abstract: Schizophrenia is considered a multifactorial disorder with complex genetic variants in response to environmental stimuli. However, the specific genetic contribution to schizophrenia risk is largely unknown. The transcription factor early growth response gene 3 (EGR3) can be activated rapidly after stimuli and thus may translate environmental stimuli into gene changes that influence schizophrenia risk. However, the downstream genes that may be regulated by EGR3 are not clear. While the 5-Hydroxytryptamine receptor 2A (5HT2AR) - encoding gene Htr2a has been implicated in the etiology of schizophrenia, the mechanisms by which Htr2a influences susceptibility to this illness are poorly understood. We previously found that in addition to schizophrenia-like abnormalities, Egr3 -/- mice have approximately 70% deduction of 5HT2AR level in the prefrontal cortex, which underlines their resistant to the sedating effect of clozapine. These findings indicate that the two schizophrenia candidate genes are in the same biological pathway that integrates multiple components resulting in schizophrenia. This dissertation is aimed to identify the mechanisms by which Egr3 regulates the expression of Htr2a in response to environmental stimuli like stress. To determine if Egr3 alters Htr2a transcription under stress, I examined messenger ribonucleic acid (mRNA) levels of these two genes in wildtype (WT) and Egr3 -/- mice after 6hrs of sleep deprivation (SD). I found both genes are increased in WT mice after SD compared with controls. In addition, Egr3 is required for Htr2a induction because SD fails to induce Htr2a expression in Egr3 -/- mice. Next, I performed chromatin immunoprecipitation (ChIP) to determine if EGR3 binds to Htr2a promoter in vivo. I found a significant increase of EGR3 binding to Htr2a distal promoter 2hrs after seizure. To determine the functionality of this binding, I co-transfected the CMV- EGR3 vector or CMV- vector alone with the Htr2a distal promoter reporter clone. I found overexpression of EGR3 activates the Htr2a distal promoter-driven luciferase gene. Although the ChIP assay shows no direct binding of EGR3 to Htr2a proximal promoter, I found EGR3 overexpression activates Htr2a proximal promoter-driven luciferase gene. These findings suggest that EGR3 regulates Htr2a probably through both direct and indirect ways. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2017

<p> A neuron&rsquo;s unique physiological waveform arises from its palette of ion channels and receptors, as superimposed on its geometrical structure. The crustacean stomatogastric ganglion (STG), a small rhythmic motor circuit, exhibits fourteen identified neuron types with highly-conserved physiological waveforms and complex morphologies. In this thesis, I examine how morphology shapes neuronal physiology in the STG. Using high-resolution neuronal reconstructions and a suite of computational tools, I quantify numerous morphological features of four STG neuron types. This work revealed remarkable animal-to-animal variability in neuronal morphology. I also demonstrate that STG neurons do not adhere to current hypotheses regarding wiring optimization principles. I then studied the physiological consequences of animal-to-animal morphological variability in one neuron type, the Gastric Mill (GM) neuron. Utilizing focal photo-uncaging of glutamate in tandem with electrophysiological techniques, I characterize passive voltage signal propagation. I find that GM neurons, despite their complex structures, operate much like single compartments. Taken together, these studies suggest that relatively compact electrotonic structures may effectively compensate for the observed morphological variability observed across animals. A final study describes the development of photoactivatable peptides for probing the subcellular actions of endogenous neuromodulatory substances in individual STG neurons. This work culminates in the synthesis of two photoactivatable peptides endogenous to the STG: TNRNFLRF-NH₂ and CabTRP1a. Although the compounds could be successfully photolyzed, they did not yield consistent responses when photoactivated in the biological preparation. The design process and preliminary experimental results are discussed. Altogether, this thesis serves as a case study of neuronal morphology and passive physiology in the STG and sheds light on our current conceptual framework for understanding how morphology maps to function in diverse nervous systems.</p><p>

