DecodingBrainEnergyMetabolisminadultsatriskforAlzheimer’sdiseaseafterneurotherapeuticinterventionsSpecific Aims Mild Cognitive Impairment (MCI) is a clinical transitory stage from normal aging to Alzheimer’s disease (AD) in which individuals have memory complaints but they are functionally independent and not demented[P]. The pathophysiological changes in the brain of AD patients start at least a decade before the presentation of the symptoms. Hence, research is focused on understanding the molecular changes that trigger the pathological changes in the brain in order to develop appropriate intervention modality for MCI and AD. One such area is the field of neuroenergetics that focuses on brain energy metabolism (BEM) by visualizing the molecular level energy and membrane metabolites changes in the in Vivo human brain using 31P (phosphorous) magnetic resonance spectroscopy (MRS) at ultrahigh magnetic field 7Tesla.The high magnetic field addresses the potential resolution limitations of MRS studies.In this research proposal, we will investigate the differences in the ratio of brain high-energy phosphate metabolites like adenosine triphosphate (ATP)/phosphocreatine (Pcr) (ATP/PCr); intracellular inorganic phosphate (Pi)/ phosphocreatine (Pcr) (Pi/PCr); and energy phospholipid like phosphomonoesters (PME)/phosphodiesterase (PDE) (PME/PDE) on the performance of combined executive functions and memory in two groups: MCI and cognitively normal adults. We will also investigate the effect of select genes Apo lipoprotein E ε4 (APOEε4) and ATP-binding cassette A7(ABCA7) on brain energy and membrane metabolites in MCI.Aim1: Brain Metabolic Differentiation between two groups (Mild Cognitive Impairment versus Cognitively normal adults) Investigate the differences in the ratios of brain energy (ATP/PCr; Pi/PCr) and phospholipids (PME/PDE) metabolites in mild cognitive impairment and cognitively normal adults using volume coil 31P MRS at 7Tesla. 1.1 The ratios of energy metabolites (ATP/PCr and Pi/PCr) will be increased in mild cognitive impairment when

compared to cognitively normal adults. 1.2 The ratio of energy phospholipid metabolites (PME/PDE) will be decreased in mild cognitive impairment

when compared to cognitively normal adults. Aim2: Relationship of Brain Metabolic response and Cognitive domains to neurotherapeutic interventions Investigate the relationship between the changes in the ratio of brain energy (ATP/PCr; Pi/PCr) and phospholipids (PME/PDE) metabolites with changes in the performance of complex executive functions and memory after gist-based cognitive reasoning training in mild cognitive impairment using volume coil 31P MRS at 7Tesla. 2.1 The changes in the ratios of energy metabolites (ATP/PCr and Pi/PCr ) will be negatively correlated with

the changes in the performance of complex executive functions and memory after gist-based cognitive reasoning training in mild cognitive impairment

2.2 The changes in the ratio of phospholipids metabolites (PME/PDE) will be positively correlated with the changes in the performance of complex executive functions and memory after gist-based cognitive reasoning training in mild cognitive impairment.

Aim3: Association of genetic variants with Brain Metabolic Response in Mild Cognitive Impairment Determine the association of genetic variants of the APOE and ABCA7 and the ratios of brain energy (ATP/PCr; Pi/PCr) and phospholipids (PME/PDE) metabolites in MCI. 3.1The presence of the ε4 allele of the human APOE gene will be associated with increase in the ratios of energy metabolites (ATP/PCr, Pi/PCr) when compared to the absence ε4 allele of the human APOE gene in mild cognitive impairment. 3.2The presence of the ε4 allele of the human APOE gene will be associated with decrease in the ratios of energy phospholipids (PME/PDE) metabolites when compared to the absence ε4 allele of the human APOE gene in mild cognitive impairment, 3.3 The loss of function ABCA7 gene variants rs3764650 and/or rs4147929 will be associated with increase in the ratios of energy metabolites (ATP/PCr, Pi/PCr) when compared to the presence of the either gene variant of ABCA7 in MCI. 3.4 The loss of function ABCA7 gene variants rs3764650 and/or rs4147929 will be associated with decrease in the ratios of energy phospholipids (PME/PDE) metabolites when compared to the presence of the either gene variant of ABCA7 in MCI.

