To understand the function and origins of sleep, sleep needs to be studied across many different species. Although it is well conserved throughout mammals, 95% of papers are restricted to just three species, Homo sapiens, Mus musculus, and Rattus norvegicus. We aimed to characterize sleep and wake in a Murid rodent Acomys cahirinus in greater detail alongside the well-studied laboratory house mouse (Mus musculus) and wild M. musculus using a well validated, non-invasive, piezoelectric system for sleep and activity monitoring. We confirmed A. cahirinus, M. musculus, and wild M. musculus to be primarily nocturnal, but with clearly distinct behavioral patterns. Specifically, the activity of A. cahirinus sharply increases right at dark onset, which is common in nocturnal species, but surprisingly, decreases sharply just one hour later. Using infra-red camera recordings in single and group cage conditions, we found that A. cahirinus is more active early in the night period than late night period in single and group cages, and this decreased activity in the latter half of the night is much greater compared to M. musculus. In order to better understand these differences in activity, we investigated the sleep architecture of A. cahirinus using electroencephalogram (EEG) recordings. Our data show that A. cahirinus have a few key differences in sleep from M. musculus. A. cahirinus have significantly longer daily sleep periods and exhibit a much higher amount of REM sleep. A. cahirinus are awake at dark onset, but sleep more than M. musculus after the middle of the night. Most strikingly, A. cahirinus do not close their eyes virtually at all while sleeping, day or night. In order to test whether the sleep patterns of A. cahirinus are affected by or responsive to different light input, we set up a light flashing experiment during the daytime. While sleep amount did not change significantly during light flashing, A. cahirinus spent significantly less time in REM compared to baseline. In contrast, M. musculus had no difference in REM sleep percentage. Histological studies showed A. cahirinus have thinner retinal layers, but much thicker corneas than M. musculus. Electroretinography (ERG) results, specifically b-wave amplitudes, are significantly different between these two species. While eye closure and sleep have not been systematically studied across mammals, our observation is clearly a rare behavior. This raises further questions about A. cahirinus sleep architecture, the adaptive value of eyes open sleep to A. cahirius and whether they may have limited visual processing even during normal sleep.

Direct current (DC) potential is an objective measure of the functional state of the human organism. It is a sensitive and accurate indicator of short- and long-term adaptations to stress, adaptive capacities, and it is an important marker of athlete readiness. Sleep is posited to be the most efficacious strategy for improving recovery to enhance sport performance, and adequate sleep is considered vital to normal psycho-physiological function. Thus, optimal sleep may enhance the functional state, in turn enhancing an athlete’s adaptability to training stress. However, little is known about the relationship between sleep and DC potential. Therefore, the purpose of this study was to examine the effect of acute (one-night) and extended (two-night) sleep quantity and quality on DC potentials in collegiate American football players. Twenty-four Division 1 American football players (Age: 20.6 ± 1.30 yr; Height: 183.4 ± 6.40 cm; Body mass: 114.40 ± 24.60 kg) wore a wrist-worn actigraphy band seven days per week over the course of 136 days, which spanned the pre-season training camp and competitive season, to measure sleep quantity and quality. DC potential was assessed six days per week using the Omegawave Ltd (Espoo, Finland) athlete monitoring system either 30 minutes upon waking or 75-120 minutes prior to the onset of the football training session. Sleep quantity was stratified into duration categories and sleep quality was stratified within sleep latency, number of awakenings, and sleep efficiency variables. Sleep quantity and quality were evaluated using acute (one night) and extended (rolling average of two consecutive nights) sleep outcomes. Within subject comparisons of DC potential were made across sleep quantity and quality categories using repeated-measures analysis of variance to examine the influence of acute and extended sleep quantity and quality on DC potential outcomes. The level of significance was set at p ≤ 0.025. Statistically significant main effects were identified for acute sleep (F3,16 = 4.68, p < .02, η2p = 0.47) and extended sleep durations (F2,17 = 7.71, p < 0.005, η2p = 0.48). Specifically, for acute sleep durations, there was a 17.1% increase in DC potentials (3.59, p < 0.01, Cohen’s d = 0.52, SE 1.18) for sleep durations ≥ 7 hours to < 9 hours, compared to sleeping < 6. For extended sleep, there was a 20% increase in DC potentials (4.53, p < 0.002, Cohen’s d = 0.68, SE = 1.13) when recording a two-day sleep average of ≥ 7.5 hours and < 9 hours, compared to an extended sleep duration of < 6 hours. A statistically significant main effect was also identified for extended wake episodes (F2,19 = 4.5, p = 0.025, η2p = 0.32). For extended sleep periods with > 4 wake episodes there was a 12% increase in DC potentials (2.57 ± 2.24mV, p < 0.25, Cohen’s d = 0.34) compared to extended sleep periods with 2-3 wake episodes. There was not a significant effect of acute (p ≥ 0.20) sleep quality or extended latency (p > 0.18) and efficiency (p > 0.08) on DC potentials. These findings suggest that sleep quantity affects DC bio-potentials and thus the functional state of the athlete. Specifically, sleep durations between 7.00/7.50 to 9 hours correspond with higher measures of DC potentials compared to lesser durations. Given the effect of sleep quantity on biological markers for training adaptability, practitioners should prioritize sleep in the training process and educate athletes on proper sleep hygiene and sleep quantity to enhance their readiness to train.

