest that differences in experiences of childhood trauma are not associated with neural activation in the amygdala in response to negative images, nor self-reported emotion regulation via cognitive reappraisal, in healthy young adults with a history of harmful alcohol use. Future studies should examine these variables in young adults with harmful alcohol use.

IDENTIFYING NEURAL ENSEMBLES IN THE NUCLEUS ACCUMBENS ACTIVE DURING COCAINE AND CHOCOLATE PLACE PREFERENCE

KL Sandum1, LT Flom1, S Hodgins1, J Crouse1, S Johnson-Noya1, CJ Litif1, AC Bobadilla1

1Department of Pharmaceutical Sciences, University of Wyoming

Neuronal ensembles in the nucleus accumbens core (NAcore), an integration center of rewards and motivation, play a role in substance abuse disorder and relapse. Seeking ensembles are sparse networks of neurons activated during reward-seeking behaviors that are thought to drive relapse behavior. The purpose of this study is to map NAcore ensembles involved in seeking cocaine and compare them to those involved in seeking natural rewards (chocolate), as well as examining these ensembles when animals have been exposed to both natural rewards and drug rewards. To test this hypothesis, we conditioned cohorts of 12-16 genetically modified Fos iCreERT2/Ai14 male and female mice with chocolate or cocaine and quantified the ensembles activated during reward seeking using the conditioned place preference model. Both cocaine- and chocolate-seeking behavior activated about one percent of the neurons in the NAcore. Next, mice were conditioned to receive both chocolate and cocaine in different compartments and the opportunity to seek either during test day in absence of reward. These mice showed a preference for cocaine and an ensemble size similar to those of mice exposed to a single reward. Moving forward, we hope to determine if activating these NAcore ensembles using a cre-dependent Gq-DREADD virus is sufficient to recreate reward-seeking behavior. Further characterizing these reward-specific ensembles will allow us to better understand the neuronal circuits driving reward seeking and relapse.

D1-SIGNALING IN THE DORSOLATERAL STRIATUM PROVIDES RESILIENCE AGAINST INESCAPABLE STRESSORS

Lareina M. Alvarez1, Miles Q. Dryden1, Margaret K. Tanner1, Alyssa Hohorst1, Jessica D. Westerman1, Esteban C. Loetz1, Benjamin N. Greenwood1.

1Department of Psychology, University of Colorado Denver

Stress-related disorders such as depression and anxiety are the most common mental health disorders in the world. A major predisposing factor to stress-related disorders is stress that is perceived as uncontrollable or inescapable. Behaviors similar to depression and anxiety are observed in rats that are exposed to inescapable tail shock (IS). These symptoms include reduced social exploration and exaggerated fear learning. Exercise can protect against the development of stress-related disorders in humans, and similarly, wheel running protects rats from developing the typical behavioral consequences of IS. A deficit in dopamine (DA) signaling could contribute to stress-related disorders, so it is particularly relevant that exercise produces a hyperdopaminergic state in which DA release is sensitized to non-exercise stimuli. Wheel running, for example, potentiates DA efflux and activation of D1 receptor-expressing neurons in the dorsal striatum during IS. D1 neurons in the dorsal striatum are associated with pleasure, movement, and stress resilience. The purpose of this study is to determine if increasing D1 signaling in the dorsal striatum (both dorsolateral, DLS, and dorsomedial, DMS, subregions) is sufficient to mimic the effects of exercise and enable stress resilience in sedentary rats. We pharmacologically increased D1 signaling by injecting the D1 agonist SKF38393 into the DLS or DMS (5 µm/µl; 1 µL/side ) immediately prior to IS in adult, male and female, Sprague Dawley rats. Experiments are ongoing, but preliminary results indicate that increasing D1 signaling in the DLS during IS provides resilience against IS. These data suggest that DLS D1 receptors could be a novel target to increase stress resilience.

TRAMAUTIC BRAIN INJURY & COGNITIVE COMPLAINTS IN DOMESTIC VOILENE OFFENDERS

Maddy Pontius1, Marylin Willison1, Ayana Thaanum1, Samuel Martin1, Brooke Stencel1, and Kim Gorgens1

1Graduate School of Professional Psychology - University of Denver

A traumatic brain injury (TBI) is a disruption in the normal functioning of the brain caused by a blow to the head. TBI can lead to health and behavioral problems that may make individuals more vulnerable to becoming involved in the criminal justice system. Justice-involved individuals have high rates of TBI, where the average rate of TBI among justice-involved individuals is 54%, compared to approximately 8.5% in the general population (Gorgens et al., 2021). In the criminal justice system, TBI is associated with poorer outcomes including greater risk for reconviction and treatment failure (Gorgens, et al., 2021). Despite the alarming rates of TBI among justice-involved individuals, there is no previous research on the rates of significant TBI among domestic violence (DV) offenders, a population where treatment outcomes are notoriously poor (Eckhardt et al., 2008). The current study used data collected by the Domestic Violence Offender Management Board to determine the prevalence rate of significant reported TBI history among persons with a DV offense history. Data sets from 54 DV offenders diverted to mental health treatment were collected. Results indicated that 39% of DV offenders reported a significant history of TBI. In this sample, the most commonly reported mechanisms of injury were motor vehicle accidents (33.3%) and assault (22.2%), compared to the general population where unintentional falls (35%) and motor vehicle accidents (17%) are the leading mechanisms of injury (AANS, 2020). The most commonly self-reported complaints were in the domains of emotional problems, physical and sensorimotor problems, memory concerns, and attention problems. This is the first study of its kind to highlight the overrepresentation of TBI in an offender population vulnerable to poor outcomes. These poor outcomes, coupled with self-reported complaints, emphasize the need for more brain injury screening and specialized interventions in DV offenders.

BRAIN ACTIVITY ASSOCIATED WITH A PROBALISTIC LEARNING TASK

Maiele Mignard*1, Max Henneke*1, Anastasia G. Sares1, Jazmin Diaz1, Emily T. Sturm1, Andrea Méndez1, John R. Duffy1, Lauren Sarabia2, Eve Delao2, Tessa Mitchell2, Raana Manavi1, Jason Tregellas2, Michael L. Thomas1.

1Department of Psychology, Colorado State University

2Department of Psychiatry, University of Colorado Anschutz Medical Campus

Reward learning is an important aspect of cognition that can be impacted in psychiatric disorders. We measured brain activity during a probabilistic learning task (PLT) which assesses reward learning. This is a translational version of the PLT, bridging animal and human clinical research. We aimed to identify brain regions implicated in the PLT to elucidate the neural mechanisms of decision-making in humans.

Our sample included 26 individuals (mean age 45): 14 cognitively healthy and 12 with schizophrenia (SZ). Participants completed the PLT while undergoing functional MRI; they were instructed to select one of two shapes, choosing the shape with a higher probability of receiving correct feedback. A voxel-wise group t-test was performed on fMRI data to identify changes in brain activity from baseline (fixation). Equitable thresholding and clustering limited the false positive rate to 5%.

Performing the PLT was associated with less activity in the inferior portion of the left angular gyrus and bilaterally in the cingulate, medial prefrontal cortex, precuneus, cuneus, and temporal pole. Most of these regions are associated with the default mode network. There was increased activation in the cerebellum and areas associated with salience and reward learning. These include anterior and middle insula, supramarginal gyrus and superior portion of the angular gyrus.

Results echo previous work in that probabilistic learning tasks suppress default mode network activity while recruiting attention and salience networks. Past studies link insula and inferior parietal lobule to evidence accumulation during reward learning. Cerebellar activity could reflect cognitive functions, and motor preparation and execution. A limitation is the currently small sample, which led to participants of varied neurological health being included together. Ongoing data collection and integration of animal research findings could further describe the neural correlates of decision-making in SZ.

ADULT MALE RATS EXHIBIT TIME-OF-DAY DIFFERENCES IN THE RECALL OF TRACE CONDITIONED FEAR AND EXTINCTION

MJ Hartsock1, AB Fausnaught1, AW Choi1, SN Dobby1, RL Spencer1.

1Department of Psychology and Neuroscience, University of Colorado Boulder

The circadian system profoundly influences most learning and memory processes, generating time-of-day differences in the speed of learning and the strength of memory recall. Work in rats and humans has demonstrated circadian modulation of delayed conditioned fear extinction, a prefrontal-cortex-dependent learning and memory process important for the treatment of fear-based mental disorders. In delayed conditioned fear extinction, a cue (often a tone) co-terminates with an aversive stimulus (often a shock). We have shown that circadian rhythms in delayed conditioned fear extinction recall require intact circadian function in the prefrontal cortex (Woodruff et al, eNeuro, 2018), suggesting that circadian function in the prefrontal cortex gives rise to circadian rhythms in prefrontal-cortex-dependent processes. Here, using adult male Sprague-Dawley rats, we characterize time-of-day differences in another prefrontal-cortex-dependent emotional learning and memory paradigm: trace conditioned fear. Trace conditioned fear and extinction differ from delayed conditioned fear and extinction in that trace conditioned fear and extinction include a time interval, or trace, between the termination of the cue and delivery of the aversive stimulus. In the trace paradigm, the prefrontal cortex appears to be engaged during both fear and extinction. Accordingly, in the trace paradigm, we find stronger fear recall during the inactive phase and stronger extinction recall during the active phase. These findings align with our results from the delayed conditioned fear paradigm, indicating greater fear suppression during the active phase. In addition, the behavioral differences we observe in the trace paradigm are reflected in c-Fos gene expression in the prefrontal cortex. Our findings are consistent with the circadian regulation of trace conditioned fear and extinction, providing evidence of rhythms in another prefrontal-cortex-dependent emotional learning and memory paradigm.

GENETIC DISTURBANCE OF BIMODAL SLEEP DISTRIBUTION TO REVEAL PHYSIOLOGICAL ROLES OF DAY SLEEP AND IDENTIFICATION OF GENE(S) CRITICAL TO DAY SLEEP

Nathan Pettid1, Susan Tsunoda1.

1Department of Biomedical Sciences, Colorado State University

Sleep has been shown to be a critical process for both mammals as well as invertebrates. Despite this, the overall importance of sleep and the mechanisms controlling it are still being elucidated. Drosophila melanogaster has provided an excellent model system in which to investigate mechanisms and functions of sleep. For example, the genetic tractability of Drosophila has allowed for identification of molecules and circuits that are critical for maintaining a healthy sleep balance. One notable aspect of Drosophila is that they exhibit a bimodal distribution of inactivity, with a periods of day sleep and night sleep. Recently our lab has discovered a new mutant, no-day-sleep (nds), that lacks a bimodal sleep pattern. Unlike wild-type, nds mutants does not sleep during the daytime. The current study aims to determine the role that day sleep plays and identify the gene(s), cells, and circuitry impacted in the nds mutant. We show that the loss of day sleep causes a decrease in locomotion, as well as lifespan, demonstrating the importance of day sleep. While there are factors known to increase day sleep in wild-type, such as increased age, and temperature, neither restores day sleep in the nds mutant, suggesting that the nds mutation affects a “hard-wired” mechanism critical to day sleep. Recently, we have also found that the loss of day sleep in the nds mutant occurs in females and not in males. Studies have shown that activation of the sex peptide receptor pathway (SPR) in females, during mating, results in a set of post-mating responses that include a decrease in day sleep. Our preliminary results indicate that virgin female nds mutants exhibit not only a loss of day sleep, but other post-mating responses as well, suggesting that the nds mutation may exhibit an overactivation of the SPR pathway. Future experiments will investigate additional behavioral consequences due to the loss of day sleep and identify the gene(s) and pathways impacted by the nds mutation.

LOW-COST, OPEN- SOURCE PLUS-MAZE WITH SENSOR PLATES AND AUTOMATION

Octavio G.G. Vazquez, Mackenzie Bannister, Katherine Etchepare, Madeline Bershinsky, Qian-Quan Sun

The elevated plus-maze experiment is a widely used method in behavioral neuroscience to score anxiety and stress. The maze consists of four elevated arms, two open and two closed with walls. The experiment compares the mice time spent in each of the arms for a determined duration. Current methodologies relying on video recording are both labor intensive, costly, and results often affected by lighting and color of the subjects. Here we present an open-source hardware and software to improve the accuracy and efficiency this experiment. The hardware consists of 3D prints and laser-cut parts that are easy to manufacture; the data is collected through capacitive sensor plates connected to a microcontroller; and the software is a Python based GUI that allows effective monitoring and storage of data. The entire system can be manufactured over-night and assembled in less than a day. The results of the experiments are immediately analyzed by the software and plotted. Thus, we offer this integrated package as a robust, affordable, and efficient alternative to perform this experiment.

A PROXIMITY SENSOR-BASED MULTIPURPOSE OBJECT RECOGNITION TEST SYSTEM FOR RODENT BEHAVIOR RESEARCH

OGG Vazquez1 3, MA Demirhan1 2, Mackenzie Bannister1 2, Qian-Quan Sun1 2.

1Wyoming Sensory Biology Center, University of Wyoming

2Department of Zoology and Physiology 3Department of Mechanical Engineering

We report an automated, low-cost, and open-source methodology for rodent object recognition (OR) tasks, which are widely behavior paradigms. This proximity capacitive sensor based method, combined with easily accessible 3-D manufacturing technology, provides an opportunity to explore new, and high throughput hardware for behavioral assessment systems. The system’s software allows behavior data to be simultaneously monitored and its output can be plotted promptly immediately upon the completion of the behavior test, thus saving labor and time . The performance of this package is cross-verified with a video-based automated software scoring and behavior chemogenetic manipulations. This integrated package is an affordable and accurate tool for the increasing efforts of behavior standardization and improvement on translational recognition memory research and other type of research with frequent use of novel object recognition (NOR) paradigms.

PRE-SEASON SYMPTOMS SCORES AND NEUROCOGNITIVE OUTCOMES ARE ASSOCIATED IN DIVISION 1 ATHLETES

R Washington1, E Osherow1, K Little1, L Granholm-Bentley2, B Davidson3, K Gorgens1.

1Graduate School of Professional Psychology, University of Denver

2Department of Neurosurgery, University of Colorado Anschutz, 3Department of Mechanical & Materials Engineering, University of Denver

Approximately 1 in 10 collegiate athletes have a history of concussion (Gaudet et. al, 2022). However, this estimate is likely low because concussions often go unreported. Although the NCAA instituted a pre participation baseline testing requirement for all athletes (NCAA Sports Science Institute, 2017) that includes symptom self-report and neurocognitive testing to better identify and assess the presence and severity of concussion post-injury, knowing how these tests work together and how they relate at baseline may be a next step to improving post-injury treatment. Our study examines the association between baseline self-reported symptoms and neurocognitive performance of NCAA Division 1 contact sport athletes who reported any symptoms at baseline. Neurocognitive data were collected in Memory (0-100), Simple Reaction Time (msec), Inspection Time (msec), and Impulse Control (msec) with the SWAY app (VanRavenhorst-Bell et. al, 2021). Twenty-two symptoms across the four domains were measured on a 7-point Likert scale (0 to 6) and added together to create a total symptom score (TSS). Pearson’s correlation coefficient was calculated for the TSS and each neurocognitive subtest score. Of the 208 athletes tested, 44 reported symptoms with the total symptoms score ranging from 1 to 46. Results showed no correlation between total symptoms score and memory (r=- 0.10), low positive correlation in simple reaction time (r= 0.26) and impulse control (r=0.25), and moderate negative correlation between total symptom scores and inspection time (r=-0.33) which suggests there may be a relationship between symptoms scores and slower processing speed. Determining relationships between symptoms and areas of neurocognitive performance could result in more informed decision making for athletes’ treatment.