DecodingBrainEnergyMetabolisminadultsatriskforAlzheimer’sdiseaseafterneurotherapeuticinterventions

Aim 1:

Independent variable(s):

Cog status (normal vs. MCI)

Dependent variable(s):

Energy metabolites [EM] (ATP/PCr; Pi/PCr); Phospholipid metabolites [PM] (PME/PDE)

Hypothesis:

EM up, PM down in MCI

Aim 2:

Independent variable(s):

Gist-based cognitive reasoning training vs. ?

Dependent variable(s):

EM; PM; performance of complex executive functions (?); memory (?)

Hypothesis:

EM neg. correlated w. exec. func., memory after training

PM pos. correlated w. exec. func., memory after training

Aim3:

Independent variable(s):

Presence of absence of APO ε4; ABCA7 (rs3764650 or 4147929)

Dependent variable(s):

Energy metabolites [EM] (ATP/PCr; Pi/PCr); Phospholipid metabolites [PM] (PME/PDE)

Hypothesis:

Correlation of APO ε4 w. EM, neg. correlation w. PM

Neg. correlation of ABCA7 w. EM, correlation w. PM

Diffusion tensor imaging (DTI) has been used to measure white matter microstructure in vivo. White matter integrity has been shown to decrease with age (see Sullivan & Pfefferbaum, 2006, for review). Training in juggling (Scholz et al., 2009), meditation (Tang et al., 2010), working memory (Takeuchi et al., 2010), neurofeedback (Ghaziri et al., 2013), and reasoning (Mackey et al., 2012) have all shown to improve white matter integrity in younger adults. Training in memory (Engvig et al., 2012) and various cognitive tasks (Lövdén et al., 2010) has been shown to improve white matter integrity in older adults.

Whereas the previously mentioned studies (like many others using lab-based

cognitive tasks) illustrate a targeted approach to cognitive training, video games provide a means by which users must simultaneously utilize multiple cognitive processes. As such, video games are currently being explored as a potential tool to improve cognition in older adults (see Toril et al., 2014 for review).

Of the many types of video games, real-time strategy (RTS) games may hold

particular promise due to their reliance on frontal-based executive functioning. RTS training has been shown to improve attentional control in both younger (Glass et al., 2013) and older (Basak et al., 2008) adults.

The current study employs two groups of older adult participants. One group (N = 22,

11 females, Mage = 63) was trained for 20 hours using an RTS game called Rise of Nations. The other group (N = 18, 12 females, Mage = 67) completed 20 hours of semantic knowledge training, which comprised of word searches, crossword puzzles, word ladders, and hub words. Participants completed extensive cognitive batteries both before and after training. DTI scans were collected for each participant before and after training as well.

The current study seeks to:

1. Use white matter tractography to estimate the integrity of the uncinate, cingulum,

and fornix in older adults before and after 20 hours of RTS video game training.

2. Compare white matter integrity at time 2 (post training) with white matter integrity at time 1 (pre training) to access any training related improvements.

3. Compare any changes in white matter integrity between the video game training

group and an age-matched active control group that completed 20 hours of semantic knowledge training.

4. Correlate any improvements in white matter integrity (post-pre) with improvements on behavioral tasks of cognition, particularly in tasks relating to attentional control (working memory, task-switching, inhibition, etc.).