Prenatal alcohol exposure (Fetal Alcohol Syndrome [FAS] and Fetal Alcohol Spectrum Disorders [FASD’s]) represents the leading preventable cause of intellectual disabilities in the western world, with FASDs estimated to affect approximately 2-5% of live births in the United States at an approximate annual cost of $3.6 billion (CDC, 2015; May et al., 2009). Ethanol (ETOH) exposure during development can lead to a variety of long-term behavioral impairments including problems with executive functioning, motor coordination, spatial learning, attention, and hyperactivity (Jones, 2011; Mattson & Riley, 1998). Much research has been conducted to develop pharmacological and/or environmental interventions to reduce these deficits, however, there are currently no clinically approved medications to treat the deficits related to fetal ETOH exposure. The current study used a developmental “3rd trimester” ETOH exposure model in neonatal rats to test the hypothesis that compounds targeting the nicotinic system will reduce deficits associated with ETOH exposure. Both compounds demonstrated promise in reducing some of the effects of developmental ethanol exposure, with DMXB-A treatment after ethanol exposure reducing balance deficits in females and spatial memory deficits in males. Solidago nemoralis treatment after ETOH exposure reduced learning and memory deficits in males and balance and executive functioning deficits in both sexes. With these results and previous work in this lab and others there appears to be ample evidence for their usefulness in reducing various forms of neurotoxicity. The long-term goal of this research is to evaluate the usefulness of both DMXB-A & Solidago nemoralis (SN) in treating deficits related to developmental ETOH exposure in humans and hopefully develop a treatment for these disorders.

Inflammation or nerve injury sensitizes several populations of nociceptive neurons in the dorsal horn of the spinal cord, including those that express the neuropeptide Y (NPY) Y1 receptor (Y1R). Our overall hypothesis is that after tissue or nerve injury, these Y1R-expressing neurons enter a state of latent sensitization (LS) that contributes to vulnerability to the development of chronic pain; furthermore, LS is under the tonic inhibitory control of endogenous Y1R signaling. First, we evaluated the intracellular signaling pathways that become activated in Y1R-expressing neurons and participate in LS. To do this, we established behavioral models of inflammatory or neuropathic pain, allowed pain hypersensitivity to resolve, and then during this period of pain remission we administered the Y1R receptor antagonist, BIBO3304, by intrathecal injection. As observed previously with mu-opioid receptor antagonists/inverse agonists, we found that BIBO3304 reinstated pain hypersensitivity via an N-methyl-D-aspartate receptor (NMDAR)- and adenylyl cyclase type 1 (AC1)-dependent mechanism. Our subsequent behavioral pharmacological experiments then established two signaling pathways downstream of AC1 that maintain LS. The first pathway involves protein kinase A (PKA) and transient receptor potential cation channel A1 (TRPA1) and channel V1 (TRPV1). The second pathway involves exchange proteins activated by cAMP (Epac 1 and Epac 2). We next found that nerve injury decreases the co-expression of Y1R with markers of excitatory interneurons, suggesting that Y1R-expressing neurons acquire a pain-enhancing phenotype after peripheral nerve injury. In a separate set of experiments that utilized Y1R-receptor internalization as an index of NPY release, we found that nerve injury increased stimulus-evoked NPY release. We conclude that injury induces pain-facilitatory mechanisms of LS in the dorsal horn involving PKA→TRPA1 and PKA→TRPV1 at the central terminals of primary afferent neurons. Whether Epac mechanisms are located on these same presynaptic terminals and/or at Y1R-expressing excitatory interneurons remain to be determined. We also conclude that injury-induced LS is masked by a compensatory up-regulation of spinal NPY release that tonically inhibits pain. These results present a novel mechanism of injury-induced LS and endogenous control of the transition from acute to chronic pain by the NPY-Y1R system. Our work sheds light on novel targets for the treatment of chronic pain.

