Browsing by Subject "Neuroscience"

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The action of xanomeline on human muscarinic receptors expressed in Chinese hamster ovary cells.
(2009-01) Noetzel, Meredith
Muscarinic acetylcholine receptors are part of the G protein-coupled superfamily of receptors. There are five subtypes of muscarinic receptors (M1-M5). These receptors are therapeutic targets for in a number of diseases, including Alzheimer's disease and schizophrenia. The orthosteric (primary) binding domain of muscarinic receptors is highly conserved across subtypes making it difficult to develop agonists or antagonists that bind with selectivity at a particular subtype. However, there are allosteric (secondary) binding sites on muscarinic receptors that are thought to be less well conserved between subtypes, which may lead to the development of drugs that bind in a subtype-selective manner. One potential drug of interest is xanomeline. Previous research has found that it binds in a unique manner to muscarinic receptors; it binds in a reversible manner at the orthosteric site and in a wash-resistant manner at an allosteric site. Furthermore, xanomeline is thought to be an M1/M4 selective agonist. The goal of the current research was to characterize the effects of prolonged exposure of the various receptor subtypes to xanomeline and determine the mechanisms through which these effects occur. To do this Chinese hamster ovary cells expressing the various individual muscarinic receptors were exposed to xanomeline for brief and long-term time periods and radioligand binding and functional assays were performed. Xanomeline was shown to bind in a reversible and wash-resistant manner at the M1-M4 receptor subtypes. Although the effects of acute xanomeline exposure were similar across subtypes, long-term treatment with xanomeline resulted in differential effects among subtypes. More detailed experiments were conducted at the M1 and M3 receptor subtypes to elucidate the mechanisms of the long-term effects of xanomeline. Wash-resistant xanomeline binding was able to modulate the functional properties of the receptor. The consequences of the long-term effects of wash-resistant xanomeline binding are dependent on activation of the receptor through the orthosteric binding site. It appears that these effects are a result of receptor internalization and allosteric modulation of the receptor. Thus, my research demonstrates that it is important to determine both the acute and long-term drug interactions with the receptor as part of the drug development process.
Alpha-2 adrenergic and opioid spinal analgesic synergy: utility and cellular mechanisms.
(2010-06) Overland, Aaron C.
Agonists acting at spinal α 2 -adrenergic receptors (α 2 AR) and opioid receptors (OR) produce analgesia through common intracellular signaling systems primarily mediated through inhibitory G proteins. Furthermore, co-activation of spinal α 2 AR and OR produces antinociceptive synergy. Synergistic analgesic interactions are important, as conventional opioid therapy is limited clinically due to the development of adverse side effects such as tolerance, dependence, abuse liability and opioid-induced hyperalgesia. Agonist combinations that interact synergistically may bypass these unwanted side effects by allowing decreases of analgesic dose and increasing the therapeutic index. Synergy between analgesic compounds has been shown experimentally and utilized clinically, yet the underlying cellular mechanisms mediating this phenomenon remain relatively unexplored. Elucidating the mechanisms underlying analgesic synergy may have broad clinical implications and may lead to the discovery of novel drug targets for pain management. The goal of this study was therefore to determine the cellular mechanisms mediating the synergistic interaction between agonists acting at two anatomically co-localized G protein-coupled receptors (GPCRs) in the spinal cord. In the first phase of these studies, we evaluated the ability of the selective delta-opioid receptor (DOP) agonist deltorphin II (DELT), the α 2 AR agonist clonidine (CLON) or their combination to inhibit nociceptive responses from mice in the tail flick test. We then examined the possible underlying signaling mechanisms involved through co-administration of inhibitors known to affect the above-mentioned receptor pair. Second, we looked at the ability of the DOP-selective agonist DELT, the α 2 AR agonist CLON, or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices and spinal cord synaptosomes. Third, we determined the specific signaling mediator involved in α 2 AR/DOP analgesic synergy using genetically manipulated mice. We observed that the in vivo and in vitro synergistic interaction between agonists acting at α 2 AR/DOP is specifically mediated through activation of protein kinase C epsilon. These findings suggest that this particular enzyme could represent a novel pain therapy target.