Aim 1: Independent variable(s): all aging Rise of Nations vs. semantic knowledge training; pre- vs. post-training Dependent variable(s): integrity (?) of uncinate, cingulum, fornix Hypothesis: Post-training improvement superior for Rise of Nations (??) Aim 2: Independent variable(s): Same aim… Dependent variable(s): Hypothesis: Aim3: Independent variable(s): Same aim… Dependent variable(s): Hypothesis: Aim4: Independent variable(s): Rise of Nations vs. semantic knowledge training; pre- vs. post-training Dependent variable(s): integrity (?) of uncinate, cingulum, fornix attentional control task performance (working memory, task-

switching, inhibition, etc. (?)) Hypothesis: Integrity correlated w. task performance (?) Improved by training

Over the past few decades, the combination of modern lifestyle and high fat enriched diets, the prevalence of metabolic disorders like obesity and type2 diabetes in on the rise. Cognitive deficits have been clinically observed in humans suffering from obesity and diabetes, and intensely studied in animal models fed high fat diet (HFD) from weaning. However, sex-differences are rarely systematically analyzed and compared. Previous studies from our lab with an outbred Long Evans (LE) rat HFD model showed impaired spatial memory in both sexes, correlating with significantly reduced intrinsic excitability of hippocampal CA1 pyramidal neurons (enhanced post-burst AHPs). Notable sex-differences in hormone signaling were observed, wherein HFD induced clinically relevant symptoms of type 2 diabetes in males (obesity, loss of blood glucose control, elevated insulin), but not in females (normal weight and blood glucose control, reduced insulin). Sex-differences were also found in insulin-dependent intrinsic excitability of CA1 neurons; HFD male neurons lost all insulin sensitivity while HFD female neurons had increased sensitivity. Based on the above findings, we hypothesize that HFD sex-dependently alters insulin signaling in CA1 region of the hippocampus. We thus arrive upon following aims: Aim1: What is the effect of HFD on the proteins involved in medium and slow components of AHP? Our preliminary western blot data shows significant increase in SK2 protein, a channel underlying medium AHP (mAHP) and a significant increase in hippocalcin protein, the calcium sensor associated with the channel underlying slow AHP (sAHP), in CA1 region of HFD fed rats as compared to the age-matched control diet (CD) fed rats. These results are consistent with the intracellular recording reported by our lab previously. To address this question further, we will look at the phosphorylation levels of SK2 channel using western blotting or immunoprecipitation technique. Secondly, we will analyze the translocation of SK2 protein from the cytoplasm to the cell membrane by separating cytoplasm and cell membrane using differential density centrifugation. Lastly, we will confirm the increase in hippocalcin by using immunohistochemistry technique. Aim2: Does the HFD sex-dependently alter the insulin signaling pathway in CA1 region of the hippocampus in rats? Dysregulation of insulin signaling pathway can impact the process of learning and memory. Our preliminary results show that only HFD female CA1 region have an increase in insulin receptor (IR), phosphorylated insulin receptor substrate (p-IRS) and phosphorylated Akt (p-Akt) protein as compared to age-matched CD males, CD females and HFD males. HFD male CA1 regions shows decrease in phosphorylated IRS and phosphorylated Akt. Further work needs to be done to measure expression of insulin like growth factor receptor 1 (IGF-R1) protein, and the phosphorylated form of IR and IGF-R1 through western blot. Next, we aim to perform immunofluorescent co-labelling of p-IRS or p-Akt along with pyramidal cell marker. Lastly, we will determine the translocation of glucose transporter (GLUT1/3/4) from cytoplasm to neuronal cell membrane.

Aim 1: Independent variable(s): Diet: control vs. HFD Sex (?): male vs. female Western vs. immunoprecipitation vs. immunohistochemistry (muddled) Dependent variable(s): mAHP: SK2 total phosphorylated cytoplasmic vs. membrane

sAHP: hippocalcin Hypothesis: HFD upregulates total SK2, hippocalcin HFD up/down(?) regulates phosphorylated SK2 immunoprecipation superior to westerns? IFC confirms westerns? Aim 2: Independent variable(s): Diet: control vs. HFD Sex: male vs. female Dependent variable(s): Insulin-signaling basal IR IRS AKt IGF-R1 Insulin-signaling phosphorylated IR IRS AKt IGF-R1 GLUT1/3/4 translocation cytosolic vs. membrane Hypothesis: Sex Diet Interaction