Ischemic stroke is the leading cause of disability world-wide and affects over 800,000 people per year in the United States. The majority of these strokes are ischemic due to a blockage of blood flow to the brain. Damage to the brain occurs at the onset of stroke, neuronal cell death is irreversible and therefore, quick treatment to remove blockage is critical factor in the recovery from stroke. Mechanical thrombectomy as a treatment for ischemic stroke provides an ideal opportunity to collect blood distal and proximal to the cerebral thrombus to examine neurochemical changes occurring during stroke. The purpose of this dissertation was to explore the trajectory of neurochemical changes that occur in response to ischemic stroke during the first 72 hours and the physiological response from stroke patients to improve stroke outcomes. The specific aims were to: 1) to determine whether venous blood gases predict infarct volume and/or mortality in acute ischemic stroke in young male rats; 2) determine whether venous blood gases predict infarct and edema volume, and/or mortality in acute ischemic stroke in aged male and female rats; 3) compare the presence and relative concentrations of acid/base and electrolytes in static blood distal to thrombus and in peripheral blood drawn from adults who received thrombectomy for ischemic stroke and identify associations to postreperfusion functional outcomes. Specific Aim One was addressed by evaluation of young (three-month old) Sprague-Dawley rats that underwent permanent or transient middle cerebral artery occlusion (MCAO). Pre- and post-MCAO venous samples from permanent and transient models provided pH, carbon dioxide, oxygen, bicarbonate, glucose, hematocrit, hematocrit, and electrolyte values of ionized calcium, potassium and sodium. The analyses indicated that mean differences in the blood gas and electrolytes between pre- to post-MCAO and pH and iCa2+ were predictors of infarct volume in the permanent MCAO model. The second aim was addressed by evaluation of aged (18 month old) male and female rats pre-MCAO, post-MCAO, and at 72 hours of permanent MCAO venous blood gas samples (pH, carbon dioxide, oxygen, bicarbonate, glucose, hematocrit, hematocrit, and electrolyte concentrations of ionized calcium, potassium and sodium). Changes in pH (from pre-MCAO to post-MACO and post-MCAO to 72 hours) and changes in Na+ and iCa2+ (from post-MCAO to 72 hours) were predictors of infarct volume and edema volume, respectively in both sexes. Cox regression revealed there was a 3.25 times increased risk for mortality based on changes (cut-off range within -2.00 to - 7.00) in bicarbonate levels (pre- to post-MCAO). The third aim was addressed by evaluation of acid/base balance (pH, carbon dioxide, oxygen, bicarbonate, ionized calcium, potassium and sodium) of ischemic stroke patients who underwent mechanical thrombectomy. Our results suggests sex differences matter in ischemic stroke populations. Significant differences occur within proximal blood between the sexes. Additionally, females had approximately 2.5 hour increased time between stroke symptom onset to thrombectomy completion time (described as infarct time). Changes in bicarbonate and base deficit were predictors of infarct time, but only in our female population.