ARP2/3 complex has a neuroprotective role and is required for mature dendritic spine head morphology
(2010-08) Maldonado, Marcela
Several lines of evidence suggest that Arp2/3 may play a role in regulating dendritic spine morphology. First, inhibition of the Arp2/3 activators N-WASP, Cortactin, and Wave alters spine morphology and density (Racz and Weinberg 2004, Pipel and Segal 2005, Soderling et al. 2007b, Wegner et al. 2008). Second, electron microscopy of dendritic spines revealed that the actin filaments within the spine head appear to be organized in "Y" shaped branches (Fifkova and Delay 1982). Since, Arp2/3 is the only complex known to make "Y" branched actin filaments, the presence of such branches in spine heads strongly suggests that Arp2/3 is involved in actin polymerization in spines. Consequently, the general hypothesis for my doctoral work is that if Arp2/3 is a major regulator of dendritic spine morphology, then Arp2/3 will be enriched in dendritic spines heads and inhibition of Arp2/3 activity will alter the number and/or morphology of dendritic spines. Our results show Arp2/3 localization within the dendritic spine heads of cultured hippocampal neurons. However, we observed Arp2/3 redistribution within dendritic shafts in response to induced synaptic activity. Temporal inhibition of Arp2/3 function during dendritic spine development showed severe morphological consequence in mature cultures. Finally, in collaboration with Dr. Robert Meller of Dow Neurobiology Laboratory at Legacy Research, Legacy Health in Portland Oregon, we show that Arp2/3 has a neuroprotective role of in ischemia tolerance.
Behavioral and neurobiological consequences of intermittent exposure to addictive drugs.
(2010-02) Rothwell, Patrick Eldredge
These studies were undertaken to better understand how repeated exposure to addictive drugs leads to adaptations in brain function and behavior related to the development of addiction. They are predicated on evidence that the mere presence of a drug in the body is not the sole determinant of adaptation - rather, the pattern of drug exposure is a key variable, with intermittent exposure making the brain reward system increasingly sensitive to drugs and leaving individuals susceptible to relapse. These experiments were designed to examine whether events occurring during the offset of drug action may contribute to the unique effects of intermittent drug exposure. The first series of experiments develops a set of behavioral measures that can be used to resolve and quantify a state of acute withdrawal caused by the offset of drug action. The second series of experiments utilizes these measures to investigate whether recurrent episodes of acute withdrawal contribute to the development of psychomotor sensitization - a specific consequence of intermittent drug exposure related to adaptations in the brain reward system. The final series of experiments describes a specific synaptic adaptation in a key component of the brain reward system (the nucleus accumbens) that is caused by intermittent drug exposure, related to the development of psychomotor sensitization, and reversed by experiences linked to relapse. The results of these studies suggests new and provocative interactions between neural circuits mediating reward and aversion, which may help identify and explain forms of neural plasticity that underlie the development of drug addiction.
The blockade of endocytosis of GluR2-containing AMPARs in the nucleus accumbens and its effect on reinstatement for cocaine-seeking behavior
(2016-08-11) Back, Johanna J
Neural plasticity related to drug addiction can be modeled by behavioral sensitization in animals through the chronic administration of drugs. Long term-de-potentiation (LTD) in the nucleus accumbens (NAc) is proposed to be involved in neural plasticity resulting from addictive drugs. The exact mechanism of LTD remains unknown; however, previous experiments have linked LTD with cocaine re-exposure and the endocytosis of GluR2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) in the NAc. In this experiment, we looked at the relapse related behavior resulting from this neural plasticity. Mice, after ten days of self-administration of cocaine and seven days of extinction, were given infusions of an inactive or active TAT peptide designed to block the endocytosis of the AMPARs. We hypothesized that this would block LTD in the NAc and therefore decrease cocaine seeking behavior seen after relapse. Reinstatement then took place with the injection of cocaine to induce relapse. The results of this experiment showed a difference in response to the active and the inactive peptide. The mice with the active peptide TAT infusions showed successful response inhibition, a decrease in reinstatement of cocaine seeking behavior.