SpecificAimsRachelWilhelm

Neurobiologicalmarkersofplasticityandintrinsicexcitabilityinthehippocampusalterwithage,asdoesperformanceonspatialmemorytasks,butthosemarkersneedtobecharacterizedinrelationtobehavioraltaskssothattherapeutictargetscanbemore precisely tuned. Unreinforced exploratory behavior of young rats in simple mazes reflect hippocampal-dependentspontaneousalternationbehavior(SAB)invisitstomazearms,meaningthatratsdistributetheirmovementaroundallregionsofanovelenvironmentinsteadofrepeatedlyreturningtothemostrecentlyvisitedarm.TheuseofspatiallearningandmemorystrategiessuchasSABsupportstheformationofacognitivemap,whichmaximizesthediscoveryofpotentialrewards.Thisalsomakes for a quick assessment of spatialmemory in aging, with behavioral impairments thatmay reflect altered plasticitymechanisms. Learning and memory induce plasticity, increasing intrinsic excitability (reducing Ca2+-dependentafterhyperpolarizations[AHPs])inCA1pyramidalneuronsinthehippocampus.Neuronsfromsenescentanimalswithimpairedmemoryshowenhancedlong-durationslowAHPs(sAHPs).TheionchannelsmediatingsAHPsareunknown,butthecalciumbindingproteinhippocalcinhasbeenshowntogatesAHPsinrodentCA1.Synapticplasticityduringmemoryconsolidationalsorapidly upregulates activity-dependent expression of Arc/Arg3.1 (activity-regulated cytoskeletal gene). Arc expression iscorrelatedwithenhancedintrinsicexcitability(reducedAHPs)andlongtermpotentiation(LTP)inratCA1neurons.Theroleoftheseplasticitymechanismsinimpairedspatialmemoryinagingwillbeinvestigated.

SpecificAim1Determinetheeffectofageand/orSABonactivity-dependentproteinexpressionofArcandhippocalcin inCA1andCA3pyramidalneurons.CrossbreedsofFisher-344xBrownNorway(FBN)ratswillbeassignedtocross-sectionalagecohortsofyoung (4-5mo),middle (14-15mo), andold (24-25mo), and allowed to freely explore a 4-armplusmaze for 12minutes.Spontaneousalternations(4differentarmentriesoutofeachsetof5)andnumberofperseverations(morethan1repeatedarmentryineachsetof5)willberecordedtocalculatescoresfor%alternationsand%perseverations.Preliminarydatarevealsthat variance in SABperformance (% alternations) increases significantlywith age. Animals performing > 2 SD’s below thepercentageofalternationsofyounganimalswillbeoperationallydefinedasSABimpaired,whileanimalsthatperformwithin1SDofyoungratswillbedefinedasSABunimpaired.Ratswillbeanesthetizedandsacrificed45minaftertheSABtask,andbrainswill be flash frozen with ice cold isopentane (2-methylbutane) prior to sectioning in the cryostat. Western blot andimmunofluorescent(IF)analysisoftissuesampleswillbeusedtoquantifyArcandhippocalcinproteinexpression.HippocalcinexpressioninCA1ishypothesizedincreasewithage,whileArcishypothesizedtodecrease.SABimpairedanimalsareexpectedto have higher hippocalcin expression and lower Arc protein in CA1 compared to controls and animals who exhibit noimpairmentinSAB.Additionally,itishypothesizethathippocalcinexpressioninCA3ofmiddleandoldratswillnotsignificantlydifferfromCA3hippocalcinexpressioninyounganimalsregardlessofSABperformance.Weexpecttoobserveanage-related,butnotbehavior-dependent,increaseinArcexpressioninCA3neurons,andsignificantlyhigherArcexpressioninCA3comparedtoCA1inagedanimals.