Vital autonomic and cardiovascular functions are susceptible to dysfunction after spinal cord injury (SCI), with cardiovascular dysregulation contributing to morbidity and mortality in the SCI population. Autonomic dysreflexia (AD) is a condition that develops after injury to the sixth thoracic spinal segment or higher and is characterized by potentially dangerous and volatile surges in arterial pressure often accompanied with irregular heart rate, headache, sweating, flushing of the skin, and nasal congestion. These symptoms occur in response to abnormal outflow of sympathetic activity from the decentralized spinal cord typically triggered by noxious, yet unperceived nociceptive stimulation beneath the level of lesion. Maladaptive plasticity of primary afferents and spinal interneurons influencing sympathetic preganglionic neurons is known to contribute to the development of AD. However, there are currently no treatments capable of targeting this underlying pathophysiology. The goal of this work was to test pharmacological agents for their potential to modify intraspinal plasticity associated with AD in order to prophylactically prevent the development of this condition altogether. We first tested whether the drug rapamycin (RAP), a well-studied inhibitor of the growth promoting kinase “mammalian target of rapamycin” (mTOR), could prevent aberrant sprouting of primary c-fiber afferents in association with reduced indices of AD severity. Naïve and T4-transected rats undergoing 24/7 cardiovascular monitoring were treated with rapamycin (i.p.) for 4 weeks before tissue collection. RAP attenuated intraspinal mTOR activity after injury, however it also caused toxic weight loss. RAP treated SCI rats developed abnormally high blood pressure both at rest and during colorectal distension (CRD) induced AD, as well as more frequent bouts of spontaneous AD (sAD). These cardiovascular alterations occurred without altered intraspinal c-fiber sprouting. Our finding that rapamycin exacerbates cardiovascular dysfunction after SCI underscores the importance of screening potential pharmacological agents for cardiovascular side effects and suggests that the mTOR pathway plays a limited or dispensable role in c-fiber sprouting after SCI. We next examined the effects of the antinociceptive drug gabapentin (GBP) on AD development. Our previous work demonstrated that a single acute administration of GBP can reduce the severity of AD. The mechanism of action, however, remains unclear. Emerging evidence suggests that GBP may act by blocking de novo synaptogenesis. We investigated whether continuous GBP treatment could attenuate the development of AD by modifying synaptic connectivity between primary afferents and ascending propriospinal neurons. SCI rats were treated with GBP every six hours for four weeks. We found that GBP reduced blood pressure during CRD stimulation and prevented bradycardia typically observed during AD. However, GBP treated rats also had a higher sAD frequency and failed to return to pre-injury body weight. Moreover, SCI reduced the density of putative excitatory (VGLUT2+) and inhibitory (VGAT+) synaptic puncta in the lumbosacral cord, although GBP did not alter these parameters. Our results suggest that continuous GBP treatment alters hemodynamic control after SCI and that decreased synaptic connectivity may contribute to the development of AD. These studies demonstrate the need for further research to better understand the cellular signaling driving maladaptive plasticity after SCI as well as the complex and dynamic changes in intraspinal synaptic connectivity contributing to the development of AD. Moreover, GBP treatment may offer clinical benefit by reducing blood pressure during AD, however the optimal dosage must be identified to avoid undesired side-effects.

Nitric Oxide (NO) is a powerful endogenous free radical that has numerous biological functions including vasodilation and serves as a post synaptic second messenger in the central nervous system (CNS). Numerous studies implicate NO• involvement in CNS disorders such as schizophrenia and drug abuse. These studies address the direct in vivo determination of an FDA-approved NO• donor (nitroglycerin) on extracellular levels of NO• in the frontal cortex and core of the nucleus accumbens in a lightly anesthetized rat. State-of-the-art in vivo amperometric recording techniques coupled with a novel 4-channel low noise pre-amplifier system and new generation microelectrode arrays (MEAs) will be used to record extracellular levels of NO• at 100Hz before and during nasal administration of either placebo (1) or nitroglycerin. This studies will determine the feasibility of measuring NO• in the CNS while administering the NO• donor nasally and determine the amplitude and kinetic time course effects of a nasally delivered NO• donor in two rat brain areas, the frontal cortex and core of the nucleus accumbens.

Addiction is characterized by a progressive loss of executive control over drug-seeking and consumption, and may be associated with a behavioral shift from instrumental goal-directed actions to stimulus-response habits. Sex differences in drug addiction have been linked to changing hormone levels across the estrous cycle, and females exhibit a particular vulnerability to psychostimulants such as cocaine and amphetamines. Psychostimulants and estrogen both influence dopaminergic activity in the dorsal striatum, a region of the brain in which dopamine activity is thought to mediate the shift from action to habit. In the present set of experiments, we examined the roles of sex, amphetamine, and degree of training on habit formation in rats. To test habit formation in each experiment, animals were trained on a variable interval (VI) schedule of reinforcement to nose-poke for sucrose pellet reinforcers, then the sucrose was devalued in half of the animals by pairing its presentation with injections of lithium chloride (LiCl) to induce nausea. Animals for whom the sucrose was paired with LiCl acquired a conditioned taste aversion for the sucrose reinforcer. When tested in extinction, paired animals who remained goal-directed should inhibit their responding for the devalued sucrose, whereas animals in habit should be insensitive to the devaluation and respond at a similar rate as their non-devalued counterparts. Experiment 1 examined the role of sex in habit formation in which intact male and female rats received identical training, devaluation, and testing in two separate within-sex experiments. After 240 reinforcer exposures females exhibited habitual behavior whereas males remained goal-directed. In Experiment 2, female rats were ovariectomized (OVX) and half were given cyclic estrogen replacement. All animals received either pre-exposure to methamphetamine (METH) or vehicle. Following exposure to 120 reinforcers, a level of training that had previously been shown to be subthreshold to habit formation in males, all female groups demonstrated goal-directed responding at test, revealing a lack of effect of hormone replacement or drug pre-treatment on habit formation in OVX females at this level of reinforcer exposure. Experiment 3 aimed to determine the degree of nose-poke training that would be sub-threshold to habit formation in intact females, and two groups were given different amounts of training. Both groups exhibited habitual responding, indicating that habit threshold in females is lower than hypothesized. Overall, these experiments suggest that females shift into habitual behavior earlier in training than males, and further experiments need to be conducted to determine how factors such as hormone milieu and psychostimulant exposure influence this progression.

