Karen S. Wilcox - US grants

[unreadable] DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is a devastating type of seizure disorder that is difficult to control with existing antiepileptic drugs. In order to better understand the process of epileptogenesis and to develop innovative therapeutic approaches for the management of TLE, animal models have been developed that exhibit the hallmarks of this seizure disorder: an initial insult to the CNS, a variable latent period, and the eventual development of spontaneous seizures of temporal lobe origin. The overall goal of this proposal is to determine the role of the medial entorhinal cortex (mEC) in the development of chronic epilepsy in the kainic acid model of TLE. This proposal will test the hypothesis that altered circuitry and changes in excitatory synaptic function of neurons in the superficial layers of the mEC contribute, at least in part, to hyperexcitability and synchronization in the mEC-hippocampal (HC) neural circuit in the kainic acid model of TLE. This hypothesis will be tested by using the whole cell patch clamp technique in the combined mEC-HC brain slice preparation obtained from control rats and those subjected to the kainic acid-induced status epilepticus (SE) model of TLE (Aims 2 & 3) to perform the following specific aims: 1) Compare and contrast the kinetics, short-term plasticity, and pharmacological modulation of excitatory postsynaptic currents (EPSCs) in Layer III PYR cells and Layer II STEL cells of the mEC in brain slices obtained from normal animals. 2) Determine if there are progressive changes in the electroresponsive membrane properties of STEL cells in the mEC at various time points following kainate-induced SE. Determine if there are alterations in the kinetics, short-term plasticity, and pharmacological modulation of EPSCs in Layer II STEL cells as a function of time following kainate-induced SE. 3) Determine if there is an enhanced probability of monosynaptic connections between STEL cells following KA treatment. By surveying the development of functional changes in principle neurons of the mEC in an animal model of TLE, we will gain insight into why PYR cells degenerate, why STEL cells become hyperexcitable, and what the functional impact of these changes are for the mEC-HC circuit. These experiments will set the stage for the development of future therapeutic interventions for the treatment of pharmacoresistant epilepsy. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE), a devastating seizure disorder that is difficult to control with anticonvulsant drugs, often develops following an initial insult to the CNS. In order to better understand the process of epileptogenesis and to develop innovative therapeutic approaches for the management of TLE, animal models have been developed that exhibit some of the hallmarks of this seizure disorder: a period of status epilepticus (SE) which serves as the initial insult to the CNS, a variable latent period during which seizures do not occur, and the eventual development of recurrent, spontaneous seizures of temporal lobe origin. Decades of research in such models have generated a vast amount of knowledge on the role of neurons in the hippocampus (HC) in TLE. However very little is currently known about the functional role in TLE of astrocytes: the other major cell type within the brain. Recently our laboratory utilized the kainic acid (KA) model of SE to investigate such `reactive' astrocytes. In this commonly used animal model of TLE, we made the novel discovery that astrocytes dramatically increase the expression of the ionotropic kainate receptor subunit, KA1. We hypothesize that activation of these newly expressed kainate receptors induces excitatory gliotransmission that depolarizes nearby CA1 pyramidal cells. Such excitatory gliotransmission in the hippocampus may have significant implications with respect to synchronization and seizure generation and this exploratory proposal will perform two specific aims to test this overall hypothesis. Specific Aim 1 will use immunohistochemical and immunoblotting techniques to determine if KA- induced SE results in a long-term increase in expression of kainate receptors on astrocytes. Specific Aim 2 will use the whole cell patch clamp technique to determine if activation of kainate receptors expressed on astrocytes induces gliotransmission that activates excitatory amino acid receptors in CA1 pyramidal cells in brain slices obtained from animals following KA-induced SE. It is anticipated that an increased understanding of the role of astrocytes in TLE will provide innovative molecular targets for the treatment of this frequently therapy-resistant seizure disorder. PUBLIC HEALTH RELEVANCE: Increasing evidence suggests that non-neuronal glial cells may contribute to seizure generation and epilepsy. Therefore, the proposed research will evaluate changes in a specific type of excitatory amino acid receptor that occur in glial cells in an animal model of epilepsy. It is anticipated that this work will reveal new potential therapeutic targets for the treatment of epilepsy. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): Translational Science and specific Challenge Topic, 15- NS-104: Early-Stage Therapy Development. The major goal of the proposed work is to develop novel technical and theoretical means to understand the mechanisms underlying temporal lobe epilepsy (TLE) and to assess new classes of possible treatments for this devastating disorder. TLE is often difficult to treat using currently available approaches and entails an economic cost of $12 billion dollars per year within the United States alone. The proposed work is transformative on several fronts. First, the work focuses on the putative role of glial support cells (specifically, astrocytes) in TLE. Although there is a convincing body of evidence that astrocytes are involved in epileptic dysfunction, this evidence has not yet gained wide acceptance, leaving approaches that focus on astrocytes underappreciated and underutilized. Second, the proposed approach depends on a novel imaging technique, called targeted path scanning (TPS), which allows recordings of neuronal and glial calcium transients in up to 100 cells simultaneously, with single-cell spatial resolution and excellent temporal resolution. The TPS approach allows the proposed research program to study mechanisms and putative treatments of TLE in interacting neuronal and glial networks, with spatiotemporal resolution that permits simultaneous analysis at both the cellular and network levels. The proposed study has two specific aims. Aim 1 addresses whether the properties of astrocytic population calcium transients are altered in brain slices derived from animals that have been subjected to the kainic acid (KA) model of TLE. These transients will be characterized, and compared with data from age-matched controls, in slices from animals during both the latent period (after induction of status epilepticus but before spontaneous seizures) and after spontaneous seizures have begun. Relevant properties to be studied include temporal frequency, magnitude, and spatial extent throughout the astrocytic network. Aim 2 focuses on interactions between astrocytic calcium transients and spike-driven calcium transients in nearby neurons. Specific questions to be addressed in this aim include: Are calcium transients in astrocytes and neurons spatially and/or temporally correlated? If so, which cell type in a given area leads the other? How do known anti-epileptic drugs affect calcium transients in astrocytes, calcium transients in neurons, and the potential interactions between the two cell types? Finally, can this ground-breaking technology be used as a network- based assay for the identification of novel anticonvulsant molecules for the treatment of pharmacoresistant epilepsy? The proposed project-a pioneering effort between a pharmacologist/epileptologist and a bioengineer-is translational in its focus and intent. The major goal of the proposed work is to develop new theories and approaches that could be invaluable in discovering new pharmacological treatments for the devastating seizure disorder, temporal lobe epilepsy. PUBLIC HEALTH RELEVANCE: Temporal lobe epilepsy is a seizure disorder with devastating effects, particularly in the large number of patients for whom current treatments are ineffective. The purpose of the proposed work is to use ground- breaking imaging technology to study this disease in interacting networks of both nerve cells and glial metabolic support cells. A likely outcome of the proposed work will be entirely new ways to assess potential pharmacological therapies for epilepsy.