SpecificAim2Determinetheeffectofageand/orSABonLTPandAHPsinCA1andCA3pyramidalneurons.Freshbrainslicesincubatedinartificialcerebralspinalfluid(aCSF)willbepreparedfromanimalsanesthetizedandsacrificed45minaftertheSABtaskforeachoftheagedcohorts.PipetteswillbefilledwithaCSFandLTPwillbeinducedwithhighfrequencystimulationinthestratumradiatum of CA1 and CA3 neurons. Amplitude of the dendritic field excitatory postsynaptic potentials (fEPSPs) after LTPcomparedtobaselinewillbemeasured.IntracellularsharpmicroelectrodesfilledwithKClwillbeusedtocharacterizeduration,amplitude,andintegratedareaofAHPsinthestratumpyramidaleofCA1andCA3neurons.ItishypothesizedthatCA1neuronsofoldandmiddlecohortswithimpairedSABwillhavegreaterAHPsandlessLTPthanneuronsfromunimpairedandyoungerrats.Anage,butnotSABrelatedincreaseinLTP,andsmallerAHPisexpectedinCA3pyramidalneurons.

Aim 1:

Independent variable(s): Age (mo): 4, 14, 24 Region: CA1 vs. CA3 Method (?): western vs. IF Dependent variable(s): SAB performance Arc expression mAHP: (?) sAHP: Hippocalcin expression Hypothesis: CA1: increased age, inc. in hippocalcin, dec. in Arc decreased SAB performance, inc. in hippocalcin, dec. in Arc CA3: no age-related effect on hippocalcin age-related effect on Arc regional: CA3 Arc > CA1 Arc in aging (?) Aim 2: Independent variable(s): Age (mo): 4, 14, 24 Region: CA1 vs. CA3 Method: LTP vs. AHP Dependent variable(s): fEPSP slope mAHP amplitude, duration sAHP amplitude, duration Hypothesis: CA1: neg. correlation between SAB performance and mAHP/sAHP measures pos. correlation between SAB performance and fEPSP slope CA3: ? smaller AHP in CA3 than CA1 ?

Sleep plays an important role in establishing optimal health, such as reducing the risk of cardiovascular disease (Wolk, Gami, Garcia-Touchard, & Somers, 2005). Unfortunately, approximately one-third of the adult population frequently complains of poor sleep quality (i.e., difficulties falling or staying asleep) (Mellinger, Balter, Uhlenhuth, 1985). In the context of romantic relationships, the majority of married and cohabiting couples share a bed (Troxel, 2010). Relationship difficulties (e.g., conflict) often co-occur with poor sleep quality, while feelings of relationship security are associated with improved sleep quality (Troxel, 2010). An optimal sleep environment is one that increases feelings of security and decreases physiological arousal (i.e., anxiety) and hypervigilance (i.e., the acute awareness of surroundings) (Dahl, 1996). Within the context of romantic relationships, feelings of security are derived from relationships in which partners are close and responsive to each other’s needs (Collins & Feeney, 2004) and are negatively associated with anxiety about the relationship (Murray et al., 2005).

Self-disclosure, or the sharing of personal thoughts and feelings, facilitates closeness (Reis & Shaver, 1998), which can further promote feelings of security and improve sleep quality. Preliminary evidence from a daily diary study of married couples suggested that sleep quality (i.e., waking and sleep duration) improved for wives on days they reported disclosing daily hassles (i.e., work stress) more than their average, while higher overall levels of husband’s disclosure were only associated with their decreased waking. (Kane, Slatcher, Reynolds, Repetti, & Robles, 2014). Thus, the ways in which disclosure affects sleep may differ by gender. Disclosers may also shape their partners’ sleep quality. Indeed, husbands took longer to fall asleep when wives disclosed daily hassles more than average (Kane et al., 2014). Further, it is unknown whether self-disclosures cause improvements in subjective (e.g., self-report) and objective (e.g., wrist actigraphy) measures of sleep quality, thus warranting experimental testing in the laboratory. Additionally, existing research has yet to identify whether variation in disclosure content (e.g., daily stressors versus past romantic experiences) may exert differential effects on sleep quality. Self-reported pilot data from a sample of undergraduate students suggested that talking about past partners (e.g., attacking an ex-partner’s character) was negatively correlated with feelings of trust toward the current partner for both men and women (Adelson, Dixon, & Goodnight, unpublished). A relational environment low in trust and security may lead to heightened anxiety (Perrone-McGovern et al., 2014) that is directly counter to a conducive sleep environment.