Cannabinoid modulation of nociception and nociceptor activity during inflammation.
(2009-06) Potenzieri, Carl Robert
Previous studies have demonstrated that peripherally-administered cannabinoids at the site of injury produce antinociception in animal models of acute and persistent pain. Peripheral cannabinoid one (CB1) receptor-mediated antinociception has been attributed to CB1 receptors located on nociceptive DRG neurons and their peripheral nerve terminals. Although these studies suggest that activation of peripheral CB1 receptors located on nociceptive nerve terminals produces antinociception, how cannabinoids modulate nociceptor activity is not known. The overall aim of this thesis was to relate the behavioral antinociceptive effects of locally-administered cannabinoids with changes in the response properties of nociceptors during non-inflamed and inflamed conditions. It was hypothesized that activation of peripheral CB1 receptors attenuated nociception and nociceptor activity only during inflammation. In behavioral studies, intraplantar administration of complete Freund's adjuvant (CFA), but not saline, produced mechanical allodynia, mechanical hyperalgesia, and heat hyperalgesia. Activation of peripheral CB1 receptors produced antiallodynia and antihyperalgesia following inflammation, but did not alter nociception during non-inflamed conditions. In electrophysiological studies, only cutaneous nociceptors (Adelta and C) from inflamed skin were sensitized, and not Abeta mechanoreceptors. Local administration of CB1 receptor agonists attenuated mechanically-evoked responses of Adelta nociceptors from inflamed skin, but did not alter the evoked responses of Adelta nociceptors from non-inflamed skin. The responses of C nociceptors and Abeta mechanoreceptors from either non-inflamed or inflamed skin were not altered following local administration of cannabinoids. Our results demonstrated that peripherally-mediated cannabinoid antinociception through CB1 receptors is mediated, at least in part, by attenuation of Adelta nociceptor activity. The results from the present studies suggest that peripherally-acting CB1 receptor agonists could be administered alone or co-administered with other analgesic drugs to treat acute and persistent pain in humans and animals.
Characterization of blood flow in the retinal vascular network
(2015-01) Kornfield, Tess Ellen
The primary goal of the work presented here is to understand how blood flow is regulated in the retinal vascular network in response to neuronal activity. In order to accurately quantify blood flow, we developed a multitude of streamlined techniques capable of measuring many properties of blood flow. These techniques were used to investigate retinal functional hyperemia, defined as the increase in local blood flow that occurs in response to nearby neuronal activity. We did a comprehensive survey of all retinal vessels to investigate the magnitude and timing of the functional hyperemia response as it presents in the different compartments of the retinal vascular network. We found that arterioles are primarily responsible for generating functional hyperemia in the retina and that, with prolonged stimulation, blood flow through the three vascular layers in the retina is differentially regulated. This result implies the presence of active capillary dilation. The work in this dissertation informs our understanding of blood flow regulation within the retinal vascular network.
Characterization of Parkinsonian Neuropathophysiology and its Modulation by Deep Brain Stimulation in the Behaving, Nonhuman Primate Model
(2016) Weinstock, Zachary;
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by debilitating motor disturbances. It is believed that the signs and symptoms of PD are caused by idiopathic cell death in the basal ganglia (BG), a network of subcortical nuclei with a well-established connection to motor control. Although there is no cure for PD, deep brain stimulation (DBS) of the internal Globus pallidus, a constituent nucleus of the BG, offers hope for patients who don’t respond well to conventional medications. However, the mechanisms underlying the therapeutic effects of DBS remains unclear, a fact largely attributed to a poorly characterized pathophysiology. Here we identify characteristic, electrophysiolgical biomarkers of PD that preempt the emergence of its behavioral signs and show that DBS works to shift cortical activity back towards a more “normal” state. Using a behaving non-human primate model of PD, we observed a disruption in the normal firing patterns and frequencies of both single motor units and neuronal populations in the primary motor cortex (M1) and supplementary motor areas (SMA) following the induction of parkinsonism. During DBS, we observed an increase in task-related neuromodulation. Taken together, our results hint at a therapeutic mechanism for DBS whereby signaling in M1 and SMA is made more salient and shifted towards “normal” activity. We anticipate that our findings will serve to guide future research and instruct the development of more effective, adaptive DBS technologies.