Naked mole-rats live in large colonies and exhibit a strict reproductive hierarchy. Each colony has 1 breeding female and 1-3 breeding males; all other individuals are non-reproductive subordinates. Subordinates show a remarkable lack of sex differences in behavior and anatomy, but can become reproductive if removed from the colony. The striated perineal muscles and their innervating motoneurons, which are sexually dimorphic in all other mammals examined, are not dimorphic in subordinate naked mole-rats. Here I asked whether sexual differentiation of this neuromuscular system occurs when subordinates become breeders. Sex differences in perineal motoneurons were not observed, regardless of social status. To my surprise counts of motoneurons in Onuf’s nucleus were increased in breeders of both sexes. This was accompanied by a reciprocal decrease in cells in Onuf’s nucleus that were characterized by small soma size. The neuronal changes correlate with increased perineal muscle volumes in breeders. Although not exhibiting typical motoneuron morphology, some small cells fit a neurochemical or functional definition of a motoneuron. I propose that small cells are recruited to the pool of large Onuf’s nucleus motoneurons when subordinate naked mole-rats become breeders. I then looked at naked mole-rats of varying status (subordinates, paired animals that have never reproduced, intact breeders, and gonadectomized breeders) to determine which cues elicit changes in perineal muscles and small cells in Onuf’s nucleus. I found that pairing is sufficient to cause decreases in the population of small cells in Onuf’s nucleus, while production of litters is necessary for increasing in perineal muscle size. The gonads were not necessary to maintain changes in small cells or perineal muscles. I hypothesized that the lack of sex differences in naked mole-rats might be related to their unusual social structure. To test this, I compared the genitalia and perineal muscles in three African mole-rat species: the naked mole-rat, the solitary silvery mole-rat, and the Damaraland mole-rat, a species considered to be eusocial, but with less reproductive skew than naked mole-rats. My findings support a relationship between social structure, mating system, and sexual differentiation.

The development of complex neural networks relies on a careful balance of environmental cues to guide and shape both ends of the eventual connection. However, the correct wiring of circuits whose components share molecular profiles depends on a more elaborate phenomenon, competition. Despite being highly studied, there is still a lack of understanding as to the mechanism that allows molecularly identical cells to form exclusive connections with their targets. To address this complex question, we turned to a simple circuit within the genetically tractable fly. Responsible for the escape reflex, the Giant Fiber System is comprised of bilaterally symmetrical axons that innervate the ipsilateral "jump" motorneuron, TTMn in a 1:1 ratio. However, if a TTMn is unilaterally ablated prior to circuit formation, this ratio is disrupted and the deprived axon forms its presynaptic terminal on the opposite side. Midline crossing by the deprived axon led to exploration of a known pathway in giant fiber development, midline repulsion via Slit and Roundabout. Axons in which Roundabout levels were reduced through a natural pathway antagonist, Commissurelesss, crossed the midline freely, confirming a native, if normally restricted ability to do so. However, unlike the overlapping giant fiber terminals seen following ablation, these axons retained their wild exclusivity, elaborating their terminals toward a single TTMn. This supported our initial aim of uncovering a competitive force in giant fiber target selection. In addition to repulsion, I also examined the attractive pathway of Netrin and Frazzled for a possible role in target identification. Varying the levels of Frazzled receptors led to increased midline crossing and overlapped terminals, suggesting a connection between this attractive receptor and the repulsion pathway first examined. Frazzled has been shown to induce commissureless expression independent of its ligand, making it an important linchpin in the regulation of giant fiber guidance and competition. In fact, when allowed to traverse the midline, giant fibers responded to a Netrin increase with overlapping synaptic terminals. In this dissertation, I present a model in which giant fibers possess competitive machinery, driven by Netrin and triggered by Frazzled, underneath the naturally restrictive midline repulsion.

