PHD, Kent State University, 2024, College of Arts and Sciences / School of Biomedical Sciences

As of 2022, three million people in the US, and sixteen million worldwide were estimated to suffer from opioid use disorder (OUD). Despite widespread efforts to increase the public availability of medical therapies for OUD, only 2.28% of people suffering from OUD will seek out and be able to sustain abstinence for at least five years. The core objectives of this work were to 1) evaluate the dose- and sex-dependent effects of fentanyl to induce rewarding states, 2) the extent to which D-Cysteine ethylester (D-CYSee) alters affective state and the acquisition of fentanyl-induced reward seeking, 3) how the timing and concentration of fentanyl administration impacts the intrinsic Ca2+ activity of neurons and astroglia from the prefrontal cortex (PFC), and 4) the extent to which D-CYSee alters intrinsic Ca2+ activity in both the presence and absence of fentanyl. To evaluate the effects of fentanyl in the presence and absence of D-CYSee on Ca2+ signaling dynamics in PFC neurons and astrocytes, this work details the development of new methods in real-time fluorescent imaging of intrinsic Ca2+ activity using a non-genetic chemical indicator in cells isolated from the rat PFC in combination with post-hoc live-cell labeling for neurons and astroglia, and a customizable cell-type informed statistical analysis pipeline with backend support for data visualization and meta-analysis. Furthermore, a general characterization of the intrinsic Ca2+ activity in this PFC preparation was conducted; first by examining the involvement of extracellular Ca2+ sources and sodium channel conductance's, followed by a deeper evaluation of the role(s) of voltage-gated L, T, & N/P/Q-Type Ca2+ channels and an assessment of NMDA, AMPA receptor, and GABAA receptor signaling in the expression of intrinsic Ca2+ activity. The findings here support: 1) that fentanyl induces reward seeking in a concentration- and sex-dependent manner, 2) that D-CYSee could be an effective co-treatment with prescribed opioi (open full item for complete abstract)

Committee: Devin Mueller, Ph.D. (Advisor); Derek S. Damron, Ph.D. (Advisor); Stephen J. Lewis, Ph.D. (Committee Member); Colleen Novak, Ph.D. (Committee Member); Robert Clements, Ph.D. (Committee Member); Rafaela S. C. Takeshita, D.Sc., (Other) Subjects: Behavioral Psychology; Behavioral Sciences; Cellular Biology; Neurosciences

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2022, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Cortical function comprises the gestalt of sensory processing mechanisms in the brain giving rise to higher-order perception, cognition, and behavior that underlies consciousness. However, traditional feed-forward theories of neuroscience fail to capture the complexity of functional interactions within the neural connectome leading to these phenomena. Prevailing theory is now positing a predictive processing framework to better describe the architecture of the cortex. C. elegans permits high-content behavioral phenomics and recording of wholebrain activity across all 302 neurons simultaneously. As such, C. elegans presents a tractable model to connect behavior to neurobiology and to test the assumptions of cortical theory. To that end, we performed whole-brain imaging in tandem with phenomic fingerprinting of behaving C. elegans in response to both aversive stimuli eliciting backwards locomotion and food-like stimuli eliciting attraction. Calcium imaging profiles revealed highly dynamic neurons with activity patterns wherein a feed-forward model does not fully explain neuronal nor behavioral response to stimuli. Suggestive of a complex sensory integration, we sought to apply a datadriven systems approach to characterize these nonlinear interactions and assess the complexity of the functional connectome in C. elegans. Ultimately, we suggest a loci for a novel correlate of the predictive processing framework emerging as a distributed computation of the neuronal network after stimulus-dependent rewiring of functional circuitry, posing an avenue for further study. As schizophrenia, autism, major depression, Alzheimer's disease, and age-related cognitive decline are linked to impairments in sensorimotor processing, this work represents an important step forward in advancing our understanding of the cortex and pathophysiology of neuropsychiatric disease.