ROLE OF OVARIAN HORMONES AND RUNNING DISTANCE IN RAPID EXERCISE-INDUCED STRESS RESILIENCE IN FEMALES

RA Abdul1, MK Tanner2, AA Hohorst1, N Jamil1, EC Loetz2, BN Greenwood2

1Department of Integrative Biology, 2Psychology, University of Colorado Denver

Stress-related disorders affect females more than males. Physical activity enables stress resilience in both sexes, but little research has characterized exercise-induced stress resilience in females. We observed that female rats are more responsive to the stress-buffering effects of exercise than are males. In female rats, three weeks of voluntary wheel running (VWR) is sufficient to prevent anxiety-like behavioral effects of inescapable stress (IS), while male rats require six weeks of VWR. Although the expression of exercise-induced stress resilience occurs regardless of estrous phase at the time of IS or behavioral testing, the role of ovarian hormones during the three weeks of exercise in mediating resilience is unknown. The goal of this study is to determine the role of ovarian hormones in accelerated exercise-induced stress resilience in females. Ovariectomized and sham (control) rats had access to locked wheels or running wheels for three weeks. After three weeks, rats were exposed to IS and were assessed for behaviors resembling anxiety twenty-four hours later. Females run more than males, and this could also contribute to rapid stress resilience from exercise in females. We limited the wheel access in another group of females to three hours per day to determine the degree to which increased running distance of females contributes to their rapid stress resilience. Results demonstrate three weeks of running prevents IS-induced behavior sequalae independent of the presence of ovarian hormones. Additionally, even though limited access to running wheels reduced running distance of females to less than that of males, three weeks of limited VWR also prevented the behavioral effects of IS. These data suggest that sex differences in the acquisition of exercise-induced stress resilience are independent of ovarian hormones and running distance.

FAMILY HISTORY OF HARMFUL ALCOHOL USE AND NUCLEUS ACCUMBENS ACTIVATION DURING MONETARY-INCENTIVE DELAY TASK

RH Hakimi1, KT Kirk-Provencher1, JL Gowin1.

1Department of Radiology, School of Medicine, University of Colorado Anschutz Medical Campus

Background: Young adults with family history of harmful alcohol use (FH+) are likely to have altered neural activity within the reward-behavior center of the nucleus accumbens during reward-based tasks. We hypothesize that FH+ participants will differ from family history negative (FH-) participants in neural processing of rewarding and threatening stimuli and on emotional self-report measures associated with a reward-based task.

Methods: The sample consisted of 75 young adults (18-23 years old, 44% male, 76.0% White) who completed a modified Monetary Incentive Delay task with a threat of scream component (MID-S) during an fMRI scan. During the scan, participants were presented with cues representing the potential to gain $5, lose $5, or gain $0, as well as whether they were in the “safe” or “threat” of scream condition. Afterwards, participants rated on a scale of 0-10 how excited, nervous, and likeable each cue and condition combination felt.

Results: There was a significant main effect of cue on neural activation in the right (F= 137.33, p < .001) and left (F= 139.60, p < .001) nucleus accumbens, but no main effect of group (both F < 1, p > .1). There was no association between self-reported liking of cues and brain activation in response to cues to gain $5 in either the safe or threat condition (all r < .15, p > .10). A series of independent sample t-tests revealed that the FH+ and FH- groups differed on their level of excitement (t = -2.87, p = 0.01) and how much they liked (t = -2.51, p = 0.02) the gain $5 cue and “safe” condition combination, as well as their level of excitement (t = -2.55, p = 0.01) on the gain $5 cue and “threat” condition combination on the self-report measure.

Conclusion: The results appear to suggest that family history positive and negative groups do not differ in neural activation within the nucleus accumbens. They may differ in self-reported excitement in response to cues to gain money.

THE INFLUENCE OF IMPLICIT BIASES ON THE PERCEPTION OF FACE MASKS: AN ERP STUDY

RH Swain1, RM Negron1, AJ O'Hare1.

1Department of Psychological Science, Weber State University, Ogden, UT.

Face masks help prevent the spread of respiratory infections. However, face masks cover important facial features that are key for the perception of emotions, and studies have shown that covering important facial features increases the likelihood of incorrectly identifying facial expressions. What remains to be explored is if face masks are perceived as negative or positive social stimuli. 64-channel EEG was recorded while participants completed a flanker implicit association test (IAT) to measure implicit biases associated with face masks. Negative or positive words appeared above or below face stimuli presented with or without a face mask. An interaction between face masks and word valence was found, such that negative words were categorized more quickly when paired with an unmasked face, and positive words were categorized more quickly when paired with a masked face. Robust N170 responses were found for all face stimuli, however, unmasked faces evoked larger P300 responses compared to masked faces. Finally, masked faces were rated as more pleasant than unmasked faces, increases in social anxiety were associated with increases in negative ratings of unmasked male faces, and increases in negative affect were associated with slower reaction times to negative words overall. These data indicate that perceiving faces with face masks does influence both our unconscious and conscious perception of others.

SEX DIFFERENCES IN IMPAIRED COGNITION AFTER SLEEP DISRUPTION IN A SUBCLINICAL ALZHEIMER DISEASE MODEL

S Valencia-Sanchez1, C Borski2, M Griffioen1,2, KL Kastengren2, C Link1, MR Opp1, C Hoeffer1,2

1Department of Integrative Physiology and the 2Institute of Behavioral Genetics, University of Colorado Boulder

Alzheimer’s disease (AD) is the most common cause of senile dementia worldwide. Plaques and neurofibrillary tangles form the neuropathological hallmark of this disease. Plaques, mostly formed by A-β, are insoluble extracellular protein aggregates. As AD progresses, brain regions critical for cognition and memory are affected, resulting in a complex cognitive decline. Sleep disruption is persistent in patients suffering from AD. Sleep impairment becomes evident before clinical and cognitive manifestations, which worsen as the disease progresses. The causal connection between these two phenomena is not clearly understood. To determine the extent to which chronic sleep disruption accelerates the onset of cognitive decline in AD preclinical subjects, we used an inducible APP mouse model (tTA/APPsi) that allows us to temporally control the expression of APP in brain, which in turn permits the dissociation between the effects on behavior of chronic sleep fragmentation and those of the underlying pathology. For this study, sleep of adult male and female mice was fragmented (2 months) after three months of APP subclinical expression. Subsequently, behavior of experimental animals and their respective controls was evaluated with open field arena (OFA), elevated plus maze (EPM), and fear conditioning tests (FC). Basic locomotor assessment through OFA and EPM showed no statistically significant differences across the experimental groups, nor in the expression of anxiety-like behavior or exploratory drive. Nevertheless, the FC test revealed a sex-specific effect on cued long-term memory (LTM). LTM impairment was potentiated in female mice compared to all other experimental groups. These results demonstrate differential sensitivities of cognitive behaviors of mice following the combination of subclinical APP expression and sleep fragmentation. Furthermore, our data suggest the utility of this inducible mouse model to elucidate mechanisms linking these two phenomena.

DOES NALTREXONE ALTER REWARD-RELATED PROCESSING IN THE NUCLEUS ACCUMBENS IN YOUNG ADULTS WITH MODERATE ALCOHOL USE?

SL Rosenblatt1, KT Kirk-Provencher1, JL Gowin1.

1Department of Radiology, School of Medicine, University of Colorado Anschutz Medical Campus

Background: The nucleus accumbens is involved in reward activation in the brain. Naltrexone, a mu-opioid receptor antagonist, reduces alcohol craving in light and heavy drinkers, yet little is known about the effects of naltrexone on reward activation in the nucleus accumbens. We hypothesize that participants will show reduced reward-related activation in the nucleus accumbens during the Monetary Incentive Delay (MID) task when taking Naltrexone relative to taking a placebo.

Method: The sample included 11 healthy, young adults with moderate alcohol use (Mage = 24.21, 73% Female, 91% White, 9% Biracial). The study used a within-subjects design where participants were randomized to taking Naltrexone for one week and a placebo for one week. Participants completed the MID task during a fMRI scan for both the Naltrexone and placebo weeks. During the task, cues were presented indicating whether the participant will gain $5, lose $5, or gain $0.

Results: Paired samples t-tests revealed that neural activation in the nucleus accumbens was greater during the gain $5 cue relative to the gain $0 cue in the left (t[21] = 4.80, p < .001) and right (t[21] = 5.01, p < .001). During the positive cue, there was no significant difference in the neural activation between the Naltrexone and placebo conditions in the left (t[10]= 0.28, p= .782) or right (t[10]= 0.63, p = .545) nucleus accumbens.

Conclusion: Results show that the MID Task worked as expected in increasing neural activation in the nucleus accumbens when presented with the opportunity to win $5 relative to earning $0. Further, in a sample of young adults with moderate alcohol use, Naltrexone relative to a placebo did not result in reduced reward-related activation in the nucleus accumbens when presented with the opportunity to win $5. Future studies should examine these associations in a larger sample of young adults with Alcohol Use Disorder.

CLINICAL SYMPTOMS, COGNITIVE FUNCTIONING, AND BRAIN HEALTH IN AGRICULTURAL WORKERS

SR Kellam1, JM Diaz1, ET Sturm1, M Henneke2, E Pehlke1, JR Duffy1, A Mendez-Colmenares1, AZ Burzynska2,3, L Stallones1, ML Thomas1.

1Department of Psychology, Colorado State University

2Molecular, Cellular, and Integrative Neurosciences, Colorado State University 3Human Development and Family Studies, Colorado State University

Agricultural work-related risks to brain health include exposure to pesticides, heavy metals, and organic dust. Despite this, there is a gap in our understanding of the underlying brain systems impacted by these risks. Our aim was to investigate clinical and cognitive domains as well as functional brain activity in those exposed to agricultural work-related risks. The sample comprised 17 agricultural workers and a comparison group of 45 non-agricultural workers. All participants identified as White and non-Hispanic. Participants completed a series of cognitive and behavioral assessments and underwent functional magnetic resonance imaging (fMRI). Analysis of cognitive scores showed that agricultural workers showed significant deficits in episodic memory and scored higher on self-reported anger, cognitive concerns, and social participation measures. The fMRI data showed increased BOLD activity around the orbitofrontal cortex and bilaterally in the entorhinal cortex for the agricultural group. The agricultural group also showed decreased BOLD activity in the cerebellum and basal ganglia. To our knowledge, this study provides the first-ever evidence of differences in brain activity associated with a history of working in agriculture. Brain imaging data analyses showed increased activation in areas associated with motor functioning, cognitive control, and emotion. These findings are limited by small sample size, lack of diversity in the sample, and coarsely defined risk factors. Despite these limitations, the results are consistent with an overall concern that risks associated with agricultural work can lead to cognitive and psychiatric harm via impacts on brain health.

MODELING TRAIT EFFECTS ON THE RELATIONSHIP BETWEEN NEURAL PROCESSING AND RESPONSE TIMES ON A FLANKER TASK IN NEUROTYPICAL ADULTS

Susan M. Mingils1, Patricia L. Davies1,3, Mei-Heng Lin2, William J. Gavin3.

1Occupational Therapy Department, Colorado State University

2Center for Molecular and Behavioral Neuroscience, Rutgers University – Newark

3Molecular, Cellular, and Integrative Neurosciences Program, Colorado State University

Bivariate correlations between brain processes and behaviors are often weak and account for only a small portion of variance. Further, measuring a single event-related potential (ERP) component may ignore the systematic nature of brain processing. Previous studies support the feasibility of path analysis to examine brain-behavior relationships. Path analysis can model a series of ERP components representing phases in a neural processing stream to predict a behavior and can account for variance in each measure due to participant traits. This study investigated the relationship between stimulus-related neural processing and behavioral response times (RT) and whether the relationships differed between correct and incorrect responses in adults. This study also examined the effects of trait variables (age and sex) on successive ERP components and RT. Participants included 60 neurotypical 18–29-year-old adults who performed a speeded flanker task during electroencephalography (EEG) recording on two separate visits 6–21 days apart. The baseline-to-peak amplitude of N1, P2, N2, and P3 and RTs for correct and incorrect responses were calculated. Four path models (N1->P2->N2->P3->RTs) were conducted separately for correct and incorrect trials for each of the two visits while controlling for traits. Model fit indices showed acceptable to excellent model fit. The model for incorrect responses at session 1 had the best model fit (RMSEA=0.00, SRMR=0.01, CFI=1.00, TLI=1.13). Only the model for correct trials at session 1 showed P3 significantly predicting RT (beta = -0.57, p < 0.001). Age significantly predicted ERP components for correct trials. Sex significantly predicted P2 for all models except incorrect trials at session 2. Sex significantly predicted RT for all four models, with females having slower RTs overall. Results show the importance of examining interrelationships between phases of neural processing and trait variables when measuring brain-behavior relationships.

MDT-PL CIRCUIT REQUIRED FOR CONTROL-INDUCED DOMINANCE

VK LaFehr1, GW Costanza-Chavez1, JA Zeigler1, GJ Potter1, PT Coleman1, HN Martin1, SF Maier1, MV Baratta1.

1Department of Psychology and Neuroscience, University of Colorado Boulder.

Instrumental control over a stressor (escapable stress, ES) confers protective effects that prevent the neurochemical and behavioral changes that result from a lack of control over a stressor (inescapable stress, IS). It is well documented that the medial dorsal thalamus (MDT) provides the largest excitatory input to the prelimbic cortex (PL). Here, we show that the PL and its input from the MDT are required for later dominance. Warm Spot Test (WST) uses a triad of rats that compete for a single warm spot on a cold cage floor. Previous findings show that ES leads to dominance in WST. We investigated the necessity of the prelimbic cortex in ES-facilitated dominance. We found that intra-PL microinfusion of muscimol (GABA-A agonist, 50 ng/hemisphere) during ES in male rats prevented the facilitation of dominance. We further investigated the necessity of the MDT→PL circuit in ES-facilitated dominance during tube test. Tube test utilizes a plastic tube where two rats are placed at opposite sides and compete to push one another out of the tube. We applied chemogenetic inhibition using the Gi-coupled hM4Di-DREADD in male rats to inhibit glutamatergic neurons in the MDT → PL pathway during ES and IS. We measured dominant behavior by assessing number of tube test wins, where a winner is defined as the rat who pushes the other rat out of the tube first. We found that the inhibition of the MDT→PL at the time of stress does not lead to ES dominance over no-stress animals. mCherry chemogenetic controls in the ES group exhibited dominance over the no-stress group. We found no difference in dominance between IS and no stress groups, whether the MDT → PL was inhibited or not. Our data show that the MDT→PL is necessary for control-induced dominance.

EFFECTS OF IMMUNIZATION WITH MYCOBACTERIUM VACCAE ATCC 15483 ON COGNITIVE FUNCTION IN A TWO-HIT STRESSOR MODEL OF CHRONIC DISRUPTION OF RHYTHMS AND HIGH-FAT DIET.