Consequences of dietarily- and genetically-induced iron deficiency on the neurodevelopment and experience-dependent plasticity of the hippocampus.
(2008-12) Carlson, Erik Sean
Iron deficiency (ID) is the most prevalent micronutrient deficiency in the world, affecting infants in both industrialized and developing countries. Children are at risk for ID during toddlerhood, and infants are at risk for ID during late gestation and early infancy due to severe maternal ID and pregnancies complicated by intrauterine growth restriction or diabetes mellitus. In both of these populations, there is evidence for both acute (during the period of ID) and long-term (after iron repletion) cognitive abnormalities. Animal models of early ID also show cognitive abnormalities, with iron deficient rat pups performing worse on spatial and recognition memory tasks. One caveat of these models is that dietary ID in the rat brain is liable to affect multiple brain systems and it is difficult to parse out the specific contribution of ID to the abnormal development seen in infants and rats. Furthermore, it is unclear whether the effect on the hippocampus is due to the lack of iron or from other processes occurring in conjunction with ID (e.g. maternal stress, hypoxia, anemia). Additionally, the effects of ID during gestation appear to target recognition memory circuitry, as opposed to other brain structures. In other words, dietary models of iron deficiency are not entirely congruent with the human disease being modeled. For this dissertation I have 3 objectives: 1) To quantitate differences due to dietary ID in mRNAs and proteins salient to hippocampal development and function during ID, and after iron repletion in rat. 2) To delineate the role of the murine Slc11a2 (divalent metal ion transporter-1) in neuronal iron uptake during hippocampal development, to determine the molecular, iii neurometabolomic, and behavioral alterations occurring due to hippocampal-specific ID during development. 3) To determine the effects of a timed reduction of hippocampal iron uptake on iron metabolism and function by characterizing a mouse carrying a hippocampus-specific tet–responsive transgene of a dominant negative TfR-1 (transferrin receptor-1). Thus, the specific aims focus on the role of iron in the developing hippocampus and ultimately to determine the contribution of hippocampal dysfunction to the overall clinical spectrum of abnormal cognitive behavior seen with early ID.
Contributions of visual area V4 to rapid shape detection
(2014-09) Weiner, Katherine L.
Vision in foveate animals is an active process that requires rapid and constant decision-making. For example, whenever a new object of potential interest appears in the visual field, we quickly decide whether or not to inspect it by directing our eyes to the object's location. We studied the contribution of primate area V4 to these types of rapid foveation decisions. We trained Macaca mulatta to saccade to a peripherally presented shape embedded in dynamic noise as soon as the shape appeared. While the monkeys performed the task, we recorded from neurons in area V4, a visual area thought to be involved in form perception. We found that approximately half of the randomly sampled V4 neurons not only rapidly and precisely represented the appearance of this shape, but they were also predictive of the animal's saccades. In approximately seven percent of our recorded sample, individual neurons were able to predict both the delay and precision of the animal's shape detection performance, suggesting that a subset of V4 neurons may have been directly and causally contributing to task performance. To examine how groups of such neurons might act in concert, we recorded from 5 to 29 V4 neurons simultaneously during the task. While modest correlations were present between pairs of cells during visual stimulation, their magnitude did not change significantly subsequent to the appearance of a shape. We quantified the reliability and temporal precision of pairs and larger populations of neurons to signal the appearance of the shape using mutual information analyses. We found that removing correlations by shuffling across trials did not affect the reliability or timing with which pairs, or larger groups of cells, signaled the presence of a shape. Our findings suggest that a small fraction of neurons in area V4 contribute independently to rapid shape detection.
Creating and applying a cognitive change model: a transdisciplinary (education, cognitive psychology, neuroscience) approach
(2013-05) Meyer, John Edward
This study uses qualitative data and literature from various disciplines to shed light on the complex phenomenon of cognitive change, especially as it occurs within educators. The resulting understandings are used to develop both verbal and visual models to illustrate the dynamics of such a transformative mental change. The qualitative data represents reflections of individual participants in a collaborative leadership problem-solving virtual environment designed to elicit cognitive conflict and potentially resulting in new understandings about power. This data was analyzed iteratively with research and literature from education, cognitive psychology, and neuroscience to gain a complete picture of cognitive change from an individual perspective. The study tests the cognitive change model's usefulness by applying it to individual participants' experiences.