Mild traumatic brain injury (mTBI) is the disruption in the normal function of the brain caused by a bump, blow, jolt to the head, or penetrating head injury. Attempts to quantify mTBI have included inconsistent methods and created difficulty in identifying its prevalence in many populations. Among the populations most vulnerable to mTBI are the homeless, for whom there is a cycle of mTBI, substance use, and arrest. This thesis consists of three chapters. In the first chapter, an outline for the relationships between homelessness, mTBI, substance use, and arrests is provided. In the second chapter, we show that the colloquial understanding of head injuries held by our homeless cohort addresses significantly fewer instances of mTBI than a definition derived from medical consensus. This adds to the growing need for consistency of reporting criteria as well as greater public education with respect to head injury. In the third chapter, we present evidence that mTBI sustained before the age of 12 can predict substance use and arrest, though it is most useful in addressing depressants such as alcohol and sedatives. Finally, we address the limitations of our homeless cohort and the direction of future investigation in light of our findings. / 2021-06-08T00:00:00Z

The trigeminal subnucleus caudalis (Vc), which has been designated as the "medullary dorsal horn"- analogous to the spinal dorsal horn (DH), relays noxious input from the orofacial region to higher brain centers. Recent work in our lab has demonstrated numerous developmental differences in dissociated cultures of Vc and DH neurons. When compared with DH neurons, the growth of neurons located in the Vc was significantly slower, and glutamate receptor activity was relatively low. Detailed studies using Hoechst 33342 and TUNEL imaging assays, suggested that when compared with DH neurons, more mature neurons in Vc underwent cell death. Co-culture with DH cells remarkably improved the neuronal growth and increased the incidence of mEPSCs in Vc, while DH neurons co-cultured with Vc cells apparently exhibited slowed growth rate and a reduced incidence of mEPSCs. Recent work has also shown that blockade of P2X7 channels or 11β-hydroxysteroid dehydrogenase type 2 (HSD2), which catalyzes the conversion of corticosterone to an inactive metabolite, significantly promoted neuronal growth in both Vc and DH cultures. Using the conditioned medium from dissociated and organotypic slice cultures, we isolated several factor(s) using size exclusion column chromatography from both Vc and DH tissues. One of the factor(s) in the Vc conditioned medium, Vc-NF2, caused a decrease in live cells when applied to dissociated cortical neurons, while one of the factor(s) in the DH conditioned medium, DH-NF1, caused an increase in live cells when applied to dissociated cortical neurons. Based on the above findings, we conclude that the development of Vc neurons may be region-specifically regulated by locally released factor(s). These studies will provide novel insights into the mechanisms of Vc neuronal development, which could ultimately lead to better analgesic craniofacial regimes for infants or adults. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2011. / June 20, 2011. / trigeminal subnucleus caudalis, region-specific regulation, neuron, development, medullary dorsal horn / Includes bibliographical references. / Xian-Min Yu, Professor Directing Dissertation; Teng Ma, University Representative; Cathy W. Levenson, Committee Member; Robert Contreras, Committee Member; Tim Megraw, Committee Member.

The sense of taste guides nutrient consumption and affects overall health and quality of life. One of the main functions of taste is the detection of chemical stimuli that elicit the perception of canonical qualities such as sweet, salty, sour, and bitter. Here, I have used a two-response operant task to essentially ask an animal, “can you taste this?” after damage to a key brain area of the gustatory system. Through the application of psychophysical methods, psychometric taste sensitivity functions were derived for a given taste stimulus. The necessity of arguably the highest-ordered central gustatory region in the rodent, the gustatory cortex (GC), in the maintenance of normal sensitivity to prototypical tastants was assessed by placing large lesions at this brain site. The thalamocortical pathway, in which taste signals ascend from the ventral posteromedial thalamus, parvicelluar layer (VPMpc) to GC, was first hypothesized to be responsible for discriminative taste function by Carl Pfaffmann and colleagues in 1977. Although the delineations of the GC are debated, neurons that are activated when a tastant is delivered onto the tongue have been found and neuronal tracing studies confirm projections from taste thalamus reside here. Although these projections have been described, one aim here was to precisely map the connections from thalamus to GC along its anterior/posterior (AP) axis, with aid of a neuronal tract-tracer. Our prior research has revealed that bilateral GC lesions significantly impair taste sensitivity to salts and quinine but not to sucrose. The range of tastants tested has been extended here to include: a) the polysaccharide, Maltrin, a highly preferred carbohydrate discriminable from sugars, and b) citric acid, a representative compound that in humans generates sour taste. The detectability of these stimuli, as well as NaCl, was tested in rats with neurotoxin-induced (ibotenic acid) GC lesions and in sham-operated controls (SHAM). The effect of unilateral GC lesions on taste detectability was also tested because, although taste deficits are apparent in humans with unilateral damage in insular cortex, there is controversy over which hemisphere is involved. Rats were trained, via operant conditioning, and tested in a gustometer to discriminate a tastant from water in a two-response taste detection task. Psychometric sensitivity functions were derived by lowering the stimulus concentration across test sessions. A lesion mapping system was then used to determine placement and size of left (LGCX), right (RGCX), and bilateral (BGCX) lesions and, in the case of the latter, symmetry as well. Replicating our prior work, for NaCl, there was a significant rightward shift in taste sensitivity between BGCX and SHAM rats and between RGCX and SHAM rats. Similar to sucrose, taste sensitivity to Maltrin was not significantly different between lesion and SHAM groups. Although there was no significant lateral shift in taste sensitivity between surgical groups to citric acid, some declines in BGCX performance relative to SHAM rats were evident for higher concentrations. Together with prior results, complete bilateral lesions in GC do not appear to disrupt sensitivity to carbohydrate stimuli, but do appear to modestly impair sensitivity to quinine and severely impair sensitivity to salts. In the case of the latter stimulus, deficits are evident after unilateral lesions in the right GC. Nevertheless, in all of these cases the rats perform well at higher concentrations. Clearly other central brain regions appear to contribute to sensory-discriminative taste processing in rats. Similar to the controversy over where GC resides within the insular cortex, there are also conflicting reports of where these projections from VPMpc terminate within insular cortex. Here, a fluorescent tract-tracer, DiI, was systematically injected into various sites of GC along the AP axis to quantify the anatomical organization of projections arising from the VPMpc which was analytically partitioned into subfields for quantification of retrogradely labeled neurons. The results confirm previous findings showing that projections to the GC from thalamus arise from the medial portion of VPMpc, whereas projections to the areas dorsal to the conventionally defined GC arise from the more lateral portions of VPMpc. Interestingly, an anterior-posterior organization from the VPMpc to the GC was also found, supporting an orotopic topography in GC suggested in the literature that appears to be roughly maintained along the ascending gustatory pathway in the rat brain. Overall, these experiments have added to the understanding of the functional and neuroanatomical organization of the central gustatory system in the rodent model. However, there is much more work to be done to identify central regions that contribute to sensory-discriminative and other domains of taste function. / A Dissertation submitted to the Department of Psychology in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / March 30, 2018. / Gustatory Cortex, Taste Detection, Taste Psychophysics, Taste Thalamus, VPMpc / Includes bibliographical references. / Alan C. Spector, Professor Directing Dissertation; Jorge Piekarewicz, University Representative; Frank Johnson, Committee Member; Thomas Houpt, Committee Member; Sara Hart, Committee Member.

As the global burden of depression continues to rise, development of more efficacious and faster-acting antidepressant treatments has remained stagnant over several decades. This has created a dire need for a newer generation of therapeutics aimed at helping a greater percentage of the patient population in a shorter period of time. Identifying which subpopulations of patients experience optimal responses to certain treatments has become of great interest, as a means of tailoring treatment strategies for more individualized clinical outcomes. In major depressive disorder, women exhibit a lifetime prevalence roughly twice that of men, and tend to display different profiles of symptomology and antidepressant response rates when compared to men, illustrating the importance of examining sex and related variables as individual differences in the pathophysiology of depression and therapeutic response. Indeed, in recent years, consideration of sex has gained interest in depression relevant preclinical research—particularly in light of the discovery that the N-methyl d-aspartate receptor (NMDAR) antagonist, ketamine, rapidly relieves depressive symptoms and suicidal ideation, even in those with treatment-resistant depression. Notably, recent work from our group and others have revealed a higher sensitivity of females to the antidepressant effects of the NMDAR antagonist ketamine. Combined with its fast-acting and relatively sustained properties, ketamine may be a particularly interesting therapeutic