Committee: Robert Smith (Committee Chair); Sinead O'Donovan (Committee Member); Rammohan Shukla (Committee Member); Bruce Bamber (Committee Member) Subjects: Behavioral Sciences; Bioinformatics; Neurobiology; Neurosciences; Systems Science

Doctor of Philosophy, Case Western Reserve University, 2025, Neurosciences

Layer 1 (L1) of the medial prefrontal cortex (mPFC) integrates long-range inputs and exerts pivotal control over deeper cortical layers, yet the specific roles of its GABAergic interneurons (L1INs) remain incompletely understood. Here, I combined morphological, electrophysiological, and behavioral approaches to elucidate the heterogeneity and function of mPFC L1INs in mice. Biocytin labeling identified three distinct morphological subtypes: neurogliaform cells (NGCs), elongated neurogliaform cells (eNGCs), and single-bouquet cell-like (SBC-like) cells, with divergent firing patterns. NGCs and eNGCs were predominantly late-spiking (LS) neurons, whereas SBC-like cells displayed non-late-spiking (NLS) firing more commonly. These L1INs formed interconnected electrical and chemical networks and exerted broad inhibitory effects on both pyramidal neurons and interneurons, highlighting their capacity to modulate deeper-layer neuronal activity. Functionally, in vivo calcium imaging during the tail suspension test (TST) revealed that L1INs displayed distinct calcium dynamics associated with active (escape/struggle) and inactive (immobility) behavioral states. Chronic restraint stress (CRS) induced behavioral despair and significantly altered L1IN activity: the fraction of L1INs active during immobility doubled (from 22.8% to 43.0%), while those active during escape/struggle decreased (from 50.6% to 32.9%). Subsequent circuit mapping indicated that following CRS, L1INs received enhanced excitatory input from the horizontal limb of the diagonal band (HDB) and diminished input from the ventromedial thalamus (vmTH). Moreover, optogenetic activation of HDB-to-mPFC L1 projections induced behavioral despair during the TST and triggered neuregulin-1 (NRG1) release from HDB terminals. This release shifted the firing pattern of mPFC ErbB4+ L1INs from LS to NLS, mirroring CRS effects. Notably, CRISPR/Cas9-mediated knockout of NRG1 in the HDB rescued the behavioral deficits, highlighting (open full item for complete abstract)

Committee: Wen-Cheng Xiong (Advisor); Lin Mei (Advisor); Peng Zhang (Committee Member); Hillel Chiel (Committee Member); Qian Sun (Committee Chair) Subjects: Neurosciences

Master of Science (MS), Wright State University, 2024, Microbiology and Immunology

Calcium signaling is a crucial regulator in many cellular processes, from immune response modulation to gene expression and apoptosis. Here, I have investigated the dynamics of calcium entry pathways, namely, store-operated calcium entry and store-independent calcium entry using a variety of cell types such as Jurkat T cells, HEK-293 cells and murine macrophages. Using fluorescence calcium imaging and selective inhibitor compounds, I have investigated the contributions of Orai and related calcium channels to calcium entry in both immune and non-immune cell types. The results demonstrate the pharmacological modulation of store-operated and store-independent calcium entry, showing the ability of compounds such as AE-19, AE-10, and EL-113 to inhibit calcium entry. Experiments involving membrane depolarization with elevated potassium concentrations confirmed that calcium entry through the plasma membrane is membrane potential-dependent. My results highlight the medical relevance of these channels, especially in the process of tubular aggregated myopathy, immune regulation and cancer. My studies may lead to therapeutics targeting calcium signaling pathways relevant to immune and muscular disorders.

Committee: J. Ashot Kozak Ph. D. (Advisor); Dawn Wooley Ph.D. (Committee Member); Marjorie M. Markopoulos Ph.D. (Committee Member) Subjects: Cellular Biology; Immunology; Microbiology; Neurosciences; Physiology

BS, Kent State University, 2023, College of Arts and Sciences / Department of Biological Sciences