Zhang H1, Dawud LM1, Marquart BM1, Hunter EA1, Concha AE1, Zabat RN1, Gebert MJ2,3, Frank MG4, 5, Lowry CA1, 3, 4, 5, 6, 7

1Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO, 80309, USA

2Department of Ecology and Evolutionary Biology, Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado Boulder, Boulder, CO 80309, USA

3Center for Microbial Exploration, University of Colorado Boulder, Boulder, CO, 80309, USA

4Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, 80309, USA

5Center for Neuroscience, University of Colorado Boulder, Boulder, CO, 80309, USA

6Department of Physical Medicine and Rehabilitation and Center for Neuroscience, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA

7Veterans Health Administration, Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Rocky Mountain Regional Veterans Affairs Medical Center (RMRVAMC), Aurora, CO 80045, USA

Circadian disruption and consumption of a Western high-sugar/high-fat diet, particularly common in persons engaged in shift work, have been linked with increased inflammation, which is considered a risk factor for development of stress-related psychiatric disorders, including anxiety disorders, mood disorders, and trauma- and stressor-related disorders, such as posttraumatic stress disorder (PTSD). Due to limited availability of effective interventions, novel prevention and therapeutic strategies are needed. The “Old Friends” hypothesis proposes that one mechanism underlying the increase in inflammatory disease, as well as stress-related psychiatric disorders, is reduced exposure to microorganisms with which humans coevolved, in particular those microorganisms that served to limit inappropriate inflammation. One such potential “Old Friend” is Mycobacterium vaccae ATCC 15483, a bacterium that has been shown to ameliorate the systemic inflammatory, neuroinflammatory, and behavioral consequences of acute and chronic stress exposures. In this study, we aim to demonstrate that immunization with a whole-cell, heat-killed preparation of M. vaccae ATCC 15483 prior to and during a “two-hit” stressor model that simultaneously pairs a high-fat diet (HFD) with chronic disruption of rhythms (CDR) prevents HFD/CDR-induced cognitive impairment in the object location memory test (OLM) in 8-week-old male and female mice. Mice received a subcutaneous injection of a whole-cell, heat-killed preparation of M. vaccae ATCC 15483 (0.1 mg; 1 x 108 bacteria) or vehicle (sterile borate-buffered saline) on day –21 and once weekly for the following 10 weeks. On day 0, mice were assigned to either a continuous control diet (CD) or HFD and either a normal light: dark condition (NLD) or CDR condition for eight weeks. Mice were then tested in the OLM test on day 53 of the protocol. Analysis of result is ongoing and we predict that M. vaccae ATCC 15483 will ameliorate HFD/CDR-induced cognitive deficits.

INVESTIGATING THE ROLE OF REACTIVE OXYGEN SPECIES ON NEURONAL WOUNDING IN C. ELEGANS

Cesar Angel Cervantes1, Ennis Deih2, Dr. Frederic Hoerndli3.

From the Department of Biomedical Sciences, Colorado State University,

C. elegans is a very versatile instrument of study in science, notably in neuron research. Being transparent, with a simple nervous system of only 302 neurons and repeating hallmarks of age-dependent and Wallerian degeneration, C. elegans is a model organism that has been used extensively to shed light on fundamentally conserved mechanisms of neuronal injury and neurodegeneration. In this study, we investigate the role of mitochondrial reactive oxygen species (ROS) as a potential agent of neuronal damage. To do this, we have expressed KillerRed, a light-sensitive ROS generator anchored to the mitochondrial outer membrane. We study the cell-specific effect of this agent by expressing it in only one pair of neurons of C. elegans that control the reversal behavior of these animals. Using one-second pulses of 560 nm light to activate the KillerRed protein for 5, 20, and 60 minutes, we induced increased levels of ROS to damage mitochondria in the entire AVA neurons. To analyze the effect of our treatments, we measured the reversal behavior of C. elegans. Induced damage via light stimulation was found to cause an onset of decreased activation of the AVA neurons via demonstration of decreased reversal behavior starting with as little as 5 minutes of treatment. However, this reduced activation of neurons continued to decrease in activity after a 24-to-48-hour period but only to a degree. To better understand the underlying cellular mechanisms leading to this functional effect, we will analyze AVA’s morphology and neuronal activity using confocal microscopy. In the future, once the injury mechanism has been characterized, potential candidates or forward genetic screens to identify gene products leading to less injury or regeneration can be undertaken.

C.ELEGANTS NRF HOMOLOG, SKN-1, MAY PLAY A ROLE IN CANNABIDIOL NEUROPROTECTION

Abdullatif Alsulami1, Vincenzo Gilberto1, Margaret Neuheardt1, Stephanie McGrath2, Julie A. Moreno1

1 Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

2 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University Fort Collins, CO

Alzheimer’s disease (AD) is a neurodegenerative disease that is affecting an increasing number of the aged population worldwide. AD is characterized by the accumulation of amyloid- and tau hyperphosphorylation along with a failure in redox homeostasis. The hallmarks of neurodegenerative diseases include the increased generation of reactive oxygen species (ROS) generation which is tightly controlled by an antioxidant defense mechanism under physiological conditions. The nuclear factor erythroid 2-related factor (Nrf-2) is a transcription factor that is responsible for the regulation of redox balance and antioxidant detoxifying responses. Since oxidative stress is believed to contribute to age-dependent neurodegenerative diseases, it could be predicted that Nrf-2 system may function in its prevention. Furthermore, Nrf-1 regulate proteosome activity to maintain protein homeostasis. The proteosome is a branch in the protein quality control system that degrades misfolded proteins in neurodegenerative diseases, thus; upregulation of proteosome activity would ameliorate proteotoxicity. This research aims to utilize various strains of the model organism C. elegans to understand the mechanism of cannabidiol at the cellular level in stressed models. The SKN-1 gene, the Nrf homolog in C. elegans, encodes for three different isoforms, skn-1a, skn-1b, and c. Skn-1b/c, which plays a role in oxidative stress, is negatively regulated by the repressor WDR-23. In C. elegans, skn-1a plays a role in proteotoxic stress through upregulation proteosome subunits and is negatively regulated by the abundance of proteosome complex protein. Cannabidiol (CBD) is a non-psychoactive phyto-cannabinoid that has multiple beneficial effects including neuroprotection. We hypothesize that CBD-mediated survival extension is through the activation skn-1 isoforms. We are in the process of testing our hypothesis using skn-1 mutated worms treated with and without CBD and performing touch test, longevity assays, and microscopy to confirm the role of skn-1. Our preliminary data show that CBD activates skn-1a and c. Also, CBD acts independently of the repressor (wdr-23). Knowledge gained will allow for a better understanding of how CBD is helping neurons function and survive due to increased misfolded proteins.

POLYAMINE PATHWAY MODULATES CELLULAR SENESCENCE IN DOWN SYNDROME

AD Sola1, CN Pham2, T Gao2, M Hebinck2, A Revier2, DA Paredes2.

1Department of Chemistry and Biochemistry, University of Denver

2Department of Biology, University of Denver

Down syndrome (DS) is characterized by the full or partial triplication of chromosome 21 (T21). This largely results in global transcriptional dysregulation, resulting in a variety of neurodevelopmental and physical disabilities. Recent published data indicate that senescence-associated phenotypes in DS are linked to this dysregulation. The number of pathways to senescence is broad and especially not yet well-defined in DS. Polyamines (PA) are polycationic small molecules involved with cell proliferation, gene regulation, autophagy, and apoptosis, and they are reported to be beneficial for aging. While aged DS cells will become senescent at a higher rate than their normosomic counterpart, we have found that they also have significantly increased presence of each of the three main PAs (putrescine, spermidine, and spermine). Thus, there is a lack of consensus as to the role of polyamines in aging. In this study, we investigate how PA impacts cellular senescence in DS using Western blot densitometry, flow cytometry, and capillary zone electrophoresis to measure senescence, cellular death, and PA metabolism. We found that human DS (T21) fibroblasts have higher concentrations of polyamines and higher rates of senescence than healthy controls which can be reduced with α-difluoromethylornithine (DFMO), an ornithine analogue which targets and blocks Ornithine Decarboxylase, the rate-limiting enzyme in the PA pathway. Next, we characterized the presence of enzymes involved in the PA pathway upon DFMO treatment in T21 fibroblasts and DS mouse hippocampal cells. We found that the PA pathway is inherently dysregulated in DS, while regulating PA by inhibition reduced the senescence normally increased in T21 cells. This data describes how PAs and their altered production relates to aging and potentially related diseases. Further research could reveal novel therapeutic targets in cellular senescence, a common mechanism in many age-related diseases.

PAIN CAUSES HYPOTHERMIA IN CERCADIAN DEPENDENT MANNER DUE TO AN OVEL BRAINSTEM HYPOTHALAMUS CIRCUIT

AE Warfield1, P Gupta12, Q Jeffs2, MM Ruhmann1, WD Todd12.

1Department of Zoology and Physiology, University of Wyoming

2Department of Zoology and Physiology Program in Neuroscience, University of Wyoming

Chronic pain has long been thought to influence circadian rhythms due to temporal clinical observations. However, little to no work has directly addressed this observation from the perspective of how chronic pain can disrupt rhythms. Using models of inflammatory and neuropathic pain we show that persistent pain causes prolonged hypothermia at predictable times of day, and a phase advance in body temperature (Tb) rhythms. We also characterize a novel circuit connecting prodynorphin neurons in the lateral parabrachial nucleus (LPBdyn) to the suprachiasmatic nucleus (SCN), the master pacemaker of circadian rhythms, and the subparaventricular zone (SPZ), the obligate relay of the SCN, using both retrograde and anterograde methods. These LPBdyn cells are activated by both types of pain as marked by c-Fos. There is also c-Fos activation in the SPZ following acute inflammatory pain but this increased level of activation is removed in double-knockout (DKO) mice lacking transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels, implying that pain driven SPZ activation is based in neural signaling from pain afferents, not peripheral inflammation. In these same DKO mice, inflammatory pain’s effect on rhythms is also abolished. To specifically implicate LPBdyn cells in this response, we performed a genetically targeted ablation, using a cre-dependent caspase in prodynorphin-cre (pdyn-cre) mice, along with spared nerve injury (SNI). Preliminary evidence shows that without LPBdyn cells the hypothermic and phase advance effects of the persistent pain response are abolished. Finally, we placed an excitatory channel (hM3) into LPBdyn cells and upon administration of its ligand, CNO, we see a hypothermic phenotype similar to the one following SNI. These data describe a mechanism for how persistent pain can disrupt body temperature rhythms, a previously unobserved phenotype, and a novel neural circuit responsible for this phenotype.

MICROFLUIDIC INTESTINAL MODEL FOR LEAKY GUT THAT COULD LEAD TO NEURODEGENERATIVE DISEASE

AECherwin1, HNTempleton2, ATEhrlich2, CSHenry1, SATobet2.

1Department of Chemistry, Colorado State University

2Department of Biomedical Sciences, Colorado State University

Accumulating evidence suggests that Parkinson’s disease (PD) pathology can arise in the gut. A hallmark of PD is the neuronal accumulation of misfolded α-synuclein (α-syn) proteins. The enteric nervous system (ENS) facilitates bidirectional communication between the brain and the gut. The goal of this study was to gain insight into how barrier breakdown and immune activation due to collagenase exposure contributes to accumulation of α-syn aggregate pathologies in gut neurons and identify cellular mechanisms of aggregate formation and uptake in a microfluidic organotypic device (MOD) model. The MOD device incorporates full-thickness murine colon tissue, which is maintained for up to 80h ex vivo. Serum-free media is supplied to the tissue on both sides; sodium sulfite is included in the luminal channel media to reduce the oxygen concentration, thus generating a physiological oxygen gradient across the barrier. Immunohistochemistry is performed to analyze barrier integrity and indicators of neuroinflammation. Cell counts in colon are analyzed from crypt to lumen. Collagenase treated tissue showed striking alterations in goblet cells and their vesicles characterized by their mucopolysaccharides identified by the fluorescently labeled lectin Ulex Europaeus Agglutinin I (UEA). Goblet cells secrete mucus to help prevent pathogen infiltration. These results suggest that collagenase reduces goblet cell number and perhaps prevents mucopolysaccharide secretion. Collagenase also appeared to alter CGRP fiber immunoreactivity and increase CD3+ T cells and ACK-2+ mast cells. Together, these results indicate increased collagenase in the intestine as a possible initiator of PD pathogenesis. Advancing understanding of aggregate formation and uptake in the gut will help identify causal relationships between their dysregulation and the development of PD pathologies.

POLYAMINES ENHANCE TOXICITY OF ALPHA-SYNUCLEIN: IMPLICATIONS FOR PARKINSON'S DISEASE

Alexandra Penney1,2, Tyler Ball1,3, Joshua Gilbert1,2, Jemil Ahmed1,3, Sunil Kumar1,3 and Daniel Paredes1,4.

1Knoebel Institute of Healthy Aging. Ritchie School of Eng. Univ. of Denver, CO

2Department of Biological Sciences

3Department of Chemistry & Biochemistry

4Department of Electrical and Computer Sciences

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder that leads to tremors, ataxia or slowing of movements. Like many other neurodegenerative diseases, PD stems from the aggregation of a naturally present protein—alpha-synuclein (αS)—and consequential impairment of normal function in other cells. The aggregates are toxic to the dopaminergic basal ganglia cells, particularly in the substantia nigra, essentially limiting the mobility and motor control of PD patients. αS is a prion-like protein thought to help maintain homeostasis of neurotransmitter release and is an important component to the presynaptic neurons. Spermine is a naturally existing polyamine that is hypothesized to induce and increase rates of αS aggregation and has elevated levels in PD patients. In this study, we found that spermine not only increases the frequency of aggregation of αS, but also increases the toxicity of these aggregates, resulting in heavily decreased cell viability. Additionally, the pathological αS aggregates generated in the presence of spermine are resistant to the natural proteolytic degradation that would clear the cell of protein aggregates, though the mechanism by which they resist degradation is yet to be uncovered. This research sets the foundations for uncovering potential new therapeutic targets in the treatment of PD. Supported by the Movement Disorder Foundation.

MYCOBACTERIUM TUBERCULOSIS INFECTION AS A MODEL FOR INFECTIOUS DISEASE INDUCED NEURODEGENERATION

Amanda S Latham1, Charlize Geer1,2, Isla Anderson1,2, David F Ackart2, Amelia Day Hines1, Brendan Podell2, Jessica Elf1, Randall J Basaraba2, and Julie A Moreno1

1Department of Environmental Health and Radiological Sciences, Colorado State University

2Department of Microbiology, Immunology, and Pathology, Colorado State University

Approximately one billion people suffer from a neurological disorder worldwide; this encompasses the 50 million people diagnosed with a neurodegenerative disease, including Alzheimer’s Disease (AD), Parkinson’s Disease (PD), and dementia. Neurodegenerative disease cases are estimated to double over the next twenty years, further establishing the importance of research in this field. This estimated escalation in cases will occur due to the rising aging population as well as the detrimental effects of environmental and infectious agents. Although the mechanism is not fully understood, increasing evidence demonstrates a role of viral and bacterial infections as contributors to disease susceptibility, progression, and pathology. Notably, epidemiological studies show that tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb), predisposes individuals for neurodegenerative diseases and is correlated with neurological deficits. Patients with TB disease, but no diagnosis of central nervous system (CNS) infection, show decreased neuropsychological functioning, cognitive impairments, and loss of interest in social activities. They are also predisposed for the development of dementia and PD. Through studies examining the effects of low-dose Mtb exposure by aerosol, we identify pathological markers of inflammation and toxicity in the central nervous system (CNS) of outbred, Dunkin Hartley guinea pigs. This includes migration and proliferation of microglia followed by the activation of astrocytes in multiple brain regions. The misfolded proteins phosphorylated tau and amyloid beta, which are pathological biomarkers found in diseases including AD and dementia, are also detected. Importantly, these effects occur despite the absence of detectible bacteria in the brains of infected animals. Through this data, we establish that the characteristic hallmarks of neurodegenerative disease, neuroinflammation and misfolded proteins, are found in our guinea pig TB model. These studies will allow us to obtain a better understanding of the role peripheral infections play on the progression of disease in the central nervous system.

THE ROLE OF REPETITIVE ELEMENTS IN NEUROINFLAMMATION AND ALZHEIMER’S DISEASE

AN Cavalier1, ME Smith1, CM McEntee1, BM Bettcher2, TJ LaRocca1.