alternative for this sensitive population. Since its discovery, significant research efforts have been dedicated to understanding the underlying mechanisms of ketamine’s antidepressant effects by both preclinical and clinical researchers alike, with the hope of developing novel rapid-acting treatments effective in a broader range of patients. However, a comparatively small proportion of such studies have included females and/or included sex as a variable in analyses. Therefore, the aim of the current work sought to develop the current gap in understanding of how sex and hormones may contribute to the heightened sensitivity of female rats to the rapid antidepressant effects of ketamine by taking a multidisciplinary approach using behavioral, pharmacokinetic and pharmacodynamics analyses in male and female rats. We recently reported that ovarian-derived estradiol (E2) and progesterone (P4) are essential for the greater sensitivity of female rats to rapid antidepressant-like effects of ketamine compared to male rats. However, whether or not the duration of response to ketamine is modulated in a sex- and hormone-dependent manner remains unknown, in addition to the possible contribution of testosterone to such sex differences. Therefore, in the second chapter we explored this systematically by investigating the influence of testosterone, estradiol and progesterone on initiation and maintenance of hedonic response to low-dose ketamine in intact and gonadectomized male and female rats. Females, but not males, experienced a sustained increase in sucrose preference following low-dose ketamine, and did so in an E2P4-dependent manner. Whereas testosterone failed to alter male treatment response, hedonic response to low-dose ketamine was enhanced in intact males when P4 was administered concurrently with low-dose ketamine. Treatment responsiveness was associated with greater hippocampal BDNF levels in female, but not male rats 24h after ketamine administration, without activation of key downstream signaling effectors. This work provides novel evidence supporting activational roles for ovarian-, but not testicular-, derived hormones in mediating hedonic sensitivity to low-dose ketamine in female and male rats. The persistence of sex differences following gonadectomy and selective involvement of BDNF in treatment response may indicate a partial role for organizational differences in these effects. In the absence of any preclinical studies of pharmacokinetic sex differences using low-dose ketamine, it is unclear whether the effects reported in the second chapter may be the result of differences in ketamine metabolism between male and female rats, or whether functional differences in the brain are the predominant driver of behavioral sex differences. Therefore, the third chapter examined whether or not sex and hormonal status affect the metabolism of low-dose ketamine in male and female rats. Intact male rats and female rats in either diestrus (low E2, P4) or proestrus (high E2, P4) were administered low-dose ketamine, and their plasma and brains collected 5-180 minutes later to analyze levels of ketamine and its metabolites, norketamine (NK) and dehydronorketamine (DHNK). Females exhibited greater concentrations of ketamine and NK over the first 30 minutes following treatment in both the brain and plasma, largely accounted for by slower clearance rates and longer half-lives. Interestingly, despite the impact of ovarian hormones on behavioral sensitivity to ketamine, no appreciable differences in pharmacokinetic parameters existed between proestrus and diestrus female rats. Together, this work suggests that while sex differences in metabolism may influence the amount of ketamine and NK reaching target areas in the brain, the impact of circulating hormones on behavioral sensitivity is more likely an effect of actions within the brain at the time of ketamine administration. As the mechanisms underlying this sex-dependent sensitivity to ketamine’s antidepressant-like effects remain elusive, ongoing phosphoproteomics work is underway to investigate the molecular mechanisms underlying this sex-dependent sensitivity to ketamine. Preliminary results revealed striking dissimilarities in the dHPC proteome and phosphoproteome of male and female rats both at baseline, and following low-dose ketamine treatment. Notably, these differences were heavily influenced by hormonal status in female rats. While future work is needed to determine the functional significance of these findings, the collective data presented herein suggest that both biological sex and the hormonal milieu are critical modulators of ketamine’s rapid actions on drug metabolism and within the brain, and provide greater insight into potential physiological and post-translational processes underlying sex- and hormone-dependent modulation of ketamine’s therapeutic effects. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / April 18, 2018. / Depression, Ketamine, Sex differences / Includes bibliographical references. / Mohamed Kabbaj, Professor Directing Dissertation; Thomas Keller, University Representative; James Olcese, Committee Member; Branko Stefanovic, Committee Member; Zuoxin Wang, Committee Member.