The opioid epidemic is a major health crisis in the U.S., resulting in an estimated 80,816 deaths in 2021. Overdose results in Opioid-Induced Respiratory Depression (OIRD) often treated by administration of competitive opioid receptor antagonists such as naloxone. However, these drugs are not effective against highly potent synthetic opioids (e.g., fentanyl) and are poorly suited for the prevention and/or treatment of opioid-craving or addiction. D-cysteine ethyl ester (D-CYSee) has been shown to prevent OIRD and disrupt the acquisition of fentanyl-induced seeking behaviors in rats. The Hippocampus (Hipp) has been established as a vital region for the encoding of drug-associated cues and contexts. Thus, the effects of D-CYSee and fentanyl on intrinsic calcium activity in heterogeneous cell cultures derived from the Hipp of P0 Sprague Dawley rat pups were observed. Males and females were cultured separately so sex differences in the cellular response to fentanyl and D-CYSee could be determined. Cells were cultured for 12-days, loaded with a fluorescent Ca2+ probe, Cal-520 AM, and imaged on an inverted microscope. It was found that fentanyl induced a transient Ca2+ peak in neurons derived from males but not females and in astrocytes derived from both sexes. Pretreatment with D-CYSee prevented this peak in neurons but only modulated it in astrocytes. This demonstrated not only a cellular basis for D-CYSee's ability to prevent drug seeking behavior, but also a clear sexual dimorphism in cellular responding to exogenous opioids within the hippocampus.

Committee: Derek Damron (Advisor); Elda Hegmann (Committee Member); Aleisha Moore (Committee Member); Soumitra Basu (Committee Member) Subjects: Biology; Cellular Biology; Neurosciences; Pharmacology

Doctor of Philosophy (PhD), Wright State University, 2019, Biomedical Sciences PhD

Recovery from peripheral nerve injury (PNI) is dependent on the restoration of proprioception, a sensory modality that provides feedback to fine-tune movement. After PNI, persistent proprioceptive abnormalities limit improvement, leaving patients with disability, social and economic hardships, and decreased quality of life. The goal of this study is to better characterize the mechanisms responsible for proprioceptive deficits caused by PNI. We investigate calcium homeostasis in healthy proprioceptive neurons by using a transgenic mouse model to target expression of a genetically-encoded calcium indicator (GECI) to neurons containing parvalbumin (PV), a calcium-binding protein present in proprioceptors and low- threshold mechanoreceptors. We then utilize this technique to assess calcium homeostasis in two models of PNI known to produce proprioceptive deficits: sciatic nerve crush and sciatic nerve transection and resuture. Our results delineate the average parameters of calcium transients from PV DRG neurons at a population level, describe the diversity in calcium dynamics between cells, animals, and sexes, and illustrate that GECI calcium transients provide sufficient resolution to discern information about the activity of specific subclasses of calcium regulatory mechanisms. Injury experiments expose abnormalities in calcium homeostasis at multiple time-points following PNI and also reveal differences in calcium handling between injury models. Thus, this study establishes aberrant calcium homeostasis as an additional source of proprioceptive dysfunction following PNI. By enhancing our understanding of the mechanisms that prevent complete recovery from PNI, we can better inform research strategies aimed at treatment and improve outcomes for patients who have suffered PNI.

Committee: David R. Ladle Ph.D. (Advisor); Mill W. Miller Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member); Mark M. Rich M.D., Ph.D. (Committee Member); Keiichiro Susuki M.D., Ph.D. (Committee Member) Subjects: Neurosciences

Doctor of Philosophy, University of Toledo, 2018, Biology (Cell-Molecular Biology)

Neuromodulators have the capacity to alter neuronal excitability and synaptic strengths throughout the nervous system, allowing animals to switch between different behavioral states. The mechanisms by which neuromodulators change neuronal physiology, and the impact of those changes on circuit output and overall behavior, remain poorly understood. Neuromodulator-dependent changes in neuronal activity patterns are frequently measured using calcium reporters, since calcium imaging can easily be performed on intact functioning nervous systems. With only 302 neurons, the nematode Caenorhabditis elegans provides a relatively simple, yet powerful system to understand neuromodulation at the level of individual neurons. C. elegans is repelled by 1-octanol, and these aversive responses are modulated by monoamines and neuropeptides. Previously, we identified that serotonin (5-HT) suppresses 1-octanol Ca++ responses and potentiates depolarizations in the ASH in response to 1-octanol. This suggested that the net effect of 5-HT is disinhibitory. Here, I further dissect the pathway used by 5-HT to disinhibit the ASH and identify a Ca++-activated K+ channel known as SLO-1 acting downstream of Ca++. Furthermore, SLO-1 plays a critical role in regulating ASH Ca++ dynamics, with important consequences for aversive behavior and 5-HT modulation. Intriguingly, mutants lacking the SLO-1 accessory proteins DYB-1, BKIP-1 and ISLO-1 have similar phenotypes, suggesting that SLO-1 may need to be localized to specific Ca++ microdomains to properly modulate ASH responses. Finally, I show that other monoamines and neuropeptides effect the ASH Ca++ dynamics, suggesting that Ca++-dependent regulation of ASH signaling and excitability may be a central regulatory theme within the neuron.