D-serine: a study of its function and regulation in the retina.
(2009-10) Gustafson, Eric Charles
Activation of the NMDA-type glutamate receptor requires the simultaneous binding of both glutamate and a coagonist, either glycine or D-serine. In the inner retina, glutamate released from bipolar cells excites NMDA receptors on retinal ganglion cells and some amacrine cells. The identity of the coagonist, however, has remained unknown. Early on, the relatively high levels of glycine and its use in the retina as an inhibitory transmitter by a subset of amacrine cells led many to believe that glycine was the endogenous coagonist. The discovery that D-serine and its synthesizing enzyme, serine racemase, are both present in the retina suggested that D-serine may play a role as well. This manuscript reports results that have examined the role of D-serine in the retinas of larval tiger salamanders and in mice. These studies suggest that D-serine is the major endogenous coagonist during light-induced responses in the inner retina of both species. In addition, the regulatory mechanisms of glycine transport and of the endogenous D-serine degrading enzyme, D-amino acid oxidase, have been shown to be essential in maintaining coagonist levels below that needed to saturate the NMDA receptor. Together, the results position D-serine as a major contributor and potential modulator of excitatory neurotransmission in the retina.
Decision making gone awry: Dorsal striatum, decision-making, and addiction
(2015-02) Regier, Paul
Millions of people use addictive substances, such as alcohol and cocaine, however only a subset of individuals become dependent on these types of substances. Addiction can be thought of as a maladaptive decision-making process, driven by distinct neural regions. As behavior shifts from goal-directed to habit-based behavior, control of this behavior by corticostriatal circuits shifts from the associative circuit, which includes the dorsomedial striatum, to the sensorimotor circuit, which includes dorsolateral striatum. Once behavior becomes more habit-based, and control shifts to the sensorimotor corticostriatal circuit, actions become difficult to devalue. Thus, behavior becomes difficult to change. In this dissertation, I explore a behavioral shift to habit-based behavior as one potential way addiction can occur. I focus on the dorsomedial and the dorsolateral striatum and the role of these two regions in goal-directed and habit-based behavior, respectively, and the role of these two regions in drug-seeking behavior. In addition, I discuss the dorsomedial and dorsolateral striatum in relation to animal models of drug addiction that differentially seek drugs, and I discuss these two regions as potential biomarkers of addiction treatment. Finally, I relate previous research as well as my own research, presented throughout the dissertation, to human drug use and consider how analogues of dorsal striatum in the human brain might play a role in human addiction and addiction treatment. In all, consideration of addiction as a maladaptive decision-making process as well as understanding the neural correlates of this process may help to generate new ways of perceiving, studying, and treating addiction.