1 Department of Health and Exercise Science, Colorado State University

2 Department of Neurology, University of Colorado Anschutz Medical Campus

Age is the primary risk factor for sporadic (i.e., not genetic/familial) Alzheimer’s disease (AD), and neuroinflammation is a key driver of brain aging and AD. Recent advances in next-generation sequencing (e.g., RNA-seq) may help identify causes of age/AD-related neuroinflammation; however, most RNA-seq studies of aging have focused on coding genes, while non-coding repetitive sequences (>50% of the human genome) have been largely ignored. Growing evidence shows that repetitive element (RE) transcript accumulation may play a role in aging by stimulating inflammation (a key feature of aging) and could also contribute to AD. REs promote inflammation through formation of double-stranded RNA and/or cytoplasmic complementary DNA, pathogen-associated patterns that stimulate innate immune responses. The mechanisms of age-related RE accumulation are unclear, but one possibility is that epigenetic changes that occur with aging/AD (e.g., reduced chromatin and DNA methylation) may increase RE accessibility. We generated RNA-seq data on blood samples from AD patients and found that RE transcripts are increased in AD vs. healthy older controls. We also found reduced expression of genes related to histone structure and DNA methyltransferase activity, supporting the idea that epigenetic RE dysregulation may play a role in AD development. To investigate epigenetic changes with age/AD, we performed ATAC-seq on the same samples to characterize chromatin structure, and we found that the majority of RE transcripts that were “up” in our RNA-seq data were also in hypochromatinized genome regions. We are currently generating whole genome bisulfite (methylation) sequencing data to determine if specific hypomethylated loci could also be a source of REs that are dysregulated with aging/AD.

KETAMINE’S RAPID ANTIDEPRESSANT EFFECTS ARE MEDIATED BY CA2+-PERMEABLE AMPA RECEPTORS IN THE HIPPOCAMPUS

Anastasiya Zaytseva1*, Evelina Bouckova1*, McKennon J. Wiles1*, Madison H. Wustrau2*, Isabella G. Schmidt1*, Hadassah Mendez-Vazquez2, Seonil Kim1,2.

1Molecular, Cellular and Integrative Neurosciences Program

2Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

*These authors contribute equally.

After demonstrating rapid and robust antidepressant efficacy, the US Food and Drug Administration (FDA) approved ketamine in 2019, sparking a surge in clinical and public interest around the world. Ketamine is shown to enhance excitatory synaptic drive in the hippocampus, which is presumed to underlie its rapid antidepressant effects. However, ketamine is a noncompetitive NMDA receptor (NMDAR) antagonist that inhibits excitatory synaptic transmission. It is thus a puzzling question how ketamine enhances glutamatergic activity to induce rapid antidepressant effects while blocking NMDARs in the hippocampus. Here, we reveal that ketamine treatment in cultured mouse hippocampal neurons significantly reduces calcineurin activity to elevate AMPA receptor (AMPAR) GluA1 subunit phosphorylation, which ultimately induces Ca2+-Permeable, GluA2-lacking, and GluA1-containing AMPARs (CP-AMPARs). Moreover, ketamine-induced CP-AMPARs enhances glutamatergic activity and synaptic plasticity in cultured hippocampal neurons. When a sub-anesthetic dose of ketamine is given to mice, it rapidly increases synaptic GluA1 levels, but not GluA2, in the hippocampus. Moreover, ketamine administration significantly reduces in vivo calcineurin activity to increase GluA1 phosphorylation in the hippocampus. Using the open field and tail suspension tests, we show that a low dose of ketamine rapidly reduces anxiety-like and depression-like behaviors in both male and female mice. However, when in vivo treatment of a CP-AMPAR antagonist abolishes the ketamine’s effects on animals’ behavior. We discover that ketamine at the low dose rapidly promotes the expression of CP-AMPARs via reduction of calcineurin activity in the hippocampus, which in turn enhances synaptic strength to induce antidepressant actions.

MUTATIONS TO THE SYNAPTOTAGMIN CALCIUM BINDING POCKET PRODUCES CONGENITAL MYASTHENIC SYNDROME-LIKE SYMPTOMS IN DROSOPHILA

Andrew Bollegar1, Morgan Litchford1, Vincent Elias1, Max Pliskin1, Casey Martin1, Caitlin T. Waring2, Joseph A. Seggio2, and Dr. Noreen Reist1.

1Department of Biomedical Sciences, Colorado State University

2 Department of Biological Sciences, Bridgewater State University

Neuronal communication is mediated by activity-dependent synaptic transmission. Depolarization of an active nerve terminal results in calcium influx that triggers the fusion of synaptic vesicles with the plasma membrane. This fusion event depends upon synaptotagmin, the presynaptic calcium sensor, binding calcium to initiate fast and synchronous vesicle fusion events. Synaptotagmin is a vesicle protein containing two calcium binding pockets, C2A and C2B, that coordinate calcium via 5 highly conserved negatively charged resides, predominantly aspartate residues. An amino acid substitution within the C2B domain of synaptotagmin 2, the isoform found at the neuromuscular junction, has been identified in a patient with congenital myasthenic syndrome (CMS). Specifically, Aspartate 301 has been replaced with a glutamate (D1E); the effect of this substitution on synaptotagmin function has not been previously examined. In this study we sought to determine whether a homologous aspartate to glutamate substitution in Drosophila would cause CMS-like deficits in the fly, thereby indicating a role for this substitution in CMS etiology. To mimic expression in the human patient, we expressed a syt-C2B-D1E mutation in synaptotagmin heterozygotes – one wild type gene and one mutant gene. To measure the physiological effect of the D1E mutation, we completed a series of electrophysiological and behavioral analyses. To ensure any deficits were a result of the mutation and not due to disruptions in protein expression levels, we conducted Western analyses. We found that evoked transmitter release and overall activity levels were decreased in syt-C2B-D1E mutants compared to controls. These findings are consistent with the decrease in neuromuscular transmission and overall weakness seen in the CMS patient. Our results indicate that the C2B-D1E substitution induces CMS-like symptoms in Drosopohila and support the hypothesis that this synaptotagmin substitution is involved in etiology of congenital myasthenic syndrome.

INTRANASAL DELIVERY OF MESENCHYMAL STROMAL CELLS DECREASE PRION-INDUCED INFLAMMATION EARLY IN DISEASE

Arielle J. D. Hay1, Genova Mumford1, Tanner Murphy1, Amelia D. Hines1, Sydney Risen2, Elizabeth Gordon1, Vincenzo S. Gilberto2, Katriana A. Popichak1, Mark D. Zabel1 and Julie A. Moreno2.

1Department of Microbiology, Immunology and Pathology

2Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins.

Prion diseases are rare protein-misfolding neurodegenerative diseases that can be genetic, sporadic, or acquired through infection. This family of diseases result from the native conformation of the cellular prion protein misfolding to the infectious form, denoted PrP-scrapie (PrPSc). The first sign of disease is neuroinflammation caused by reactive astrocytes and the activation of microglia. This is due to the accumulation of PrPSc and results in oxidative stress, disruption of neural signaling, and glial scarring. The combination of these symptoms leads to cellular dysfunction of neurons, including synaptic dysfunction and the loss of synaptic proteins, which leads to neuronal death. There is evidence that PrPSc itself may not be the neurotoxin, and that other cellular stress pathways, including the inflammation of glial cells, play a major role in disease pathogenesis. Glial cells respond to PrPSc aggregation in the brain by producing proinflammatory cytokines and chemokines, as well as neurotoxic signals that can contribute to neuronal death. Both astrocytes and microglia can be infected by PrPSc and disseminate infectious prions to neurons. We show that adipose-derived mesenchymal stem cells (AdMSCs) secrete anti-inflammatory mediators in response to prion infection and can decrease inflammation in prion-infected cell models. When AdMSCs are delivered intranasally into a mouse model of prion disease, we see a decrease in inflammatory signaling molecules, reactive astrocytes and activated microglia at early stages of disease. We are investigating the mechanism used by AdMSCs to regulate inflammatory signaling in neuronal health in the context of in vitro prion infection, as well as continuing to assess their protective role in mouse models of infectious and genetic prion disease.

THE EFFECTS OF GRAFT SOURCE AND ORIENTATION ON FUNCTIONAL RECOVERY FOLLOWING ABLATION OF BRANCHED PERIPHERAL NERVE

Bridger Sparks1, JuliAnne Allgood1, Jared Bushman1.

1Division of Pharmaceutical Sciences, University of Wyoming

Approximately 20 million people are currently suffering from a peripheral nerve injury (PNI) in the United States. A disruption through trauma, surgical mistakes, or medicinal side effects are what can cause a peripheral nerve injury (PNI). Injuries to peripheral nerves are the most common cause of persistent neuronal dysfunction in the human population as regeneration is rarely restorative. Despite the highly branched structure and organization of the peripheral nervous system, most studies show factors that affect regeneration through sharp bisections and linear ablations of peripheral nerves, very little has been investigated or documented about PNIs that ablate branch points. We have begun to study injuries and regeneration through branched segmental defects using a combination of autografts and allografts in the inbred Lewis rat strain. The injury model is a 2.5 cm complex segmental defect of the sciatic nerve that includes where the sciatic nerve branches into the peroneal and tibial nerves. After this section was removed, experimental groups included autograft in the pre-injury orientation, autografts in a switched orientation (auto-switch) where the peroneal branch within the autograft was sutured to the tibial branch and vice versa, and allografts of the femoral branch in both orientations. Functional recovery measures included compound muscle action potentials (CMAPs), and gait analysis using the Noldus Catwalk system. Data indicates that outcomes are significantly influenced by graft source and orientation at early time points but are not influenced by these factors after 36 weeks.

GENETIC REGULATION OF GLUTAMATE SIGNALIGN DURING COCAINE- AND SUCROSE-SEEKING

C Litif1, K Sandum1, S Hodgins1, L Sa1, L Flom1, AC Bobadilla1

1School of Pharmacy, University of Wyoming

Cocaine use disorder (CUD) facilitates chronic cocaine-seeking in 1.3 million Americans and is yet to have an effective treatment. This is partly due to the lack of understanding of discrete reward-specific mechanisms underpinning and driving cocaine-seeking behavior apart from other biologically necessary reward-seeking behaviors e.g., eating and drinking. Formation and maintenance of discrete neuronal networks, or ensembles, underlies cocaine-seeking. Humans (Homo s.), mice (Mus m.), and flies (Drosophila m.) share conserved glutamatergic reward-signaling that is hijacked to facilitate cocaine-seeking. Glutamatergic signaling plays a physiological role in regulating motivation to acquire biological needs through goal-directed behaviors. Within mice, we know 70% of cocaine- and sucrose-seeking ensembles are exclusive to each reward while 30% of neurons are shared. We know that glutamatergic genetic signatures underly cocaine-seeking. However, it is not well understood which glutamatergic factors regulate cocaine-seeking compared to non-drug sucrose-seeking in an individual brain. Thus, we aimed to define and differentiate reward-specific glutamatergic signaling factors using polyreward self-administration models for cocaine and sucrose acquisition assays inclusive of assessing reward-specific seeking, preference, and locomotion. We utilized fluorescently activated cell sorting and RNA sequencing to genetically characterize cFos-dependent cocaine- and sucrose-seeking ensembles in mice. We conducted genetic screening by over- and under-expressing glutamatergic factors in flies to assess their role in regulating cocaine intake preference and associated locomotion. Using a multi-model approach strengthens the likelihood of narrowing our genetic understanding of glutamatergic factors involved in aspects of CUD. Our investigations have furthered the knowledge of the underlying neurogenetics mediating cocaine-seeking to advance understanding and prevention of CUD relapse.

ADAR1 SUPPRESSION CAUSES REPETITIVE ELEMENT TRANSCRIPT ACCUMULATION AND INTERFERON SIGNALING IN HUMAN ASTROCYTES

CM McEntee1,2, AN Cavalier1,2, TJ LaRocca1,2.

1Center for Healthy Aging, Colorado State University

2Department of Health and Exercise Science, Colorado State University

Neuroinflammation is central to brain aging and many age-related neurodegenerative diseases, but upstream mechanisms of age-related neuroinflammation are not fully understood. One potentially important modulator of neuroinflammation is the enzyme adenosine deaminase acting on RNA 1 (ADAR1) which regulates the accumulation of cytoplasmic double-stranded RNA (dsRNA) through adenosine-to-inosine (A-to-I) editing to disrupt base-pairings and prevent dsRNA-induced innate immune activation. However, the role of ADAR1 in the context of age-related neuroinflammation and the sources of these dsRNA have yet to be investigated. Here, we suppressed ADAR1 in primary human astrocytes (a key cell type in neuroinflammation with aging) via siRNA transfection and used a combination of RNA-seq, immunofluorescence, and immunoblotting to analyze resulting cellular changes. We found that ADAR1 suppression resulted in 220 up-regulated and 745 down-regulated genes, and these changes in gene expression were largely related to increased cytokine and type I interferon responses. We also found increases in expression of repetitive elements (repeat sequences in the genome that may form dsRNA), and many were substrates for A-to-I editing. Our data also suggested that repetitive element-derived dsRNAs bind to melanoma differentiation-associated protein/retinoic acid-inducible gene I (MDA5/RIG-I) to stimulate the production of many pro-inflammatory cytokines (e.g., TNF-ɑ, CXCL10, IFNβ) and increase expression of ICAM-1 (a marker of pro-inflammatory astrocytes). Lastly, we found that our in vitro data may be clinically relevant as ADAR1 gene expression was reduced with aging and neurodegenerative disease (e.g., Alzheimer’s disease) in published RNA-seq datasets, and this decline was associated with an increase in expression of repetitive transcripts. Together, these observations suggest an important upstream role of ADAR1 in neuroinflammation with aging and age-related neurodegenerative disease.

EMERGENT PRION DISEASE IN SWEDISH MOOSE CAUSES REMARKABLY RAPID DISEASE IN MICE

Diana C. Lowe1,2, Julianna Sun2, Sehun Kim2, Jenna Crowell2, Emma Raisley2, Bailey Webster2, Glenn Telling1,2.

1Cell and Molecular Biology Graduate Program

2Prion Research Center2. Department of Microbiology, Immunology and Pathology. Colorado State University.

Chronic Wasting Disease (CWD) is a neurodegenerative disease of cervids (deer, elk, moose, reindeer and red deer) caused by prions, which are aberrantly folded forms of the normal prion protein (PrP). CWD has a long incubation period and is extremely contagious, constituting a growing endemic situation for cervids in North America. Zoonotic risk of CWD transmission to humans is uncertain, but animal prion diseases have shown a potential to cross the species barrier, illustrated by the outbreak of mad cow disease in the United Kingdom in the mid-1980s. In addition, CWD has recently emerged in Northern Europe and recent studies suggest, that it constitutes a sporadic, unstable type of CWD, with distinct properties from its North American counterpart, posing an unpredictable threat to wildlife in the region and potentially to humans. To study the characteristics of this new strain, we inoculated moose brain material in a mouse model of CWD diagnosing clinical signs at ~90 days, the fastest progress to disease ever reported for a model of CWD. Future studies will determine additional properties of this CWD strain which can shed light on the mechanisms of evolution of prion diseases in the wild and their zoonotic potential.