Taken together, feelings of relationship security may serve as an important mechanism between the disclosure-to-sleep quality association. Furthermore, disclosures may affect sleep quality differently as a function of disclosure type, gender, and role of disclosure target or disclosure recipient. Romantic couples (married and cohabiting) will first complete a background felt security measure in the lab. Couples members will then be randomly assigned to a disclosure type condition in the lab and then later assessed for the quality of their sleep at home that night via self-report and wrist actigraphy. The specific aims of the present investigation are:

1. To test relationship security as a mediator explaining the positive relation between disclosing a daily hassle to a romantic partner and sleep quality and a negative relation between disclosing a past partner experience to own sleep quality.

2. To examine whether the female partner’s disclosure of a daily hassle or past partner experience impairs the male partner’s sleep quality the same night.

3. To examine whether the male partner’s disclosure of a daily hassle improves the female partner’s sleep quality that night, but his disclosure of a past partner experience impairs the female partner’s sleep quality that night.

Aim 1:

Independent variable(s): Relationship felt security (?): low to high (?) Disclosure type: Daily hassle vs. Past partner experience Quality modality: Self-report vs. actigraphy Dependent variable(s): Sleep quality (?): time to sleep onset, hours (?), # of arousals (?), etc.? Hypothesis: Sleep quality pos. correlated w. felt security for hassles disclosure Sleep quality neg. correlated w. felt security for past partner disclosure Aim 2: Independent variable(s): Sex of discloser: female Disclosure type: Daily hassle vs. Past partner experience Quality modality: Self-report vs. actigraphy Dependent variable(s): Sleep quality of male (?): time to sleep onset, hours (?), # of arousals (?), etc.? Hypothesis: Daily hassle disclosure impairs sleep quality Past partner disclosure improves sleep quality Aim3: Independent variable(s): Sex of discloser: male Disclosure type: Daily hassle vs. Past partner experience Quality modality: Self-report vs. actigraphy Dependent variable(s): Sleep quality of female (?): time to sleep onset, hours (?), # of arousals (?), etc.? Hypothesis: Daily hassle disclosure improves sleep quality Past partner disclosure impairs sleep quality

Specific Aims The goal of this study is to better understand how preschoolers learn about the domain of biology in informal learning contexts (e.g., home, museums). Specifically, this study serves to examine what types of explanations preschoolers are willing to accept about biological events from their parents (e.g., explanations that are dismissive, explanations that answer the question, or explanations that repeat information from the child’s question but offer no new information). In general, beliefs about biological concepts are greatly influenced by instruction and during early childhood much of this instruction is from parents since it is with parents that children spend most of their time (Crowley et al., 2001; Gelman & Legare, 2011; Hatano & Inagaki, 1994, 1997; Jipson & Callanan, 2003). Thus, this work would help us understand how parents choose to talk to their kids about biology during early childhood and inform researchers about how we could eventually aid parents in discussing science with their young children. Aim 1: Determine what types of explanations parents offer to their preschool-age child about biological phenomena during a set of activities designed to encourage biological thinking. Specifically, we want to code what strategies parents use to answer questions posed by their children. We want to understand if parents offer their children explanations about biology in ways that are accurate, coherent, and relevant to the biological activity. Additionally, we want to determine how children then respond to the explanations they are given by their parents (e.g., do children ask follow-up questions, provide their own explanations). Aim 2: Evaluate how parental beliefs about science, religion, and learning relates to the quality (i.e., accuracy, coherence, and relevance) of explanations they provide their preschooler. Additionally, we want to examine how parent’s beliefs of their child’s understanding of biology and science more generally influences the way they talk to their child about biology. Aim 3: Evaluate preschoolers desire to seek out more information about the biological phenomena they and their parents discussed during the science activities. This will allow us to determine if children are interested in learning more about particular biological phenomena they heard about and how that may be influenced by the types of scaffolding and explanations parents offered to their child about that specific phenomena.