Development of a Neuroinformatics Pipeline and its Application to Gene-environment Interaction in Neurodegenerative Disease
(2020-12) Overgaard, Shauna
BackgroundAlzheimer’s disease is a neurodegenerative disease and the sixth-leading cause of death in the United States. While there has been a greater understanding of Alzheimer’s disease (AD) processes in the last two decades, clinical trials in AD have not been successful, suggesting that further research is needed to understand key questions pertaining to the underpinnings of the disease. Alzheimer’s disease is a complex disease with significant heterogeneity in the disease progression and expression of clinical symptoms. The presence of the ϵ4 allele of the Apolipoprotein (APOE4) increases the risk of Alzheimer’s disease and is associated with earlier onset of Alzheimer’s disease pathology. On the other hand, variability in “Resilience,” i.e., the ability to cope with Alzheimer’s disease pathology, is associated with the differences in the expression of clinical symptoms. Objective The work presented draws on the methodological strengths of health informatics, biostatistics, and neuroscience, to achieve two specific aims: (1) Deployment of a neuroinformatics pipeline for the replicable collection, manipulation, and analysis of AD data (2) Employment of the neuroinformatics pipeline to evaluate the potential impact of specific allele carriership on what is recognized as a resilience mechanisms in the context of AD. Specifically, the neuroscience questions are: (1) Does the effect of cognitive reserve on GMD differ by APOE4 genotype? (2) Does APOE4 carrier status impact clinical functioning, and is this effect mediated by global efficiency? (3) Do APOE4 carriers as compared to non-carriers demonstrate differences in network recruitment (specifically, global efficiency of the default mode network)? Methods To evaluate the complex interplay of gene-environment interactions in AD, we investigated the impact of APOE4 and education on brain structure in the first study. In our second study, we used a core construct from graph theory to compute global efficiency on single-subject 3T MRI scans and evaluated the interplay between pathology, APOE4, education, and global efficiency and their impact on clinical functioning. In our third study, we applied causal inference models to investigate the causal relationships between pathology, APOE4, education, and global efficiency, considered drivers of clinical functioning in AD. Conclusion This work uniquely contributes to health informatics through the construction of a neuroinformatics pipeline that combines multimodal biomedical data (neuroimaging, genomics, cognition, and clinical), employs database management, automated computing, graph theory, and biostatistics to answer clinical questions. This work contributes to science by proposing a method to measure and monitor brain health, providing additional insight into the mechanistic underpinnings of APOE4 allele carriership underlying AD pathology.
Differential regulation of opioid receptors during inflammation
(2009-07) Satterfield, Catherine Suzanne
Properties of the opium poppy have been exploited for centuries for the alleviation of pain and to induce euphoria. Classically thought to produce its effects solely in the central nervous system, peripheral opioid analgesic systems are now widely accepted. The activation of these systems leads to a reduction in primary afferent fiber excitability leading to the inhibition of sensory transduction. Opioid receptors function is modulated by a variety of mechanisms. An example of this is enhanced peripheral opioid receptor function following inflammation. The present study examined peripheral opioid receptor regulation in early and late stages of CFA inflammation. Additionally, a new model of UVB of inflammation was characterized. Peripheral MOR receptors are differentially regulated in late and early CFA inflammation. Peripheral MOR is not responsible for attenuated responses of nociceptors to mechanical stimuli 18 hours after CFA inflammation. DAMGO reduced mechanical responsiveness of nociceptors at 72 hours after CFA inflammation in a concentration and antagonist reversible manner indicating that MOR efficacy is enhanced during later stages of CFA inflammation. UVB produced severe but localized inflammation that differed from inflammation produced by CFA. This inflammation sensitizes nociceptor units innervating irradiated skin and results in enhanced peripheral opioid receptor efficacy.
Dissociating Cortico-Striato-Thalamo-Cortical Neural Circuitry Using Rodent Models of Cognitive Flexibility
(2021-04-12) Cooper, Dawson C
Mental illness is the single largest cause of disability worldwide. These disorders are characterized by breakdowns in neuronal communication between and among different areas of the brain. In order to restore proper functioning, treatment strategies have increasingly focused on modulating specific neuronal circuits. Deep brain stimulation (DBS) allows for targeted circuit-based neuromodulation and has shown to be a promising treatment for mental disorders. Despite its success, the mechanisms underlying its therapeutic effects remain unclear. Further investigating cortico-striato-thalamo-cortical (CSTC) circuits, often impaired in those with major depressive (MDD) or obsessive-compulsive disorder (OCD), may provide mechanistic clues. MDD and OCD can be characterized by impairments in cognitive control—the ability to organize, plan, and schedule mental operations in different environments. Cognitive control depends on distinct subregions of the prefrontal cortex (PFC) which project into the striatum. Here we show that DBS applied to the mid-striatum in an attentional set-shifting task improves cognitive flexibility in outbred rats (n=12) by significantly decreasing reaction time (p < 0.01). Furthermore, we developed a novel touchscreen two-armed bandit task which may help in determining which parts of the PFC are responsible for DBS’ effects on cognitive flexibility. Our results demonstrate that DBS is able to modulate the neural circuitry underlying cognitive flexibility and that Long-Evans rats can serve as a viable animal model in translating the two-armed bandit behavioral paradigm. Our future study will evaluate the effects of DBS in both set-shifting and the two-armed bandit. Behavioral paradigms with an increased dependency on more ventral parts of the PFC, involved in the two-armed bandit, are hypothesized to not benefit from mid-striatum DBS treatment. Our results may translate to human behavioral tasks and serve as a predictor for DBS’ effectiveness.