ROLE OF DIET ON DEVELOPMENT AND REGENERATION AFTER SPINAL CORD INJURY

EJ Purifoy1,2, K Mruk1

1School of Pharmacy, University of Wyoming

2Wyoming Research Scholars Program, University of Wyoming

Spinal cord injury is a major public health, as well as individual, burden. Injury to the spinal cord results in permanent disability including: loss of mobility, sensation, or function below the sight of injury. Regenerative models, such as zebrafish, may offer molecular insights into how regeneration of the spinal cord is possible, leading to discoveries of new therapies. A current limitation of this model is the lack of husbandry standardization across the field, which may affect the regenerative properties of zebrafish following spinal cord injury. Adequate nutrition allows for proper development of larvae and may also play a role in regeneration of the spinal cord. For this project, we tested three different diets. These diets were then compared between non-injured and fish injured at 5 and 7 days post fertilization by measuring the length of the larvae over 5 days to see the effects on growth, development, rate of spinal cord bridge, and behavior to study recovery of motor function. Our results suggest that the rotifer diet caused uninjured fish to grow significantly larger than both the liquid and powder diets. Furthermore, larvae fed a rotifer or liquid diet both had a similar rate of regeneration, whereas the commercial powder diet yielded a significantly lower regeneration. This suggests that diet and nutrition plays a huge role in normal development of zebrafish and plays a role in recovery from injury. Standardization of feeding protocols mays lead to enhanced use of this model for other regeneration studies.

TRANSMISSION PROPERTIES OF NORTH AMERICAN SHEEP SCRAPIE PRIONS IN TRANSGENIC MOUSE MODELS

EmmaKate Raisley1,2, Julianna Sun1,3, Nick Heyer1, Jifeng Bian4, Sehun Kim1, Jenna Crowell1, Jason Bartz5, Tracy Nichol6, Terry Spraker7, Juergen Richt8, Glenn Telling1,3

1Prion Research Center and the Department of Microbiology, Immunology, and Pathology, Colorado State University

2Walter Scott Jr. College of Engineering, Colorado State University

3Program in Cell and Molecular Biology, Colorado State University

4United States Department of Agriculture, Animal Plant Health Inspection Service, Veterinary Services, Washington DC, United States 5Department of Medical Microbiology and Immunology, Creighton University

6United States Department of Agriculture, Animal Plant Health Inspection Service, Veterinary Services

7College of Veterinary Medicine and Biomedical Sciences, Veterinary Diagnostic Laboratory, Colorado State University

8Center of Excellence for Emerging and Zoonotic Animal Diseases, Kansas State University

Fatal neurodegenerative diseases such as scrapie of sheep and goats as well as chronic wasting disease (CWD) of cervids are caused by novel proteinaceous infectious agents referred to as prions. The transmission of prion diseases is often very efficient; paired with their characteristic long incubation time, prion diseases have resulted in disastrous consequences across species. For example, contamination of the cattle feedstock in the UK led to a large influx in bovine spongiform encephalopathy, mad cow disease, in the late 1980s. However, it was not until over a decade later that the first cases of variant Creutzfeldt Jakob Disease in humans were identified as a result of consuming the contaminated beef. This species cross over event resulted in over 200 deaths following the outbreak. To more closely evaluate the species barriers of scrapie, transgenic mouse models overexpressing prion protein (PrP) genes were utilized. Mice overexpressing sheep PrP, referred to as TgOv have been shown to be susceptible to well established and characterized UK scrapie isolates. Since little is known about North American scrapie isolates, two distinct TgOv mouse models representing natural polymorphisms in the sheep PrP gene were challenged with North American scrapie isolates. Determining the strain characteristics of the North American scrapie isolates could offer insight on their specific properties and transmissibility. Additionally, similar transgenic mice expressing deer or elk PrP were challenged with these same isolates to test the hypothesis that North American scrapie was the origin of CWD.

LEVEL OF DISABILITY AND IDENTIFYING OBJECTIVE INTERVENTION TARGETS FOR EFFECTIVE REHABILITATION IN LONG COVID

H Pierro1, A Francis1, L Noteboom2, D Stoot3, K Gorgens, B Davidson1.

1Ritchie School of Engineering, University of Denver, Denver, CO

2Department of Psychology, University of Denver, Denver, CO

3High Definition Physical Therapy, Lonetree, CO

4Graduate School of Professional Psychology, University of Denver, Denver, CO

The medical community has yet to develop effective interventions that provide relief to patients with Long COVID. There are no standardized scales of severity or intervention targets for Long COVID. We recently found that post-concussion assessment tools are sensitive to Long COVID. However, unlike research methods, it is important that a clinical assessment for Long COVID patient be brief and effective. The purpose of this investigation was to identify neurocognitive and balance performance assessments that are best associated with symptom categories in patients with Long COVID. Self-identified participants with Long-COVID (n=33, ages 21-71) completed a symptom inventory, neurocognitive assessment, and a series of balance assessments. Severity of each symptom was assessed from 0-6 and a symptom score was calculated for the total inventory and each category—Somatic, Cognitive, Sleep, and Affective. Symptom scores were associated with subtests of the ANAM—Simple Reaction Time (SRT), Procedural Reaction Time (PRT), and Repeated Simple Reaction Time (RSRT)—and force platform measures during the Clinical Test of Sensory Interaction on Balance (CITSIB) using Pearson’s correlation coefficient. Total Symptom Score, Somatic, and Sleep symptom scores were moderately correlated to age and sex matched standard scores from the PRT (r=0.50-0.58), and low to moderate correlations to RSRT (r=0.30-0.61). Total Symptom score was moderately correlated with the major axis of the 95% ellipse during Eyes Closed Hard Surface balance. The correlations indicate a moderately strong construct validity on level, or severity, of disability due to Long COVID. Establishing simple and objective performance measures to augment the common intake symptom questionnaires may add a substantial benefit to understanding the severity of disability and provide an objective quantitative intervention target to measure before and after therapy for Long COVID patients.

MIR-137 IS A BRAIN-ENRICHED MICRORNA REQUIRED FOR NORMAL INSULIN SIGNAL TRANSDUCTION

Hana Saedi1, Susan Tsunoda1.

1Biomedical Sciences Department, Colorado State University

The insulin receptor (InR) is widely expressed in the central nervous system. Understanding the role of insulin/insulin signaling in the central nervous system has become of great interest in the past decade. The disturbance of insulin action in the central nervous system has been linked to defects in energy homeostasis, metabolic and behavioral disorders, impaired learning and memory, and neurodegenerative diseases. The insulin signaling pathway is a well-conserved nutrient-sensing pathway throughout the animal kingdom. Studies over the last decade have revealed that miRNAs play a significant role in regulating signaling pathways that contribute to neural and metabolic diseases. miRNAs are small non-coding RNAs responsible for gene regulation at the posttranscriptional level by pairing to the 3' UTR of target genes. miR-137 is a well-conserved brain-abundant miRNA that plays critical roles in cell proliferation and development. By using miRNA target prediction algorithms, multiple genes in the insulin signaling pathway are predicted to be regulated by miR-137, including the insulin receptor (InR), the protein kinase B (AKT), S6K kinases, the adiponectin receptor (AdipR), and the InR phosphatase (PTP61F), suggesting that miR-137 influences the insulin signaling pathway. Here, we present evidence that miR-137 is required for normal insulin signaling in Drosophila melanogaster. Drosophila miR-137-/- null mutants exhibit reduced brain insulin signaling activity which contributes to physiological effects such as obesity, abnormal feeding, and increased survival when challenged by starvation. In addition, our results suggest that miR-137 is required for the phosphorylation of the InR, potentially by targeting PTP61F/PTP1B, the major protein tyrosine phosphatase that negatively regulates InR. This study highlights the importance of miR-137 in regulating insulin signal transduction and reveals novel phenotypes related to energy homeostasis in the organism.

THE ROLE PLAYED BY RESIDUE 226 OF PRP IN CHRONIC WASTING DISEASE PATHOGENESIS AND STRAIN SELECTION

JL Sun, SJ Kane, S Kim, J Crowell, B Webster, E Raisley, D Lowe, GC Telling.

Prion Research Center, Colorado State University

Prions are infectious proteins causing fatal neurodegenerative diseases in humans and animals. Contagious transmission of chronic wasting disease (CWD), a prion disease affecting deer, elk, and other cervids, has caused disease in wild and captive animals in 26 States and 3 Canadian provinces. The primary sequence of the prion protein (PrP) plays a role in determining susceptibility and disease pathogenesis in both intra and interspecies transmissions of prion disease. North American deer or moose PrP encodes glutamine at residue 226 (Q226), North American elk PrP encodes glutamate (E226). To assess the effects of this difference on CWD pathogenesis, we created gene targeted (Gt) mice in which the murine PrP coding sequence was targeted and replaced with CervidPrP-Q226 or CervidPrP-E226, referred to as GtQ and GtE mice. Previous studies showed that GtQ and GtE mice were susceptible to North American CWD, and that time to disease onset was faster in GtE mice. To fully understand the mechanism underlying this difference, we conducted a longitudinal analysis of disease in GtE and GtQ in which mice were intracerebrally inoculated with elk CWD prions. Mice were collected every 15 days until terminal disease. Brain extracts were analyzed for PrP27-30 and glycoform ratio profiling by western blotting, disease-associated PrP by immunohistochemistry and histoblotting, titer determination by the cervid prion cell assay (CPCA), and the appearance of PrPSc using mAb PRC7 in ELISA format. Our analysis reveals primary structural differences at residue 226 of CerPrP have pronounced effects on the outcomes of disease in Gt mice infected with North American CWD prions. Since North American deer and moose express CerPrPC-Q226 and elk express CerPrPC-E226, this study lends insight into the natural pathogenesis of CWD in these species.

THE EFFECTS OF GRAFT SOURCE AND ORIENTATION ON OUTCOMES AFTER ABLATION OF A BRANCHED PERIPHERAL NERVE

JuliAnne Allgood1, Bridger Sparks1, Jared Bushman1.

1Division of Pharmaceutical Sciences, University of Wyoming, Laramie, WY, United States

Nearly 80 km of peripheral nerves extend and branch throughout the human body. Injuries to peripheral nerves are the most common cause of persistent neuronal dysfunction in the human population as regeneration is rarely restorative. Despite the highly branched structure and organization of the peripheral nervous system, the near totality of studies and data address only peripheral nerve injuries that are either a single sharp bisection or linear segmental defects. How regeneration may occur when the injury encompasses branch points is unknown, presumably because there have not been any effective treatments for addressing branched injuries. We have begun to study injuries and regeneration through branched segmental defects using a combination of autografts and allografts in inbred and outbred rat strains and in combination with targeted localized immunosuppressive strategies. The injury model is a 2 cm complex segmental defect of the sciatic nerve that includes where the sciatic nerve branches into the peroneal and tibial nerves. After this section was removed, experimental groups included (1) autograft in the pre-injury orientation, (2) autografts in a switched orientation (auto-switch) where the peroneal branch within the autograft was sutured to the tibial branch and visa versa, (3-4) allografts of the femoral branch in both orientations. Outcome measures included compound muscle action potentials (CMAPs), gait analysis using the Noldus Catwalk system, end point nerve morphometry, muscle mass and retrograde labeling down each branch. Data indicates that outcomes are significantly influenced by graft source and orientation at early time points but are not influenced by these factors after 36 weeks.

SEX AND REGION-SPECIFIC EFFECTS OF JUNK FOOD DIET ON PERINEURONAL NETS WITHIN THE PREFRONTAL CORTEX OF RATS

Kirkpatrick GE1,5, Shafar B2, Gaston B2, Zhang Z3, Ferrario CR, Brown4 TE5*

1Biomedical Program, University of Wyoming, Laramie, WY 82071

2School of Pharmacy, University of Wyoming, Laramie, WY 82071

3Department of Zoology & Physiology, University of Wyoming, Laramie, WY 82071

4Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109

5 Integrative Physiology and Neuroscience, Washington State, Pullman WA 91163

One of the key factors in the development of obesity is the overconsumption of palatable hypercaloric foods, which override homeostatic regulation of feeding to alter reward circuitry (Ferrario, 2020). Current literature suggests that consumption of palatable hypercaloric diets alters medial prefrontal cortex (mPFC) function, resulting in reduced executive control of eating behaviors (Lowe et al., 2019). The mPFC contains parvalbumin expressing, fast-spiking interneurons (PV+ neurons) that are surrounded by specialized extracellular matrix structures called perineuronal nets (PNNS). PNNs are known to limit synaptic plasticity and modulate PV+ neuron activity to maintain excitatory:inhibitory balance within local PFC circuits (Brown and Sorg, 2022). Our previous work showed that 21d consumption of 60% high fat diet altered the intensity of Wisteria Floribunda Agglutinin staining within the mPFC in both a sex and region-specific manner (Dingess et al., 2020). It is unknown if the changes we previously reported are widespread amongst palatable diets. To address this question, we exposed 60d male and female Sprague-Dawley rats to a “junk food” diet (14% protein, 58% carbohydrate, 28% fat, Oginsky and Ferrario, 2019) and assayed whether the presence and/or intensity of PNNs in the mPFC were altered. Rats were placed into one of two groups: ad libitum chow or ad libitum junk food. Following 30d exposure to either diet, we quantified the number and intensity of PNNs in the prelimbic (PL) PFC, infralimbic (IL) PFC, and the ventromedial orbitofrontal cortex (vmOFC). In both sexes there were no changes in PNN number in any region. In the PL-PFC there was no change in PNN intensity in males and females. In the IL-PFC, males had a significant reduction in PNN intensity (CH: 100.0±3.2, JF: 71.5±7.9), while the females showed no significant changes (CH:100.0±3.9, JF:102.5±6.0). In the vmOFC there was a significant decrease in PNN intensity for both male and female rats (Male-CH: 100.0±1.9, JF: 77.81±4.6; Female-CH: 100.0±3.5, JF: 71.6±5.4). Taken together, these results suggest that exposure to a junk food diet alters PNN remodeling in a sex and region dependent manner. In addition, the pattern of PNN changes was unique between junk food and previous findings for 60% high fat diet.

OPTIMIZING SPINAL CORD INJURY IN ZEBRAFISH LARVAE: EFFECTS OF AGE ON REGENERATION

KL Underwood1, WJ Walker1,2, PI Garrett1,3, S Linch1, K Mruk1,2,3.

1School of Pharmacy, University of Wyoming

2Zoology & Physiology Graduate Program, University of Wyoming

3Neurosciences Graduate Program, University of Wyoming

Zebrafish are an increasingly popular model for spinal cord injury (SCI) regeneration. Larval zebrafish are time and cost efficient for modeling SCI, but currently are not standardized on age of injury. Our lab has sought to determine the how the age of larval zebrafish affects SCI regeneration by transecting at three different ages relating to developmental complexity of the central nervous system. We found that larval zebrafish can regenerate at all three transection ages, but overall survival varies with the age of injury. We confirmed using a fluorescent marker that the radial glia form a bridge during regeneration. We observed an increase in fluorescence at the edges of the injury site, suggesting an increase in either number of radial glia cells or marker expression. This increase in fluorescence is stronger at older ages. There was also upregulation of extrinsic factor ctgfa at the site of injury, consistent with adult transection studies. We also found that some axons crossed the injury site before the radial glial bridge has fully formed. In addition, behavioral analyses showed that swimming is achieved before the glial bridge has formed, and that the type of swimming restored is dependent on bridge formation. These results suggest different neuronal circuits may be involved in recovery of locomotion. Overall, we found that age of injury in larval zebrafish affects different aspects of regeneration of SCI, and should be considered when designing experiments aimed at understanding regeneration.

CHARACTERIZATION OF THE HARTLEY GUINEA PIG BRAIN: A MODEL FOR SPONTANEOUS AND PROGRESSIVE NEURODEGENERATION.

Kristen Glennie1, Amanda Latham2, Arsha Moorthy1, Maureen Walsh1, Tom LaRocca1,4, Kelly Santangelo3,4, Karyn L. Hamilton1,4, and Julie A. Moreno2,4 .