DESCRIPTION (provided by applicant): Viral infections of the central nervous system (CNS) are associated with an increased risk for seizures, status epilepticus (SE), and the development of chronic epilepsy. We recently described a novel animal model of viral-induced epilepsy. Mice (C57BL/6) who receive intracerebral injections of Theiler's Murine Encephalomyelitis Virus (TMEV) display acute spontaneous seizures several days after infection, survive the initial infection and go on to develop spontaneous recurrent seizures. Preliminary data using C57BL/6 mice with various cytokines or cytokine receptors knocked-out have shown that these genetic manipulations can significantly alter the pathologic sequelae observed following TMEV injection of wildtype mice. Therefore, we propose to test our overall hypothesis that TMEV infection is associated with increased expression of TNF-a that contributes to increased neuronal excitability, acute seizures, neuropathology, and ultimately, epilepsy. The proposed experiments will lead to a greater understanding of the role of viral and immune contributions to acute seizures, altered neuronal and glial function, and epileptogenesis. We will use a multidisciplinary approach; including, chronic video-EEG monitoring and brain slice electrophysiology to: 1) test the hypothesis that the TNF-a pathway plays an important role in the development of chronic seizures following TMEV infection, and 2) test the hypothesis that TNF-a signaling mediates changes in synaptic and/or glial function in the hippocampus in TMEV infected mice. We anticipate that the results generated will provide a novel model in which to study the role of infection in the development of epilepsy. In addition, these experiments will provide important new insight into the role of TNF-a in cell death, synaptic transmission and epileptogenesis and set the stage for the development of novel therapeutic interventions for the prevention of infection-induced epilepsy.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE), a devastating seizure disorder that is difficult to control with anticonvulsant drugs, often develops following an initia insult to the CNS. In order to better understand the process of epileptogenesis and to develop innovative therapeutic approaches for the management of TLE, animal models have been developed that exhibit some of the hallmarks of this seizure disorder: a period of status epilepticus (SE) which serves as the initial insult to the CNS, a variable latent period during which seizures do not occur, and the eventual development of recurrent, spontaneous seizures of temporal lobe origin. Recently we used the kainic acid (KA) model of TLE to investigate 'reactive' astrocytes in the hippocampus (HC), a brain region known to be involved in seizure generation. There is a significant increase in gap junction coupling between astrocytes following KA-induced status epilepticus (SE). Therefore, the astrocytic network architecture is altered in brain regions associated with seizure generation. We also discovered that astrocytes express kainate receptor (KAR) subunits following SE and hypothesize that activation of KARs can result in calcium (Ca2+) transients that induce the release of signaling molecules that modulate neuronal activity in the HC. The present application will use targeted path scanning 2-photon microscopy (TPS) to simultaneously evaluate rapid Ca2+ transients in large networks of astrocytes in brain slices obtained from animals treated with KA to induce SE. We employ in utero electroporation to target a genetically encoded Ca2+ indicating protein (Lck- GCaMP3) to the rat HC so that we can use brain slices obtained from adult animals to determine 1) if activation of KARs induces somatic Ca2+ signaling in networks of reactive astrocytes in the HC and 2) if KAR- induced and/or other agonist-induced Ca2+ signaling in the fine processes of reactive astrocytes induces the release of signaling molecules that directly influence network activity in HC brain slices obtained from KA- treated rats during both the latent period and chronic epilepsy. Finally, we will use electron microscopy to determine if there are ultrastructura changes in KAR expression, gap junction coupling, and dendritic ensheathment in the astrocyte compartment of the tripartite synapse of the CA1 and CA3 regions of the HC following KA-induced SE. The combined use of TPS with the stable expression of Lck-GCaMP3 in cells of the HC is a technical achievement that will contribute to our understanding of the functional role of KAR expression in astrocytes following status epilepticus (SE), both in the latent period and in chronic epilepsy. The proposed experiments will also determine how pathologic glial/neuronal interactions, both structural and functional, influence circuit activity during the development and persistence of epilepsy. Finally it is anticipated that the proposed experiments will lead to the identification of novel molecular targets for innovative therapeutic approaches for the treatment, prevention, and/or cure of this devastating seizure disorder.