Duchenne muscular dystrophy and extraocular muscle: a potential sparing mechanism with therapeutic implications.
(2009-10) Kallestad, Kristen Marie
This project investigates the role of extraocular muscle (EOM) progenitor cells in sparing the muscles from pathology associated with Duchenne Muscular Dystrophy (DMD). Mouse models of muscular dystrophy and wild type mice were analyzed by flow cytometry and cell culture for the size, heterogeneity and functional characteristics of stem and satellite cell populations of EOM and limb muscles. EOM have a 5-fold increase in progenitor cells compared with limb muscles. Additionally, an enriched population of cells expressing the stem cell marker CD34 but no other typical stem or differentiation markers (Sca-1, CD45, CD31, pax-7, m-cadherin) exists in the EOM. We refer to this population as EOMCD34 cells. The EOMCD34 cells are present in developing muscle, but only maintained in adult EOM, surviving in very aged animals. The EOMCD34 cells are also present in EOM of DMD model animals, but not their limb muscles. EOMCD34 cells are resistant to apoptosis and proliferate in vivo. Finally, these cells are capable of forming myotubes in vitro. The EOMCD34 cells may represent a primitive stem cell population, which is capable of maintaining life-long pools of myogenic precursor cells. Since EOM continuously remodel throughout life, unlike other skeletal muscle, it is logical that the proliferative potential of their precursor cells is enhanced. Since one proposed mechanism of DMD muscle destruction is exhaustion of the reparative progenitor cells, the EOMCD34 cells might prove useful for myoblast transplant therapies for DMD.
Dynamic regulation of the NMDA receptor coagonist D-serine in the mammalian retina.
(2011-09) Sullivan, Steven J.
The N-methyl D-aspartate (NMDA) receptor coagonist D-serine is important in a number of different processes in the central nervous system, ranging from synaptic plasticity to disease states, including schizophrenia. In the retina, light-evoked responses of retinal ganglion cells are shaped in part by NMDA receptors which require a coagonist for activation. There is debate over whether glycine or D-serine is the endogenous coagonist of retinal ganglion NMDA receptors. I used a mutant mouse lacking functional serine racemase (SRKO), the only known D-serine synthesizing enzyme in mammals, to show that retinal ganglion cells depend on D-serine for NMDAR activation (chapter 1). Most changes in NMDA receptor currents during synaptic activity have been attributed to glutamate fluctuations against a steady background of coagonist, excluding the possibility of dynamic coagonist release. The retina is a particularly useful system to determine if coagonist release occurs in the nervous system, because it can be naturally stimulated with light. By saturating the glutamate binding site of NMDA receptors, I was able to measure coagonist release during light-evoked responses. Coagonist release was detected in retinal ganglion cell light responses and depended on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic AMPA receptors. Coagonist release was significantly lower in SRKO mice (chapter 2). By directly measuring extracellular D-serine using capillary electrophoresis, I demonstrated that D-serine can be released from the intact mouse retina through an AMPA receptor dependent mechanism (chapter 3). The collective works put forth in this thesis imply that activity-dependent modulation of D-serine availability may add an extra dimension to NMDA receptor coincidence detection in the central nervous system.
Early iron deficiency anemia alters structure in pyramidal neuron apical dendrites and cytoskeletal modifiers associated with dendrite development in the hippocampus.