1 Department of Health and Exercise Science, Colorado State University Fort Collins, CO

2Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

3Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University Fort Collins, CO

4Columbine Health Systems Center for Healthy Aging

The rate of Alzheimer’s Disease (AD) and Alzheimer’s Disease Related Dementia (ADRD) diagnosis is predicted to increase from 55 million people worldwide in 2020 to 139 million in 2050. Despite this rapid predicted increase in diagnosis and the debilitating symptoms that occur with AD/ADRD, there are many challenges attenuating the discovery of effective cures, treatments, and preventative strategies. Given the complex “inflammaging” phenotype that often contributes to neuroinflammation and ultimately AD/ADRD pathology, one of the largest barriers we face is finding an accessible model that mimics naturally-occurring, inflammation-driven, neurodegeneration in humans. Recent research, however, has demonstrated that the Dunkin Hartley guinea pig strain may address these limitations. Hartley’s naturally and rapidly develop various systemic and inflammation-driven, age-related co-morbidities characteristic of “inflammaging” in humans. Specifically, early research in the hippocampal region of the brain has found that Hartley’s develop neuroinflammation and biomarkers of neurodegeneration, which are known characteristics of progressive neuropathology. These characteristics include protein dyshomeostasis, glial proliferation, neuronal cell loss, and the formation of aggregated misfolded proteins, amyloid beta and hyperphosphorylated tau. To further elucidate these findings, we are currently quantitatively and qualitatively examining the hippocampus, frontal cortex, cerebellum, and thalamus in 5mo and 15mo Hartley guinea pig’s for markers of progressive neuropathology. These findings will further characterize the specific ‘brain aging’ phenotype – and contribute to the development of identifying the Dunkin Hartley guinea pig as a model for naturally occurring neurodegeneration.

GENETIC ALTERATION OF TAU EXPRESSION AND EPILEPTOGENESIS AFTER INTRAHIPPOCAMPAL KAINATE TREATMENT IN MICE

Madeleine C. Moseley1, Ryan A. Cloyd2, Young-Jin Kang1,3, Sang-Hun Lee1,3, Rafael Roberts3, Bret N. Smith1,3.

1Department of Biomedical Sciences, Colorado State University

2Department of Physiology, University of Kentucky College of Medicine

3Department of Neuroscience, University of Kentucky College of Medicine

Temporal lobe epilepsy (TLE) is the most common focal epilepsy in adults and is often resistant to anti-epileptic drugs. Tauopathies like hyperphosphorylated tau seen in Alzheimer’s disease (AD) has been associated with epilepsy in human and animal studies. Alternatively, lack of tau expression through genetic deletion has shown to improve seizure outcomes and reduces epilepsy-related death (i.e., SUDEP) in animal models of genetic epilepsies. The cause of SUDEP is unknown in individuals with TLE but evidence from epilepsy models indicate alterations in regions that control vagus nerve function as a contributing factor to cardiorespiratory collapse and SUDEP. Few studies have examined the impact of tau expression or hyperphosphorylation in acquired epilepsies or epilepsies not caused by other underlying genetic factors. Here, we investigated how altered tau expression (i.e., lack of tau and expression of tau hyperphosphorylation) impacts epileptogenesis of acquired TLE using the intrahippocampal kainate (IHK) model. We used the htau mouse model of tauopathy that produces mice that either express no tau of any type (i.e., tau-/- mice) or non-mutant human tau that begins to become hyperphosphorylated by 1.5 months of age, but do not express native murine tau protein (i.e., htau mice). Our data suggest that status epilepticus (SE) and spontaneous seizures are less likely to develop in IHK-treated tau-/- mice compared to non-transgenic control and htau mice. Further, IHK treatment resulted in an increase in SUDEP in htau mice that was not present in tau-/- or non-transgenic control mice, suggesting an impact of tau hyperphosphorylation on SUDEP and, possibly, seizure burden. Results show IHK-treated tau-/- and htau mice exhibit resistance to changes in synaptic function associated with TLE suggesting a role of native tau in the development of TLE. Together, these results provide important information on epileptogenesis in acquired TLE in the context of tauopathy.

CBD PROTECTS UV STRESS IN A SKN-1 DEPENDENT PATHWAY IN C. ELEGANS

Margaret Neuheardt1, Abdullatif Alsulami1, Stephanie McGrath2, and Julie A. Moreno1

1Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

2Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University Fort Collins, CO

Alzheimer’s disease is a neurodegenerative disease that progresses with age and can disrupt memory and general brain function. One of the characteristics of the disease is the presence of misfolded proteins, particularly amyloid-b and tau hyperphosphorylation. These cause neuronal stress and induce the accumulation of reactive oxygen species (ROS), which have damaging effects on neurons. This research aims to use various strains of the model organism C. elegans to understand the mechanism of cannabidiol (CBD) at the cellular level. CBD is a non-psychoactive phyto-cannabinoid that has been previously shown to be neuroprotective, but the mechanism remains unknown. The nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor that regulates antioxidant responses, which is predicted to counteract the oxidative stress produced by ROS. The SKN-1 gene, which is the ortholog of Nrf in C. elegans, encodes for three different isoforms: skn-1a, skn-1b, and skn-1c. Skn-1c is a known transcription factor conserved with Nrf2, whereas the function of skn-1b remains partially unknown. We hypothesize that mediated survival extension is accomplished through activating skn-1. This effect extends the lifespan through the ROS scavenging ability of CBD. We are currently testing this hypothesis in skn-1 mutated worms, through applying ultra violet (UV) exposure to CBD treated and untreated worms. We have found that CBD does improve lifespan through the mechanism of ROS scavenging. Compared to a known ROS scavenger, N-acetyl cysteine (NAC), CBD elongates the lifespan of the worms.

HIV AND FIV GLYCOPROTEINS INCREASE CELLULAR TAU PATHOLOGY VIA CGMP-DEPENDENT KINASE II ACTIVATION

Michael J Doolittle1,*, Matheus F Sathler2,*, James A Cockrell3, India R Nadalin2, Franz Hofmann5, Sue VandeWoude4, and Seonil Kim1,2.

1Molecular, Cellular and Integrative Neurosciences Program

2Department of Biomedical Sciences

3Department of Human Development and Family Studies

4Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, 80523, USA

*These authors contribute equally.

As the development of combination antiretroviral therapy (cART) against human immunodeficiency virus (HIV) drastically reduces the number of HIV deaths, more of individuals with HIV are now entering the prime age when Alzheimer’s disease (AD)-like symptoms begin to manifest. Hyperphosphorylated tau, a known AD pathological characteristic, has been prematurely increased in the brains of HIV-infected patients as early as in their 30s and is increased with age. This thus suggests that HIV infection may lead to accelerated AD phenotypes. However, whether HIV causes AD to develop more quickly in the brain is not yet fully determined. Interestingly, we have revealed that viral glycoproteins, HIV gp120 and feline immunodeficiency virus (FIV) gp95, induce neuronal hyperexcitation via cGMP-dependent kinase II (cGKII) activation in cultured hippocampal neurons. Here, we use cultured mouse cortical neurons and demonstrate that HIV gp120 and FIV gp95 are sufficient to increase cellular tau pathology, including intracellular tau hyperphosphorylation and tau release to the extracellular space. We further reveal that viral glycoprotein-induced cellular tau pathology requires cGKII activation. Together, HIV infection likely accelerates AD-related tau pathology via cGKII activation.

EXPLORATORY USE OF RT-QUIC TO QUANTIFY AND COMPARE KINETICS OF AMYLOID FORMATION IN NORTH AMERICAN AND SCANDINAVIAN CWD PRIONS

ML Tyer1, X Shi1, J Sun1, S Kim1, S Benestad2, G Telling1.

1Department of Microbiology, Immunology and Pathology Colorado State University

2Norwegian Veterinary Institute

Chronic wasting disease (CWD) is a transmissible, universally fatal neurodegenerative disease caused by the pathogenic misfolding of prion protein. CWD is endemic to North America (NA), and most recently has been identified in Scandinavian cervids. Interestingly, isolates collected from Scandinavian CWD cases have been shown to possess unique strain characteristics. Previous research from our group has shown Scandinavian CWD is etiologically distinct from its North American counterpart, which may account for these strain differences. Here, we seek to further characterize the kinetic properties of Norwegian CWD (NorCWD) isolates through real-time quaking-induced conversion (RT-QuIC) assay, a method for quantifying the rate a prion seed can form amyloid from a standard substrate. Our preliminary results show that Nor red deer and moose isolates display variable amyloid formation rates within technical replicates, with little discernible linear range over a dilution series. This is distinct from NA elk and mule deer, which display a predictable linear range over 10-4 to 10-7 titrations. Notably, the NorCWD isolates converted substrate at a faster rate than seeds taken from NA. Taken together, these results indicate that the distinct properties of NorCWD can be recapitulated using a standard in vitro assay with recombinant Syrian hamster PrP as a substrate. These preliminary data will guide the direction of our future research investigating features influencing NorCWD pathogenicity.

IN VITRO CHARACTERIZATION OF HUMAN NEURONAL NETWORKS ON MULTIELECTRODE ARRAYS AS A MODEL TO INVESTIGATE NEURODEGENERATIVE DISEASES

MR Lemieux,1 B Freigassner1, FZ Thathey1, MR Opp1, CA Hoeffer1, CD Link1.

1Department of Integrative Physiology, University of Colorado Boulder

Multielectrode arrays (MEAs) are a powerful tool that can be used to measure in vitro electrophysiological changes. A great benefit of this system is the ability to characterize human neuronal networks without cellular disruption. Our purpose for using this model is to explore Alzheimer’s disease states to identify mechanisms that can be therapeutic targets. Here we used human induced pluripotent stem cells (hiPSCs) with a doxycycline inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with human primary astrocytes. The cells were plated in dense 12 µL droplets containing partially differentiated neurons and astrocytes at a 2:1 ratio on a 24-well plate. Each well contains an electrode field with 16 electrodes in each array. The cells matured for 32 days on the 24-well arrays and recordings were obtained using Axion Biosystem’s Maestro Edge neural spike filters (200 Hz- 3kHz) with a sampling frequency of 12.5 kHz. Recordings of activity showed network bursting with strong synchrony where over 90% of wells had a synchrony index above 0.8. The spontaneous network burst frequency averaged 0.14 Hz which researchers have claimed reminiscent of slow wave sleep in humans. Validation and characterization of these networks were further investigated by immunofluorescence on coinciding slides as well as treatments on the MEAs. Some treatments we have explored include human serum, GABAA receptor antagonist bicuculline, adenosine A2A receptor agonist CGS 21680, synthetic tau, and amyloid beta. In the future we hope to continue to discover potential Alzheimer disease molecular targets as well as explore the bidirectional relationship between lack of sleep and Alzheimer’s onset.

A RETINAL CONTRIBUTION TO OPID-INDUCED SLEEP/WAKE DYSREGULATION

N Bergum1, Jozsef Vigh1.

1Department of Biomedical Sciences, Colorado State University, Fort Collins

Opioids remain among the most potent treatment options for treating moderate to severe pain. Their long-term use, however, is associated with a plethora of negative side effects including drug dependence, the development of analgesic tolerance and sleep problems. While tolerance develops to the analgesic effects of opioids, opioid-related sleep problems do not appear to develop tolerance following chronic exposure. As opioid receptors are robustly expressed in both the pain pathway and central sleep/wake circuitry, it is unclear as to why tolerance develops in the former, but not the latter. Recent studies from our group have identified a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that transmit environmental light information to the brain’s central sleep/wake circuitry. Importantly, these cells express the µ-opioid receptor (MOR) and their activity can be directly regulated by MOR agonists. As opioid-induced sleep/wake alterations do not appear to develop tolerance, we wanted to explore the contributions of ipRGCs to this phenomenon. To address this, we employed a multidisciplinary approach to examine the functional role of MORs expressed by ipRGCs in chronic opioid-induced sleep/wake changes in mice. First, we show that morphine accumulates in the mouse retina following chronic systemic exposure. We also observed that morphine-induced locomotor activation is reduced in mice lacking MORs expressed by ipRGCs. Finally, MOR-mediated inhibition of whole cell calcium currents does not develop tolerance following chronic morphine exposure. Together, these data suggest that MORs expressed by ipRGCs contribute to chronic opioid-induced sleep/wake disturbances.

EFFECTS OF DOPAMINE D3 RECEPTOR ACTIVATION ON MEDIAL PREFRONTAL CORTEX LAYER V PYRAMIDAL NEURONS RESONANCE

N M.M Mohamed1,M.PThomas2.

1Department of Biological Education, University of Northern Colorado

Dopamine is known to play an important role in cognition, including working memory. Theta rhythms are observed in the mPFC during working memory tasks. Abnormal mPFC theta rhythms are observed in patients with working memory deficits. The purpose of our research is to understand whether dopamine alters the theta rhythm through activation of its cognate D3 receptor. In this study, we examined the effects of two selective D3R agonists on HCN currents (as measured by the hyperpolarization-induced sag in current clamp) and electrical resonance in layer V pyramidal neurons in mouse prefrontal cortical slices. Whole-cell patch clamp recordings were made from type I layer V pyramidal neurons in slices from mouse medial prefrontal cortex. Using current clamp mode, hyperpolarizing current pulses were applied and the “sag” amplitude measured before and after application of selective dopamine D3R agonists. The D3R agonists significantly inhibited the sag amplitudes of type I pyramidal neurons. In the same type I neurons, sinusoidal current was injected with the frequency swept from 0 Hz to 10 Hz over a 10-second period., Voltage responses were measured, and the resonant frequency determined, before and after the application of the D3R agonists. The D3R agonists significantly altered the resonant frequency in the type I layer V neurons.

DETECTING NEURODEGENERATIVE BIOMARKERS IN EXTRACELLULAR VESICLES FROM AGING CANINE PLASMA

Omar Yanouri1, Amelia D. Hines2, Stephanie McGrath3, Breonna Kusick3, and Julie A. Moreno2.

1Program in Molecular and Cellular Integrative Neuroscience, College of Veterinary Medicine and Biomedical Sciences, Colorado State University Fort Collins

2Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University Fort Collins

3Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University Fort Collins

Canine cognitive dysfunction (CCD) syndrome is an age-related neurodegeneration common in older dogs with many similarities to Alzheimer’s disease. CCD presents with hallmarks of pathological aging and AD in humans, such as aggregation and accumulation of misfolded protein, neuroinflammation and progressive brain atrophy. These similarities, and the fact that CCD is a naturally occurring, age-related degeneration that presents with Amyloid-β (Aβ)-plaques and hyperphosporylated tau make CCD a promising model for Alzheimer’s disease and other related dementias. The purpose of this research is to identify biomarkers of CCD present in blood plasma. Plasma was gathered from canine patients participating in a longitudinal study examining CCD progression. Samples were obtained from both young and aged populations every three months, and included canine patients who displayed clinical signs of CCD. Extracellular vesicles were isolated from the plasma samples using size exclusion chormotogrphy, and were examined for pathological aging biomarkers using immunoblot. We have analyzed samples for protein levels of such as Aβ1-42, glial fibrillary acidic protein (GFAP), and various forms of phosphorylated tau. We are currently in process of assessing our baseline samples for each of our canine cohorts’ 1.) young, 2.) aged canines and 3.) aged canines with CCD. The field is in need of an ante-mortem biomarker for CCD to better track and understand disease progression. Due to the high similarity, a CCD assay and understanding of this disease’s progression may translate to Alzheimer’s disease or related dementias, allowing for the development of new diagnostic assays or therapeutic treatments.