Aim 1: Independent variable(s): Explanation coding re. biology (?): accuracy, coherence, relevant Dependent variable(s): Children’s response to explanation: follow-up questions, own explanations Hypothesis: ? Aim 2: Independent variable(s): Parental beliefs about science, religion, learning (?) Parent’s beliefs of child’s understanding of biology and science Dependent variable(s): Explanation coding re. biology (?): accuracy, coherence, relevant Hypothesis: ? Aim 3: Independent variable(s): Explanation coding re. biology (?): accuracy, coherence, relevant Scaffolding (?) Dependent variable(s): Children’s desire to seek out more information (?) Hypothesis: ?

Our lab recently published data that are inconsistent with suggestions that noise-induced cochlear synaptopathy - a permanent reduction in afferent cochlear synapses and corresponding Wave-I amplitude of the auditory brainstem response (ABR) - could be observed in the human auditory system following common, recreational noise exposures, such as attending bars, nightclubs, or concerts. These translational suggestions stem from animal studies by Kujawa and Liberman (2009, 2015) which demonstrated a massive 40 dB temporary threshold shift (TTS) reduction in hearing sensitivity accompanied by evidence of permanent cochlear synaptopathy, induced by a 130 dB narrow-band frequency noise exposure in rodents. Because the reduction in hearing sensitivity recovered to normal after 1 week, the audiogram (a standard clinical measure of hearing sensitivity) was determined to be “normal”, and did not detect the permanent synaptopathic changes, which require advanced electrophysiologic evaluation (e.g., ABR). The warranted concerns are as follows: 1. If noise-induced cochlear synaptopathy occurs in humans, it will be undetected by the standard test battery for a clinical evaluation of hearing loss; 2. If noise-induced cochlear synaptopathy occurs in humans, as detected by advanced electrophysiologic evaluation, will any real-world symptoms emerge as a function of these changes, or will cochlear synaptopathy be an asymptomatic phenomenon? There is speculation that this “hidden hearing loss” could explain what we sometimes clinically observe as abnormal difficulty understanding speech in noisy environments, despite normal audiometric thresholds (Kujawa and Liberman 2009, 2015). Our recently published study does not reveal electrophysiologic nor functional evidence (e.g., difficulty understanding speech in noisy environments) of permanent noise-induced cochlear synaptopathy in human subjects following loud, recreational noise exposures at bars, nightclubs, and concerts. Although the noise levels of these events were frequently in excess of the 100% noise dose safety regulation set forth by the Occupational Safety and Health Administration (OSHA), our subjects’ TTSs were no where near the degree of TTS that was necessary to induce cochlear synaptopathy in rodents (40 dB); however, firearm discharge, often associated with massive TTS, could provide a more relatable noise exposure to that of the animal model, and a population of humans that are more likely to be at-risk for auditory neural injury from recreational noise exposure. Human subjects will be recruited from the UT Dallas campus in Richardson, TX, and will be required to possess a current firearm license. Firearm discharge will be performed at the DFW Gun Range and Academy. All audiologic testing will be performed in our laboratory in the Callier Center for Communication Disorders at the downtown Dallas location. Specific Aim 1: In human subjects with clinically normal hearing (thresholds better than 25 dB), we will quantify changes in audiometric threshold sensitivity, ABR Wave-I amplitude, and distortion product otoacoustic emissions (a test of cochlear cell integrity per response amplitude) before, 24 hours after, and 1 week after 20 rounds of firearm discharge. Specific Aim 2: In human subjects with clinically normal hearing (thresholds better than 25 dB), we will quantify changes in the ability to accurately identify words in the presence of background noise before, 24 hours after, and 1 week after 20 rounds of firearm discharge, as evaluated by the clinical Words-in-Noise test. Specific Aim 3: In human subjects with clinically normal hearing (thresholds better than 25 dB), we will quantify ABR Wave-I amplitude as a function of annual noise exposure history, as evaluated by the Noise Exposure Questionnaire (NEQ) survey.