The suprachiasmatic nucleus of the hypothalamus (SCN) contains a master pacemaker that controls a wide variety of circadian rhythms of physiology and behavior. Recent work has provided a thorough understanding of the molecular mechanisms by which these oscillations are generated. The rhythmic expression of clock genes is essential to pacemaker function. These clock genes are also expressed rhythmically throughout the mammalian organism including peripheral organs and brain regions outside of the SCN. Synchronization within these tissues and maintenance of phase angle between the different systems depends upon the integrity of the SCN. The present experiments will focus on the nature of the SCN-dependent signals responsible for the entrainment of peripheral oscillations. Such signals may be neural, humoral, or behavioral, and various organs may rely on different signals or a combination of such signals. The first specific aim will test the importance of neuronal vs. humoral signals in the regulation of peripheral oscillator phase. The asymmetrical haPer1 expression in the SCN of Syrian hamsters held in constant light that show split locomotor behavior will be used to determine if a similar level of asymmetry of clock gene expression in the left and right sides of the body in bilaterally paired organs is accomplished by lateralized neuronal projections of the SCN. Asymmetrical expression of physiologically important genes in these peripheral organs will also be assessed. Specific aim 2 will determine if a molecular clock oscillates within arousal-promoting neurons, specifically the hypocretin-expressing neurons of the LH/DMH and the tyrosine hydroxylase-expressing cells of the locus coeruleus. Specific Aim 3 will utilize the behaviorally split Syrian hamster to determine if lateralized projections from the SCN control the phase of clock gene expression within these arousal-promoting neurons.

Includes bibliographical references. / Background: Depression is a debilitating mood disorder, negatively affecting an individual’s health and well-being. Despite this, the aetiology of depression remains poorly understood. Consistently, depression treatments are far from satisfactory due to limited efficacy and adverse side effects often associated with them, suggesting a need to improve the current animal models of depression in order to understand the basic mechanisms of the disorder. In an attempt to elucidate the pathophysiology of depression, a rodent model of depression (maternal separation, MS) is used to study the neurobiological mechanisms implicated in depression. However, MS alone produces inconsistent findings and often additional stressors are used to exaggerate the effects of MS. To create a more robust model of MS, MS rats were exposed to chronic constant light (CCL). However, contradictory findings have been reported with CCL. Aims: This study aimed to explore the effects of additional CCL in an MS model by measuring glutamate and potassium-stimulated [3H]DA release in the nucleus accumbens (NAc), testing the effects of CCL on serotonin (5-HT) levels in the hypothalamus and prefrontal cortex (PFC) and measuring ì-opioid receptor (MOR-1) levels in the NAc and orexin receptor (OXR-1 and OXR-2) levels in the PFC. Methods: In order to achieve these aims four experimental groups were chosen, out of which two groups; non-maternally separated (NMS) rats and maternally separated (MS) rats were exposed to CCL for 3 weeks during adolescence and the remaining two groups; NMS and MS rats were not subjected to CCL. At postnatal day 80 (adulthood), rats were decapitated and brain tissue collected for analysis of glutamate- and potassium-stimulated [3H]DA release in the NAc using in vitro superfusion. Serotonin levels in the hypothalamus and PFC were determined using Enzyme-Linked ImmunoSorbent Assay (ELISA). Western blot analysis was used to measure MOR-1 levels in the NAc, OXR-1 and OXR-2 in the PFC. Results: MS caused a significant decrease in glutamate-stimulated [3H]DA release in the NAc. In the NAc shell, CCL exposure revealed a trend towards a decrease in [3H]DA release in response to both glutamate- and potassiumstimulation. Moreover, in the hypothalamus NMS and MS rats subjected to CCL had significantly increased 5-HT levels compared to NMS and MS rats without xvii CCL exposure. In the PFC CCL had a significant effect on 5-HT levels and it was revealed that NMS CCL rats had decreased 5-HT levels compared to NMS rats. Similarly, MS CCL rats had significantly decreased 5-HT levels compared to NMS. MS and CCL did not have any significant effect on MOR-1 protein levels in the NAc. On the other hand, MS rats had increased OXR-1 and OXR-2 proteins levels in the PFC compared to NMS and MS CCL rats. Conclusion: MS decreased glutamate-stimulated [3H]DA release in the NAc. Serotonin levels in the hypothalamus and PFC were altered by the effects of MS and CCL. Furthermore, MS exposure increased OXR-1 and OXR-2 protein levels in the PFC. However, MS and CCL did not alter MOR-1 protein levels in the NAc. Therefore, this study has demonstrated that CCL exaggerated the effects of MS and created a more robust model of MS.