EFFECT OF VOLTAGE-GATED K+ CHANNEL INHIBITION BY 4-AP IN SCI RECOVERY IN ZEBRAFISH

PM Hoffman, K Mruk.

University of Wyoming School of Pharmacy

Irreversible spinal cord injury (SCI) affects around 17,000 new Americans each year, with a current total surmounting to around 290,000 Americans affected. Globally, between 250,000 to 500,000 new cases occur annually. In a normal, healthy individual, K+ is crucial for proper action potential (AP) firing to carry out normal neuronal functions. 4-aminopyridine (4-AP) is a medication currently available for prescription use to alleviate symptoms of multiple-sclerosis (MS). The mechanism of action of 4-AP is inhibition of voltage gated K+ channels, specifically channels in the KV1 family, which slows cell repolarization, causing a longer firing period of the AP. Recently, 4-AP has been found to have neuroprotective properties, giving rise to more questions about potential usefulness in other neuronal disorders. Several ongoing studies are being conducted to explore other areas where 4-AP may have an effect, including stroke, traumatic brain injury, and peripheral nervous system recovery. The goal of our study is to determine if delaying K+ efflux, and thereby delaying cell hyperpolarization, has an effect on the recovery of SCI in zebrafish. Zebrafish are pro-regenerative species that share 70% of their genome with humans, making them the optimal specimen for this study. Using a transgenic line of zebrafish with glial cells labelled in GFP, we transected the spinal cord above the cloaca and followed the fish for 7 days post injury. We tracked swim patterns, specifically the type of movement (twitch vs small vs large) and the timing of movement (days post injury) to determine whether 4-AP can enhance recovery in a regenerative model. We also determined whether 4-AP can affect glial bridging post-injury in 5-day old zebrafish embryos. We found that 4-AP increases movement in later stages of recovery when compared the vehicle alone, DMSO. To optimize therapeutic potential, our next area of focus is to change dosing and dosing regimen.

CO-ACTIVATION OF SELECTIVE NICOTINIC ACETYLCHOLINE RECPETORS IMPROVES HIPPOCAMPAL BRAIN RHYTHMS AND MEMORY IN THE MOUSE OF ALZHEIMER'S DISEASE

Rahmi Lee1, Seonil Kim1.

1Department of Biomedical Sciences, Colorado State University

It has been suggested that reduced activity in GABAergic inhibitory interneurons disrupts neural oscillations in the hippocampus, which leads to memory loss in Alzheimer's disease (AD). A prominent AD pathology in the human brain is the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChR). Aβ is known to interact with these receptors and impair their function. nAChRs are expressed more in GABAergic inhibitory interneurons, thus cholinergic deficiency is a prime suspect for Aβ-induced impairment of inhibitory dysfunction in the hippocampus and cognitive decline in AD. Our previous findings, using cultured mouse hippocampal neurons show Aβ selectively interacts with α7- and α4β2-nAChRs, but not α3β4-nAChRs, and decreases activity in inhibitory interneurons, but induces hyperexcitation in excitatory neurons. We thus hypothesize that Aβ reduces hippocampal GABAergic activity by selectively inhibiting α7- and α4β2-nAChRs, resulting in hippocampal oscillatory disruption and memory loss in AD. To test our hypothesis, the AD mouse model, 5XFAD transgenic mice, and wild type (WT) littermates were treated intraperitoneally with α7- and α4β2-nAChR agonists. Saline was given to control mice. Fear conditioning was performed to see if agonists improved memory. We found that 5XFAD mice showed clear deficit in contextual memory which was successfully reversed by co-stimulation of α7- and α4β2-nAChR agonists. Stereotaxic surgery was then performed to measure local field potentials of theta and gamma oscillations, key components for learning and memory. During memory consolidation, theta, slow and fast gamma activities were significantly reduced in 5XFAD mice. Co-activation of α7- and α4β2-nAChR agonists was sufficient to restore normal rhythmic activities in 5XFAD mice.

FUNCTIONAL EVALUATION OF AAV INTRACEREBROVENTRICULAR DELIVERY OF THE CTR REPRESSOR IN ADULT MICE LACKING TDP-43 IN FOREBRAIN NEURONS

Rashmi Thapa1, Hyrum Ruby1, Sierra McOmie1, Harlie Kaligis2, Tianyu Cao3, Gabsang Lee4, Philip Wong3, Yun Li1,*.

1 Department of Zoology and Physiology, University of Wyoming, 1000 E University Avenue, Laramie, WY 82071, USA

2 Department of Cellular and Molecular Biology, Harvard College, Cambridge, MA 02138

3 Department of Pathology, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA

4 Department of Neurology, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA, * Correspondence to: Yun Li (yli30@uwyo.edu)

TAR DNA binding protein 43 kDa (TDP-43) is a conserved DNA/RNA binding nuclear protein whose major function is splicing repression of cryptic exon during RNA processing. Mislocalization of TDP-43 is commonly found in Alzheimer’s Disease Related Dementia (ADRD) such as frontotemporal dementia (FTD) and mixed etiology dementia such as Alzheimer’s disease (AD) with TDP-43 pathology. Multiple lines of evidence support the notion that loss of TDP-43 splicing repression contributes to neuron loss and cognitive deficits in FTD and AD with TDP-43 pathology. A chimeric repressor, CTR that fused the N-terminal fragment of TDP-43 with a well-characterized splicing repressor domain to replace the C-terminal fragment of TDP-43, has been found to mimic splicing repression function of TDP-43 to prevent the incorporation of cryptic exons in the transcripts of cultured cells. In addition, perinatal delivery of AAV9-CTR rescued the pathophysiology and motor deficits in mice lacking TDP-43 in motor neurons. In our present study, we intracerebroventricularly (ICV) delivered AAVPhP.eB-CTR in adult mice lacking TDP-43 in forebrain, to evaluate the rescue efficacy of CTR at cellular, neuronal activity, and behavioral levels. We performed miniscope in vivo calcium imaging to monitor the neural calcium activity in awake, behaving mice and demonstrated that AAVPhP.eB-CTR delivered at 6-month-old age, efficiently rescued the aberrant calcium activity in the forebrain neurons lacking TDP-43. We also demonstrated that while TDP-43 forebrain depleted mice displayed remarkable cognitive behavior deficits as accessed by social behavior test and novel object recognition tests, CTR treatment efficiently rescued these behavioral deficits in TDP-43 forebrain depleted mice. These observations support utilizing AAVPhP.eB-CTR delivery system as an effective therapeutic strategy for the treatment of TDP-43 pathology linked AD and ADRD.

LOSS OF δ-CATENIN FUNCTION IMPAIRS SOCIAL BEHAVIOR

Regan L Roach1, Hadassah Mendez-Vazquez1, Rahmi Lee1, Matheus Sathler1, Madeleine C Moseley1,2, Rosaline A Danzman 1,2, Jessica P Roberts2, Libby Koch3, Seonil Kim 1,2

1Department of Biomedical Sciences

2Molecular, Cellular and Integrative Neurosciences Program

3Department of Biology , Colorado State University, Fort Collins, CO 80523

Normal social behavior is vital to many species for survival. Therefore, its underlying mechanisms have long been a focus for research. However, our comprehension of the physiological, cellular, and molecular mechanisms of social behavior is still limited. Moreover, there are several neurological/psychiatric disorders such as autism spectrum disorder (ASD) that have social dysfunction as a common characteristic. With this, expanded knowledge of the mechanisms mediating social behavior will improve our understanding of such diseases. Importantly, genetic alterations in the δ-catenin gene in humans are associated with severely affected ASD patients from multiple families. δ-catenin is a postsynaptic scaffolding protein and is important for AMPA receptor (AMPAR) GluA2 subunit localization and functions in several regions of the brain. Some ASD-associated δ-catenin mutations significantly reduce excitatory synapse density in cultured neurons, indicating a loss of δ-catenin function. Moreover, our preliminary findings demonstrate that δ-catenin knockout (KO) mice exhibit social dysfunction. We also show that synaptic AMPAR subunit GluA2 levels in cortical areas are significantly lower in δ-catenin KO mice than in their wild-type (WT) littermates. This indicates that a loss of δ-catenin functions induces social deficits via impairing AMPAR-mediated synaptic functions. Additional new data show that δ-catenin KO increases glutamatergic excitation in cultured cortical excitatory cells, whereas it is decreased in inhibitory cells. This suggests that δ-catenin deficiency likely disrupts the cellular E/I balance, which will in turn disturb the neural activity in the brain, particularly in the medial prefrontal cortex (mPFC) that is known to regulate social behavior. These joint findings lead us to hypothesize that normal social behavior relies on δ-catenin-mediated prefrontal activity at the cellular and network levels.

DO EXTRACELLULAR VESICLES FROM CANINES WITH CANINE COGNITIVE DYSFUNCTION CAUSE NEUROINFLAMMATION THROUGH ASTROCYTE ACTIVATION?

Sean Boland1, Amelia Hines1, Katarina Popichak2, Stephanie McGrath3 and Julie A. Moreno1

1Department of Environmental and Radiological Health Sciences

2Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523

3Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA

Extracellular vesicles (EVs) are shown to play a pivotal role in cell-cell signaling, cellular homeostasis, and waste deposition. However, they are beginning to be explored as possible routes for initiation and/or progression of neurodegenerative diseases, including Alzheimer’s disease (AD) and other neurodegenerative diseases. EVs cargo include but are not limited to proteins, nucleic acids (primarily mRNA and miRNA), and lipids. It has been shown that EVs from normal healthy cells contain cargo that possess neuroprotective properties. Conversely, aging, damaged, or cells under neuroinflammatory conditions contain cargo that can be damaging to neurons and can lead to neuronal death. Astrocytes, the cells that maintain neuronal health, can express pro-inflammatory (A1) or anti-inflammatory (A2) phenotypes. Previous studies show they become A1 activated from activated pro-inflammatory microglia secreting Tnf-a, IL-1a, and C1q. However, cellular pathways involving EVs may cause A1 activation, without the need of microglial assistance. A1 activation and neuroinflammation is characteristic of many neurodegenerative diseases, including AD. AD laboratory models are mainly transgenic rodent models that lack a natural occurring disease. However, aging canines have a naturally occurring disease that mimics AD, canine cognitive dysfunction (CCD) syndrome. We therefore are investigating if EVs from an aged canine can stimulate human astrocytes to a pro-inflammatory (A1) phenotype. Our hypothesis is that EVs from aged canines will contain cargo that leads to A1 activation of human astrocytes, compared to EVs from aged match non-CCD positive canines. Our read-outs for this will be quantitative real-time PCR and ELISAs to determine the degree of A1 phenotype.

NON-TOXIC NANOLIGOMERS™ TARGETING KEY NEUROINFLAMMATORY PATHWAYS ARE NEUROPROTECTIVE IN PRION DISEASE

SJ Risen1, S Boland1, S Sharma, G Weisman1, A Hines1, AJD Hay1, V Gilberto1, S McGrath2, A Chatterjee3, P Nagpal3, JA Moreno.

1Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA

2Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA

3Sachi Bioworks Inc., Colorado Technology Center, Louisville, CO, USA

Neuroinflammation is a key factor in the development of neurodegenerative diseases, including prion disease. There are currently no effective treatments to halt pathogenesis and progression, which includes accumulation of misfolded proteins, neuroinflammation, and cognitive/behavioral deficits followed by irreversible neuronal death. We hypothesized that downregulation of two key neuroinflammatory targets, NF-κB and NLRP3, would be neuroprotective. To test this, we utilized translational inhibiting Nanoligomers™ in a prion-diseased mouse to assess the impact on glial inflammation, behavioral/cognition deficits, and loss of neurons. Established by PK/PD studies, Nanoligomers™ are systemic but nontoxic to mice, at doses up to 5% of body weight. Nanoligomer™ treated mice displayed decreased numbers of glia, and improved behavior and cognitive tests. Critically, the Nanoligomer™ protected the brain from prion-induced spongiotic change, neuronal loss, and significantly increased life span, indicating that Nanoligomer™ inhibition of inflammatory pathways can prevent neuronal death and slow the progression of neurodegenerative diseases.

PAIN CAUSES HYPOTHERMIA IN A CIRCADIAN DEPENDENT MANNER DUE TO AN OVEL BRAINSTEM TO HYPOTHALAMUS CIRCUIT

AE Warfield1, P Gupta12, Q Jeffs2, MM Ruhmann1, WD Todd12

1Department of Zoology and Physiology, University of Wyoming

2Department of Zoology and Physiology Program in Neuroscience, University of Wyoming

Chronic pain has long been thought to influence circadian rhythms due to temporal clinical observations. However, little to no work has directly addressed this observation from the perspective of how chronic pain can disrupt rhythms. Using models of inflammatory and neuropathic pain we show that persistent pain causes prolonged hypothermia at predictable times of day, and a phase advance in body temperature (Tb) rhythms. We also characterize a novel circuit connecting prodynorphin neurons in the lateral parabrachial nucleus (LPBdyn) to the suprachiasmatic nucleus (SCN), the master pacemaker of circadian rhythms, and the subparaventricular zone (SPZ), the obligate relay of the SCN, using both retrograde and anterograde methods. These LPBdyn cells are activated by both types of pain as marked by c-Fos. There is also c-Fos activation in the SPZ following acute inflammatory pain but this increased level of activation is removed in double-knockout (DKO) mice lacking transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels, implying that pain driven SPZ activation is based in neural signaling from pain afferents, not peripheral inflammation. In these same DKO mice, inflammatory pain’s effect on rhythms is also abolished. To specifically implicate LPBdyn cells in this response, we performed a genetically targeted ablation, using a cre-dependent caspase in prodynorphin-cre (pdyn-cre) mice, along with spared nerve injury (SNI). Preliminary evidence shows that without LPBdyn cells the hypothermic and phase advance effects of the persistent pain response are abolished. Finally, we placed an excitatory channel (hM3) into LPBdyn cells and upon administration of its ligand, CNO, we see a hypothermic phenotype similar to the one following SNI. These data describe a mechanism for how persistent pain can disrupt body temperature rhythms, a previously unobserved phenotype, and a novel neural circuit responsible for this phenotype.

PREFRONTAL-MEDULLARY CIRCUIT INHIBITION DYSREGULATES PHYSIOLOGICAL RESPONSES TO METABOLIC STRESS

C Dearing1, C McCartney1, E Lukinic1, S Pace1, B Myers1.

1Department of Biomedical Sciences, Colorado State University

Chronic stress increases risk for metabolic disorders, including diabetes. However, the neurobiological basis of chronic stress impacts on glucose homeostasis have not been defined. The current study tested the hypothesis that the prefrontal infralimbic cortex (IL) – rostral ventrolateral medullary (RVLM) circuit is necessary to prevent glucose intolerance. To this end, female rats with Cre-dependent expression of tetanus toxin in RVLM-projecting IL neurons were chronically stressed for 2 weeks or remained unstressed. These rats were then acutely challenged with a fasted glucose tolerance test (GTT). Endocrine metabolic function was evaluated during GTT by measuring blood glucose, insulin, glucagon, and corticosterone, the primary rodent glucocorticoid. Following chronic stress, circuit-intact females had impaired glucoregulation characterized by decreased glucose clearance, elevated corticosterone, and insulin insensitivity. Inhibition of the IL-RVLM circuit also impaired glucose tolerance regardless of stress status. However, in unstressed animals with circuit inhibition, this impairment was characterized by elevated glucagon with no compensatory insulin response. Chronically stressed females with circuit inhibition showed broader autonomic dysregulation and disruption of counter-regulatory mechanisms involved in glucose homeostasis. Studies in males are ongoing but indicate that chronic stress exposure improves glucose clearance, while circuit inhibition during chronic stress leads to glucose intolerance. Collectively, these data indicate the IL-RVLM circuit is necessary for maintaining glucose homeostasis following chronic stress.