Chronic musculoskeletal pain is a leading cause of disability worldwide yet the mechanisms of chronification and neural responses to effective treatment remain elusive. Non-invasive imaging techniques are useful for investigating brain alterations associated with health and disease. Thus the overall goal of this dissertation was to investigate the white (WM) and grey matter (GM) structural differences in patients with musculoskeletal pain before and after psychotherapeutic intervention: cognitive behavioral therapy (CBT). To aid in the interpretation of clinical findings, we used a novel porcine model of low back pain-like pathophysiology and developed a post-mortem, in situ, neuroimaging approach to facilitate translational investigation. The first objective of this dissertation (Chapter 2) was to identify structural brain alterations in chronic pain patients compared to healthy controls. To achieve this, we examined GM volume and diffusivity as well as WM metrics of complexity, density, and connectivity. Consistent with the literature, we observed robust differences in GM volume across a number of brain regions in chronic pain patients, however, findings of increased GM volume in several regions are in contrast to previous reports. We also identified WM changes, with pain patients exhibiting reduced WM density in tracts that project to descending pain modulatory regions as well as increased connectivity to default mode network structures, and bidirectional alterations in complexity. These findings may reflect network level dysfunction in patients with chronic pain. The second aim (Chapter 3) was to investigate reversibility or neuroplasticity of structural alterations in the chronic pain brain following CBT compared to an active control group. Longitudinal evaluation was carried out at baseline, following 11-week intervention, and a four-month follow-up. Similarly, we conducted structural brain assessments including GM morphometry and WM complexity and connectivity. We did not observe GM volumetric or WM connectivity changes, but we did discover differences in WM complexity after therapy and at follow-up visits. To facilitate mechanistic investigation of pain related brain changes, we used a novel porcine model of low back pain-like pathophysiology (Chapter 6). This model replicates hallmarks of chronic pain, such as soft tissue injury and movement alteration. We also developed a novel protocol to perform translational post-mortem, in situ, neuroimaging in our porcine model to reproduce WM and GM findings observed in humans, followed by a unique perfusion and immersion fixation protocol to enable histological assessment (Chapter 4). In conclusion, our clinical data suggest robust structural brain alterations in patients with chronic pain as compared to healthy individuals and in response to therapeutic intervention. However, the mechanism of these brain changes remains unknown. Therefore, we propose to use a porcine model of musculoskeletal pain with a novel neuroimaging protocol to promote mechanistic investigation and expand our interpretation of clinical findings.

Background: The 5-hydroxytrptamine receptor 4 (5-HT4 receptor) is heavily expressed on colonic epithelial cells and has been targeted as a therapeutic for functional bowel symptoms and pain; however, adverse cardiac events related to 5-HT4 agonist treatment limited their therapeutic use. Previous studies in the Mawe laboratory have demonstrated that intraluminal application of a 5-HT4 agonist exerts protective epithelial actions in animal models of colitis, and accelerates recovery from colitis. The aim of this study was to test the effects of a luminally restricted 5-HT4 agonist in a mouse model of experimental colitis. Methods: The luminally restricted 5-HT4 agonist (Takeda Pharmaceuticals; 10 mg/kg) was administered to mice during active dextran sodium sulfate (DSS) induced colitis. Colitis activity was evaluated using disease activity index, a fecal lipocalin-2 assay, and histological damage scoring. Epithelial proliferation and colonic motility were also measured as readouts of the potential protective actions and colonic function, respectively. Results: Oral gavage and intracolonic delivery of this luminally restricted 5-HT4 agonist had no detectable effect on recovery from colitis or colonic motility as compared to vehicle. Additionally, in positive control experiments, we failed to see an effect of the 5-HT4 agonist, tegaserod, on colitis severity or colonic motility in any of the measures tested. Conclusions: In conclusion, it is unclear if the luminally restricted 5-HT4 agonist has any effect on recovery from DSS colitis. Given inconsistencies with the model and lack of an effect of tegaserod, additional studies will be required, possibly involving different doses and time points, to fully assess the actions of this luminally restricted compound in colitis recovery.