Committee: Bruce Bamber (Committee Chair); David Giovannucci (Committee Member); Richard Komuniecki (Committee Member); Guofa Liu (Committee Member); Scott Molitor (Committee Member); Robert Steven (Committee Member) Subjects: Biology

Master of Science (MS), Wright State University, 2017, Physiology and Neuroscience

The carotid bodies (CB), the primary peripheral chemoreceptors, respond to changes in blood gases with neurotransmitter release, thereby increasing carotid sinus nerve firing frequency and ultimately correcting the pattern of breathing. It has previously been demonstrated that acute application of the adipokine leptin caused perturbations of intracellular calcium and membrane ion movement in isolated CB Type I cells (Pye et al, 2015) and augmented the response of the intact CB to hypoxia (Pye et al, 2016). This study's aim was to examine, in-vivo, if elevated leptin modulated CB function and breathing. Rats were fed high-fat chow or control chow for 16-weeks. High-fat fed (HFF) animals gained significantly more weight compared to control fed (CF) animals (n=18; p<.001; 512.56 g ± 14.70 g vs. 444.11 g ± 7.09 g). HFF animals also had significantly higher serum leptin levels compared to CF (n=18; p<.0001; 3.05 ng/mL ± 0.24 ng/mL vs. 1.29 ng/mL ± 0.12 ng/mL). Whole-body plethysmography was used to test the acute hypoxic ventilatory response (HVR) in unrestrained, conscious animals. HFF animals had an attenuated 2nd-phase of the HVR when compared to CF (n=18; p<.05; 710.1 ± 41.9 mL kg-1 min-1 vs. 855.4 ± 44.05 mL kg-1 min-1). CB Type I cells were isolated and intracellular calcium measured; no significant differences in the cellular hypoxic responses between groups were observed. These data show differences in the 2nd-phase of the HVR caused by high fat feeding are unlikely to be caused by an action of leptin on the Type I cells. However the possibility remains that leptin may have in-vivo postsynaptic effects on the carotid sinus nerve; this remains to be investigated.

Committee: Christopher Wyatt Ph.D. (Advisor); Eric Bennett Ph.D. (Other); David Ladle Ph.D. (Committee Member); Mark Rich M.D./Ph.D. (Committee Member); Robert Fyffe Ph.D. (Other) Subjects: Biology; Cellular Biology; Neurosciences; Physiology

Doctor of Philosophy, University of Toledo, 2015, Biology (Cell-Molecular Biology)

Neuromodulation in sensory circuits is critical because it allows an organism to respond appropriately to a given stimulus. Sensory systems are modulated by monoamine neurotransmitters as well as neuropeptides, which act in concert to regulate sensory circuits to give rise to complex behavioral states. One common technique for studying sensory circuits is brain activity mapping, where circuits are probed with fluorescent indicators whose readouts are related directly to neuronal activity. In the present work, we focus on the modulation of a pair of sensory neurons, the ASHs, in the nematode Caenorhabditis elegans. ASHs are polymodal, nociceptive neurons that are extensively modulated by monoamines and neuropeptides. Using a combination of genetics, Ca2+ imaging, electrophysiology, and behavioral assays, we have identified a complex instance where the monoamine serotonin (5-HT) stimulates aversive behaviors and neuronal depolarization, but decreases sensory-evoked Ca2+ signals, indicating that the recorded Ca2+ levels do not positively correlate with neuronal activity. Mechanistically, 5-HT is likely acting through the SER-5 receptor and Gαq signaling in ASHs to downregulate Ca2+ directly by initiating a Ca2+-driven negative feedback loop targeting the L-type Ca2+ channel EGL-19. Together, these studies reveal a complex inhibitory feedback mechanism for sensory modulation, and have broad implications for activity mapping of complex neural circuits.