LEPTIN ADMINISTRATION DURING EARLY-FATTENING IN 13-LINED GROUND SQUIRRELS (ICTIDOMYS TRIDECEMLINEATUS) DISRUPTS BODY WEIGHT AND FOOD INTAKE IN A SEX-SPECIFIC MANNER.

CK Deal1, CT Williams!

1Department of Biology, Colorado State University

The control of feeding in mammalian hibernators is poorly understood but is known to involve several peripheral signals that access the brain to control appetitive and consummatory behaviors. Mammalian hibernators are characterized by a period of drastic fattening and feeding in the fall, becoming clinically obese, followed by a short period prior to hibernation immergence where animals cease feeding and lose body weight even if food is available. To improve our understanding of the mechanisms involved in this seasonal change in adiposity and appetite, we took advantage of the 13-lined ground squirrel (Ictidomys tridecemlineatus; 13LGS) as a model to manipulate potential controls of feeding. We utilized fattening 13LGS, implanting males and females with osmotic pumps containing either the meal cessation hormone, leptin, or saline for a period of 7 days, measuring daily food intake and bi-daily body weight. Serum was collected prior to, and after pump implantation periods to measure leptin levels and blood glucose. Our results uncover a unique, sex-specific response to leptin and its effect on body weight during fattening. Leptin treated animals gained significantly less body weight and consumed less food over the 7-day implant period than saline animals, in-line with the catabolic role of leptin. This effect is more pronounced in males, where leptin disrupted fattening more than in females; males consumed less food than females. Glucose levels increased upon leptin administration compared to saline only in males, suggesting impaired glucose tolerance. Serum leptin levels were highest in post leptin implant male and female animals, indicating osmotic pump effectiveness. How this satiating molecule converges on distinct brain regions that control energy balance in seasonal mammals is unclear; RNA-sequencing of these regions will uncover transcript level differences that may explain this enigmatic seasonal switch in feeding and body weight.

SEX DIFFERENCES IN PREFRONTAL-HYPOTHALAMIC CONTROL OF BEHAVIOR AND STRESS RESPONDING

D SCHAEUBLE, T WALLACE, B MYERS.

Department of Biomedical Sciences, Colorado State University

Depression and cardiovascular disease are both augmented by daily life stress. Yet, the biological mechanisms that translate psychological stress to affective and physiological outcomes are unknown. Previously, we demonstrated that stimulation of the stress-responsive ventral medial prefrontal cortex (vmPFC) has sexually divergent outcomes on behavior and physiology. Importantly, the vmPFC does not innervate brain regions directly responsible for initiating autonomic or neuroendocrine stress responses; thus, we hypothesize that intermediate synapses integrate cortical information to initiate stress responding. The posterior hypothalamus (PH) directly innervates stress effector regions and receives substantial innervation from the vmPFC. In the current study, we use circuit-specific approaches to examine whether vmPFC synapses in the PH coordinate stress responding. Here we examine the effects of optogenetic vmPFC-circuit stimulation in males and females on behavior and stress responding. Additionally, we use an intersectional genetic approach to knockdown synaptobrevin in PH-projecting vmPFC neurons. Our collective results show that male vmPFC-PH circuitry promotes positive valence and is both sufficient and necessary to reduce sympathetic-mediated stress responses. In females, the vmPFC-PH circuit is not necessary for stress responding but is sufficient to elevate neuroendocrine responses. Altogether, these data suggest cortical regulation of stress-reactivity is regulated by hypothalamic projections in a sex-specific manner.

CELLULAR COMPOSITION OF THE BRAINSTEM IN LIGHT OF AURICULAR VAGAL NERVE STIMULATION IN MICE.

EA Castellanos1, SA Tobet12.

1Department of Biomedical Sciences, Colorado State University

2Department of Biomedical Engineering, Colorado State University

The vagus nerve (VN) is a major component of the autonomic nervous system. It influences multiple components of visceral sensation, motor activity, and homeostasis. This nerve passes through the neck into the thorax and abdomen, and to the ear as its auricular branch. Electrical stimulation of the vagus auricular branch has been proposed as a non-invasive alternative neuromodulatory therapy for treating disorders, such as depression. Unlike the invasive method of cervical vagal nerve stimulation where surgery is done to stimulate the entire nerve, activation of the auricular branch stimulates a small portion of the VN. The cervical branch of the VN synapses through the nodose ganglia, directly going to the caudal portion of the nucleus of the solitary tract (NTS), while the auricular branch of the vagus synapses through the jugular ganglion where there are afferent inputs to the NTS. To begin mapping the chemoarchitecture of the likely auricular vagus targets in the brainstem, mice brains labeled with initial neuropeptide targets: calcitonin gene-related peptide (CGRP), Glutamate Decarboxylase 67 (GAD67), and Tyrosine Hydroxylase (TH). The distribution of each substance was seen in various brainstem nuclei involved in autonomic regulation, like the NTS. Ongoing studies are examining other neurochemical markers along with mapping cFOS activation as a function of auricular vagal nerve stimulation and the underlying chemoarchitecture. Therefore, this study reinforces previous literature of cellular components in the brainstem and will establish activity of the auricular branch of the vagus nerve.

PREFRONTAL PROJECTIONS TO THE VENTROLATERAL MEDULLA: IMPLICATIONS FOR FEMALE METABOLIC REGULATION

Ema Lukinic 1 Sebastian Pace 1 Carley Dearing 1 Carlie McCartney 1 Brent Myers

1Department of Biomedical Sciences, Colorado State University, Fort Collins CO

The acute stress response has helped humans develop the fight or flight response used in dangerous situations. However, when the effects of the stress response persist over time, it becomes chronic stress, and adverse health effects can arise (Lu et al. 2021). Chronic stress is a contributor to metabolic dysfunction, leading to issues with cardiovascular health, such as heart disease, hypertension, and heart attacks (Yaribeygai et al. 2017). Recent work has shown that female rats exhibit greater metabolic dysregulation following chronic stress (Dearing et al. 2021), but the neurobiological basis for these changes is unknown. The infralimbic cortex (IL) is a region in the prefrontal cortex in charge of cognitive stress appraisal, whereas the rostral ventrolateral medulla (RVLM) located in the brainstem influences sympathetic outflow to the periphery. We evaluated the necessity of the IL-RVLM circuit to mitigate the negative health outcomes that are associated with chronic stress. First, 6 female rats were injected with the tract tracer to evaluate the projections from the IL to the RVLM. Then to evaluate circuit function, retrograde-transported Cre was injected in the RVLM while two Cre-dependent viral constructs, tetanus toxic light chain (TelC) and green fluorescent protein (GFP), were injected in the IL of 21 female rats. These injections inhibited the IL-RVLM circuit projections and allowed us to evaluate how the rats respond to a metabolic stressor after two weeks of chronic variable stress (CVS). The results showed that neurons in the IL project to catecholaminergic neurons in the RVLM of female rats. Additionally, the stress-naive and inhibited circuit condition had increased corticosterone and decreased glucose clearance following acute glycemic stress. The CVS group showed that there was broad glucose dysregulation regardless of circuit inhibition. These results allow us to understand the neurologic basis of how stress impacts sex specific responses.

ANDROGEN TREATMENT OF MALE FGF8-DEFICIENT MICE ALTERS EXPRESSION OF NEUROTROPHIC GENE IN THE PREOPTIC AREA

Evan Egger1, Kelsey Sanders1, Pei-San Tsai1.

1Department of Integrative Physiology, University of Colorado Boulder

Gonadotropin-releasing hormone (GnRH) neurons are responsible for the development and regulation of reproductive function in all vertebrates. The disruption of the GnRH system can lead to severe reproductive impairment and infertility in both human and mice. Interestingly, a fraction of these reproductive impairment cases can be reversed with the chronic treatment of androgens, suggesting androgens may upregulate genes neurotrophic to GnRH neurons. The objective of this study was to use a candidate gene approach to examine if the expression of four genes previously found to be neurotrophic to GnRH neurons was altered by androgen treatment. These genes included a prototypic fibroblast growth factor ligand (FGF2), two FGF receptors (FGFR1 and FGFR3), and brain-derived neurotrophic factor (BDNF). Male wildtype (WT) and heterozygous FGF8 hypomorphic (FGF8 Het) mice were used. The latter harbored FGF8 deficiency which resulted in ~50% reduction in GnRH neurons. WT and FGF8 Het mice were injected subcutaneously with 500 μg testosterone enanthate (TE) every week from postnatal day (PN) 60-88 and sacrificed on PN95. Somatic, testicular, and seminal vesicle (SV) mass was measured, and the expression of four candidate genes was quantified in the preoptic area (POA) by quantitative PCR (qPCR). TE treatment significantly increased the growth trajectory of FGF8 Het mice, suppressed testicular mass, and enhanced SV mass. Surprisingly, TE significantly downregulated FGF2 expression but did not alter the expression of the remaining three genes. Several explanations could account for these observations, including the potential role of aromatization, the possible involvement of translational regulation, and the heterogeneity of cell populations in the POA. Together, these results suggested either the involvement of additional genes or neurotrophic mechanisms that may underlie the benefits of androgen treatment on the GnRH system. Supported by NIH R01 HD083260 to PST.

DREADD-ING STRESS: USING CHEMOGENETICS TO BYPASS VARIABILITY AND GO RIGHT TO THE SOURCE WITH CRF ACTIVATION

KR Montgomery1, MS Bridi2, LM Folts1, R Marx-Rattner2, HC Zierden2, TL Bale1.

1Department of Psychiatry, University of Colorado Anschutz Medical Campus

2Department of Pharmacology, University of Maryland School of Medicine

High lifetime stress is one of the strongest predictors of neuropsychiatric disease development. Additional environmental and biological factors, including sex, interact with chronic stress to increase risk. Many neuropsychiatric disorders also have sex-biased presentations, with males having increased incidence of autism and schizophrenia and females having elevated risk for PTSD, depression, and anxiety. Understanding how risk factors interact is essential for developing novel therapeutics. Non-homeostatic stressors are processed by limbic structures that ultimately converge onto the paraventricular nucleus of the hypothalamus (PVN) where activation of corticotropin-releasing factor (CRF) neurons initiates the hypothalamic-pituitary-adrenal (HPA) axis. Current rodent stress models rely on sensory exposures to activate the HPA response; however, the robustness of the stress response varies with the individual animal’s perception of the stressor. Here, we tested the hypothesis that chemogenetic activation of CRF neurons mimics the effects of sensory stressors while bypassing the variability of perception. We used the Gq-coupled DREADD receptor hM3Dq to activate CRF neurons by administering the DREADD ligand clozapine-N-oxide (CNO) to CRF-Cre+/hM3Dq+ (DREADD+) mice. To determine if chronic CNO administration induces a chronic stress-like state in DREADD+ animals, we administered CNO daily to male and female DREADD+ and DREADD- mice and monitored physiological changes and behavioral assessments indicative of a chronic stress state. We found that chronic CNO induced weight loss and thymus atrophy and elevated the HPA response to acute restraint stress in DREADD+ males but not females. In contrast, female, but not male, DREADD+ mice had significantly elevated freezing behavior following chronic CNO. These results demonstrate that chronic CRF activation has both sex and region-specific effects that may underlie different vulnerabilities to neuropsychiatric conditions.

PREFRONTAL-MEDULLARY CIRCUIT ACTIVATION ATTENUATES STRESS REACTIVITY

SA Pace1, E Lukinic1, T Wallace1, D Schaeuble1, J Moore1, B Myers1.

1Biomedical Sciences, Colorado State University

Organismal adaptation to stress relies on brainstem catecholaminergic neurons. Notably, catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) drive sympathetic activity to enable adaptations like corticosterone release and glycemic mobilization. However, it is unclear how brain regions involved in the cognitive appraisal of stress regulate the RVLM. Our prior studies found that the rodent ventromedial prefrontal cortex (vmPFC) integrates behavioral and physiological responses to stress. Thus, a potential vmPFC-to-RVLM connection would represent a crucial link between stress appraisal and sympathetic reactivity. Here, we investigated a direct vmPFC-to-RVLM circuit by utilizing anterograde and retrograde tract tracers. These tracing studies demonstrated that stress-reactive vmPFC neurons project to the RVLM in male and female rats. To understand the function of this vmPFC-to-RVLM circuit, we injected a viral vector coding for channelrhodopsin-2 (ChR2) in the vmPFC of males and females. Next, a fiber optic cannula was implanted dorsal to the RVLM to evoke vmPFC synaptic glutamate release during restraint stress with blood sampled to determine stress reactivity. Rats expressing ChR2 on vmPFC terminals had decreased corticosterone responses to stress relative to control rats in both sexes (males, p < 0.01; females, p < 0.05). Thus, both male and female rats have a direct circuit from the vmPFC to the RVLM capable of limiting glucocorticoid stress responses. Post-experiment tissue analysis revealed vmPFC-to-RVLM stimulation preferentially activated non-catecholaminergic RVLM neurons in both sexes (males, p < 0.05; females, p < 0.01). Moreover, vmPFC appositions were identified onto inhibitory RVLM neurons in both sexes. Therefore, vmPFC projections may activate RVLM inhibitory cells to limit stress reactivity. Ultimately, excitatory/inhibitory balance at vmPFC synapses in the RVLM may be critical for the health consequences of stress.

Supported by F31 HL162571 to S.A. Pace and R01 HL150559 to B. Myers

TRANSCRIPTOMIC ANALYSIS OF THE PREOPTIC AREA IN FGF-DEFICIENT MICE HOUSED WITH SAME SEX OR OPPOSITE SEX PARTNERS

TN Akonom1, MA Allen2, PS Tsai1.

1Department of Integrative Physiology, University of Colorado at Boulder

2Biofronteirs Institute, University of Colorado at Boulder

The GnRH neuronal population is as an essential part of the reproductive axis in all vertebrates. These neurons rely on fibroblast growth factor (FGF) signaling for their growth and maturation. Transgenic mice with a functional disruption of FGF receptors (dnFGFR mice) exhibit a postnatal decline in GnRH neuron count, delayed puberty, impaired fertility, and early reproductive senescence. However, this decline can be reversed by housing with an opposite-sex (OS) partner. The goal of this study is to identify, using RNA-Sequencing (RNA-Seq), differentially expressed genes that could participate in the rescue of GnRH neurons. Male control and dnFGFR mice were paired with a same-sex (SS) or OS partner after weaning on postnatal (PN) day 20. At PN140 and PN320, preceding and concurrent with the observed increase in GnRH neuron count, animals were sacrificed and the preoptic area (POA) was isolated for total RNA extraction. Total RNA from the POA including both GnRH neurons and neighboring cells was subject to library preparation and high-throughput sequencing with paired end read length of 150 bp. A total of ~40 million reads were generated per sample, and ~80% were mapped to the GRCm38 RefSeq genome. Differential gene expression analysis showed a genotype-dependent response in that only dnFGFR mice at PN320 exhibited differentially expressed genes (adjusted p < 0.1) in response to OS housing. A total of 22 genes were upregulated and 50 downregulated between dnFGFR SS- and OS-housed mice. Gene Ontology (GO) enrichment analysis did not capture pathways related to these genes, but gene set enrichment analysis (GSEA) identified biological processes related to cytoplasmic translation and specific forms of cellular metabolism. These data show that the effect of OS housing on dnFGFR mice is measurable on a transcriptomic level and suggest promising targets for future research. Supported by NIH R01 HD083260 to PST.