Aim 1: Independent variable(s): 20 rounds down range time: pre-, 1 d, 1 wk after Dependent variable(s): audiometric threshold sensitivity ABR wave-1 amplitude Distortion product optoacoustic emission amplitude Hypothesis: impairments from baseline at 1 d, gone at 1 wk (?) Aim 2: Independent variable(s): 20 rounds down range time: pre-, 1 d, 1 wk after Dependent variable(s): words-in-noise test performance Hypothesis: impairments from baseline at 1 d, gone at 1 wk (?) Aim3: Independent variable(s): Annual noise exposure history: NEQ survey Dependent variable(s): ABR wave-1 amplitude Hypothesis: Correlation between noise exposure and ABR wave-1 amplitude (?)

Aim 1: To verify potential sex-dependent reversal of cognitive deficit, caused by a high-fat diet (HFD), in response to intranasal application of insulin. Male and female Long Evans (LE) rats will be fed a HFD for 15 weeks and will receive either insulin or saline treatment prior to a spatial memory behavioral test, Spontaneous Alternation Task (SAT). We hypothesize that, due to the unaltered sensitivity to insulin by female CA1 pyramidal neurons after a diet induced cognitive impairment, HFD females, but not males, will improve their performance on the task if given intranasal insulin application.

Aim 2: To verify possible peripheral influence of the intranasal application of insulin on the blood. LE rats fed a HFD for 15 weeks will be fasted for 12 hours and blood glucose levels accessed through Insulin Tolerance Test (ITT) following insulin intranasal application. We hypothesize that the insulin application method will not influence peripheral glucose levels, targeting exclusively the central nervous system.

Aim 3: To verify sex differences in levels of insulin in both plasma samples and cerebrospinal fluid (CSF) in response to diet and treatment. Insulin will be quantified using ELISA kits to determine metabolic effects of the diet and the treatment in both sexes. We hypothesize that insulin levels in the central nervous system will demonstrate the same sex-dependent metabolic dysfunction shown in the peripheral system, with higher concentrations of insulin in CSF of HFD males and lower levels in CSF of HFD females compared to control. In addition, rats given intranasal insulin are expected to have higher concentrations of the hormone than their counterparts in treatment.

Aim 4: To verify differences in levels of corticosterone in both plasma samples and cerebrospinal fluid (CSF) in response to diet and treatment. Corticosterone will be quantified using ELISA kits to determine stress related effects of the diet and the treatment. We hypothesize that corticosterone levels in the central nervous system will reflect the diet dependent metabolic dysfunction shown in the peripheral system, with higher concentrations of corticosterone in CSF of HFD rats. In addition, rats given intranasal insulin are expected to have higher concentrations of the stress hormone than their counterparts in treatment.

Aim 1: Independent variable(s): Diet: control vs. HFD Sex: male vs. female Treatment: saline vs. insulin Dependent variable(s): SAT performance Hypothesis: HFD males and females impaired on SAT compared to controls HFD male SAT performance not improved by insulin or saline HFD female SAT performance improved by insulin by not saline Aim 2: Independent variable(s): Diet: control vs. HFD Sex: male vs. female Treatment: saline vs. insulin Time: ? Dependent variable(s): blood glucose Hypothesis: ? Aim3: Independent variable(s): Diet: control vs. HFD Sex: male vs. female Treatment: saline vs. insulin Locale: peripheral vs. central Dependent variable(s): insulin: ELISA value Hypothesis: peripheral insulin elevated in HFD males, decreased in HFD females central insulin elevated in HFD males, decreased in HFD females Aim4: Independent variable(s): Diet: control vs. HFD Sex: male vs. female Treatment: saline vs. insulin Locale: peripheral vs. central Dependent variable(s): corticosterone: ELISA value Hypothesis: HFD elevates corticosterone in males & females insulin elevates corticosterone in males & females (?) HFD/insulin treatment vs. control/insulin treatment: (?)