Committee: Bruce Bamber PhD (Committee Chair) Subjects: Biology; Neurobiology; Neurosciences

Doctor of Philosophy (PhD), Wright State University, 2015, Biomedical Sciences PhD

Obesity is the healthcare crisis of our generation. Circulating levels of the adipokine leptin are proportional to adiposity and in addition to its role as a satiety factor leptin has been shown to have a host of pleiotropic effects. Among these effects is its role as a respiratory stimulant where it has been shown to have both whole body and central stimulatory effects on breathing. Porzionato et al. (2011) showed that leptin and leptin receptors are selectively expressed in the chemosensing type I cells of the carotid body. Carotid bodies are small chemosensitive organs that act as the primary peripheral oxygen sensors, sitting bilaterally in the bifurcations of the left and right common carotid arteries. In addition to sampling blood gasses on their way to the brain they play a role in setting central sensitivity to CO2 and play a key role in sleep apnea induced hypertension. Whilst leptin receptors in the carotid body have been shown to be functional, the acute physiological effects of leptin on type I cell function has not been assessed. Work presented here shows that acutely administered leptin: (1) increases intracellular Ca2+ that is dependent on extracellular Ca2+ and PI3Kinase in type I cells; (2) increases membrane conductance of type I cells in a BKCa dependent manner, without altering membrane potential – suggesting that BKCa channels are not open at resting membrane potential in CB type I cells; (3) does not increase acid or hypoxia induced membrane depolarization in isolated CB type I cells, but does increase baseline activity and the size of hypoxic responses in arterially perfused ex-vivo carotid body preparations. This work demonstrates that leptin affects CB function acutely. Investigating the effects of chronic hyperleptinemia on carotid body activity would appear to be warranted based on these findings.

Committee: Christopher Wyatt Ph.D. (Advisor); Thomas Brown Ph.D. (Committee Member); Madhavi Kadakia Ph.D. (Committee Member); Robert Putnam Ph.D. (Committee Member); Nicholas Reo Ph.D. (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, University of Toledo, 2015, Biology (Cell-Molecular Biology)

Dysfunctional integration of sensory information is associated with numerous pathological conditions in the human nervous system, including autism spectrum disorders, attention deficit hyperactivity disorder (ADHD), and schizophrenia. In the model organism Caenorhabditis elegans, the noxious odorant 1-octanol is primarily sensed by the ASH sensory neurons, and presentation of the odorant causes animals to initiate an aversive response that is modulated by the presence of food or serotonin (5- HT). However, in the present study we found that presentation of 1-octanol to animals activates, inhibits, and has no effect on the glutamatergic ASH, AWC, and ASE right (ASER) sensory neurons, respectively. The ASHs, AWCs, and ASER all synapse onto a common postsynaptic pair of interneurons, the AIB interneurons. The AIB interneurons profoundly affect animal behavior. Based on indirect evidence, the leading hypothesis states that some of the upstream glutamatergic sensory neurons signal onto the AIBs through receptors containing the AMPA-type subunit GLR- 1. Using whole-cell patch clamp electrophysiology and calcium imaging, we found that glutamate evokes both an anionic current mediated by the glutamate-gated Cl- (GluCl) channel subunit AVR-14, and a cationic GLR-1-dependent current in the AIBs. Similarly, previous work has shown that tonic glutamate release from the AWCs and ASER onto GLR-1 and AVR-14 on the AIBs, respectively, differentially modulate 1- octanol-evoked aversive behaviors. Additionally, the L-type voltage-gated calcium channel (VGCC) subunit EGL-19 amplifies glutamate-evoked increases in AIB intracellular Ca2+ (iCa2+). In addition to glutamate, acetylcholine also evokes an inhibitory current in the AIBs that is partially mediated by the acetylcholine-gated Cl- (AChCl) channel subunit ACC-1. Interestingly, the AIA interneurons are cholinergic, direct much of their synaptic output onto the AIBs, are thought to inhibit the AIBs, and are also innervated by the A (open full item for complete abstract)