(2009-09) Brunette, Katyarina Efimenko
Using a dietary model in Sprague Dawley rats of gestational/neonatal iron deficiency, our lab has demonstrated structural, biochemical, electrophysiological and behavioral changes in the developing hippocampus. The high energy demands of the developing hippocampus make it particularly vulnerable to iron deficits. At gestational day two, the pregnant dams were given iron deficient (ID) chow to induce approximately 50 % brain iron deficiency by postnatal day (P)15. Our dietary model allowed us to observe changes during ID at P15 and P30 and also after iron repletion at P70. Dendritic changes have been demonstrated with MAP-2 staining, but this stain only allowed for measurement of the first portion of the apical dendrite. Therefore, the Golgi stain was used to allow for tracing of the entire apical dendritic trees, with quantification using Sholl analysis to observe the growth trajectory. The objective was to determine how apical dendrite growth is altered, short term and long term, by early iron deficiency. Approximately 20 neurons were traced from each of the three time points and two dietary conditions. Four or more animals were used from each group. Results show early iron deficiency altered dendritic developmental trajectory. Distance to peak branching was shorter in the ID and formerly ID animals as well as thinner third generation branches at P15. Also at P70 in FID animals, peak branching density was decreased. Decreased transcript levels were seen in the IDA and formerly IDA animals. Altered transcript levels of various cytoplasmic and transmembrane proteins critical to structural growth (RhoA, Rac1, Cdc42, Cypin, Cofillin, Profilin, Crmp1, Cxcr4) support altered morphology and contribute to reduced plasticity of the system. Early iron deficiency affects apical dendrite development and results in long term decreased cytoskeletal plasticity, these findings may underlie some of the functional deficits seen in this condition.
The Effect of Developmental Iron Deficiency on Gene Expression, Tet Proteins, and Dna Hydroxymethylation In the Rodent Brain
(2020-06) Barks, Amanda
Fetal-neonatal iron deficiency (ID) has a lasting negative impact on neurodevelopment, resulting in significant cognitive, socio-emotional, and learning and memory deficits in adulthood, as well as increased risk for neuropsychiatric disease. Given that ID is the most common micronutrient deficiency worldwide, and that pregnant women and young children are disproportionately affected, it presents a significant public health concern. Preclinical models have demonstrated that the developing central nervous system (CNS) is particularly affected by ID, and that the deleterious neurodevelopmental effects and neuropsychiatric risks that follow are associated with dysregulation of CNS gene expression. Dysregulated genes map to signaling pathways and networks critical for neurodevelopment and neuronal function, suggesting that these critical functions are compromised by ID. If developmental ID is corrected by iron repletion within a critical period, correction of neurodevelopmental deficits is possible. However, if iron repletion occurs outside of the critical period, the phenotypic and gene expression changes persist into adulthood despite correction of the deficiency. While changes in gene expression can be understood as the proximate cause of the ID neurocognitive phenotype, it is still unclear what the ultimate cause is. As such, there is a gap in our understanding of how developmental ID establishes and maintains gene expression changes in the CNS. A potential mechanism by which iron could enact these changes is through Ten-Eleven Translocation (TET) enzymes, a family of iron-dependent hydroxylases that generate the epigenetic modification 5-hydroxymethylcytosine (5hmC), or DNA hydroxymethylation. Epigenetic modifications such as DNA hydroxymethylation have the ability to stably influence gene expression throughout the lifespan, and are known to be labile to environmental influences. Of particular relevance, 5hmC is more abundant in the brain than any other tissue, and it increases in enrichment as neurodevelopment progresses, particularly in genes critical for neuronal development and function. The central hypothesis of my thesis research is that dysregulation of TET enzymatic activity and 5hmC by fetal-neonatal ID drives gene expression changes in brain that contribute to the long-term neurocognitive phenotype of developmental ID. To test this hypothesis, the following aims were proposed: 1) Determine the effect of fetal-neonatal ID on TET activity and 5hmC in two regions of the developing rat brain, the hippocampus and the cerebellum, and 2) Determine whether treatment of developmental ID with dietary iron repletion can reverse the changes to this epigenetic system. Completion of these aims contributes to the long-term goal of understanding the cellular and molecular underpinnings of CNS dysfunction and increased neuropsychiatric disease risk following developmental ID. Because the standard therapy of iron repletion incompletely rescues the neurodevelopmental phenotype of ID, there is a need for better therapeutic options. By better understanding the underlying mechanisms of ID-related hippocampal dysfunction, it may be possible to identify new therapeutic targets for more effective treatment of iron deficiency.

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