Committee: Bruce Bamber PhD (Committee Chair); Richard Komuniecki PhD (Committee Member); Robert Steven PhD (Committee Member); Guofa Liu PhD (Committee Member); Scott Molitor PhD (Committee Member); John Bellizzi PhD (Committee Member) Subjects: Neurobiology

Master of Science (MS), Wright State University, 2015, Physiology and Neuroscience

Somatostatin (SST) is a neuropeptide hormone that regulates the release of secondary hormones. Evidence suggests SST plays a neuromodulatory role due to its distribution throughout the central nervous system. Interestingly, SST has been suggested to affect the carotid body, the small peripheral chemoreceptors that regulate breathing. It has been shown that the peripheral chemoreflex sensitivity to CO2 and hypoxia is reduced by SST in humans (Pedersen et al., 1999; Pandit et al., 2014). SST has also been found to inhibit whole cell Ca2+ currents recorded from adult rat carotid body type I cells (e Silva & Lewis, 1995), but the mechanism by which this occurs is unknown. This study aimed to identify the types of SST receptors (SSTR) on type I cells and confirm the mechanism by which their activation inhibits Ca2+ influx. Specific antibodies were used to identify SSTR1-5 on type I cells, and results showed that SSTR1-5 were present on the membrane and cytoplasmically in type I cells. To record intracellular Ca2+ entry, type I cells were loaded with FURA-2 (5 µM) and depolarized in response to stimuli, including 80 mM K+ and hypoxia. Type I cells applied with 2, 10, and 20 minute applications of 1 µM SST had no significant inhibition on voltage-gated Ca2+ entry compared to controls. Also, a 2 minute application of 1 µM SST did not significantly inhibit Ca2+ influx in adult rat type I cells when compared to controls. These results indicate that 1 µM SST does not significantly inhibit K+- nor hypoxic-evoked Ca2+ influx in isolated type I cells from carotid bodies. Thus the mechanism by which SST inhibits the acute ventilatory response to hypoxia and hypercapnia is not likely via inhibition of voltage-gated Ca2+ influx nor via inhibition of the chemosensory response of type I cells.

Committee: Christopher Wyatt Ph.D. (Advisor); Adrian Corbett Ph.D. (Committee Member); Sherif Elbasiouny Ph.D. (Committee Member) Subjects: Neurosciences; Pharmacology; Physiology

Master of Science (MS), Bowling Green State University, 2010, Biological Sciences

Nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) is the sole pathway for direct communication between the cytoplasm and the nucleoplasm of eukaryotic cells. NPC allows unregulated passive diffusion of small molecules (< 20 kDa – 40kDa) and facilitated translocation of larger molecules (up to 500 MDa). More and more evidence suggests that calcium stored in the lumen of nuclear envelope and endoplasmic reticulum may further regulate nucleocytoplasmic transport. However with challenges in direct measurements of transport kinetics in the NPC, the calcium-regulated mechanism is still poorly understood. Here single-molecule fluorescence microscopy was used to characterize the nuclear pore permeability of passive diffusion and facilitated translocation under various calcium store concentrations. By snapshots of real-time transient movements of small molecules (10 kDa dextran) and large molecules (97 kDa protein -importin β1 (Imp β), through the NPCs, novel features under real-time trafficking conditions were observed that escaped detection by ensemble measurements. It was found that: i) transport rates cannot be used to reflect the change of nuclear pore permeability, which was mistakenly used in previous ensemble experiments; ii) transport rates for both passive diffusion and facilitated translocation can be significantly affected by the store calcium concentrations; and iii) nuclear pore permeability for passive diffusion is affected more by the amount of stored calcium than that for facilitated translocation.

Committee: Dr. Weidong Yang (Advisor); Dr. Carol Heckman (Committee Member); Dr. Paul Morris (Committee Member) Subjects: Biology; Cellular Biology