? DESCRIPTION (provided by applicant): The Anticonvulsant Drug Development (ADD) Program will host a symposium entitled Therapy Development in the Era of Team Science and Big Data: What Will the Future Bring to the Patient with Epilepsy? This symposium will be held from May 17th, 2015 - May 20th, 2015 in Park City, UT and will be open to the public. Despite FDA approval of over 16 anticonvulsant drugs in the last two decades, nearly 1/3 of all patients with epilepsy fail to achieve full seizure control. In addition, it is currently not possible to prvent the development of epilepsy in patients at high risk following a CNS insult. Therefore, there is an acute need for more effective therapies for the patient with intractable epilepsy and for persons at risk. The goal of the symposium is to explore how innovative breakthroughs in basic science can be leveraged by public, federal, and private partnerships to rapidly develop new treatments for the patient with epilepsy. A creative conference structure, ample discussion time, and inclusion of junior investigators in all aspects of the meeting will insure that new knowledge, new collaborations, and new insight into state of the art therapy development will be significant outcomes of the symposium. This application requests funds to partially support travel for the invited faculty and junior investigators and to provide funds for conference services, printing, and audio-visual technologies.

NINDS EPILEPSY THERAPY SCREENING PROGRAM

Drug addiction is a significant societal issue, with more than 20 million individuals being diagnosed with a substance use disorder (SUD) in the past year. While current treatments show some success, more than 50% of patients treated for SUD relapse within one year. Corticostriatal circuitry and plastic changes therein are involved in the transition from drug abuse to addiction. In particular, the dorsal lateral striatum (DLS) has emerged as a key brain region regulating habitual control over behavior, including that underlying addiction to stimulants. Astrocytes play fundamental roles in brain neuroplasticity through several mechanisms, including regulation of extracellular glutamate levels. Presently, nothing is known about functional changes in astrocytic networks in DLS of animals exhibiting habitual vs. goal-directed control over drug-seeking behavior.! The proposed experiments will therefore directly determine the function of astrocytes in DLS of rats exhibiting habitual vs. goal-directed control over cocaine seeking, and relative to yoked-saline controls. In Aim 1 we will test the hypothesis that transition to habitual control over cocaine-seeking behavior is concomitant with a decrease in astrocytic glutamate transport via decreased GLT-1 expression and function in DLS. Recent data provide the first indication that expression of GLT1, the primary glutamate transporter in astrocytes, is significantly decreased in dorsal striatum following extended cocaine self-administration in rats. However, whether the function of glutamate transporters in astrocytes of the dorsal striatum changes in concert with the development of habitual control over cocaine seeking is completely unknown. Therefore, we will use patch clamp electrophysiology to record slow transporter currents in astrocytes (Aim 1a) and AAV-mediated expression of the iGluSnFR protein and 2-photon microscopy to measure the levels and dwell time of glutamate in the extrasynaptic space (Aim 1b) in DLS in brain slices prepared from rats trained to self- administer cocaine and exhibiting habitual vs. goal-directed control over cocaine seeking, as well as from yoked-saline controls. In Aim 2, we will test the hypothesis that these changes in glutamate transport result in significantly altered extrasynaptic and synaptic NMDA receptor-mediated transmission in spiny efferent neurons in DLS. We will use whole-cell patch clamp to assess changes in extrasynaptic NMDA receptor-mediated currents by examining the amplitude of tonic NMDA receptor-mediated currents, as well as the amplitudes and decay kinetics of evoked NMDA receptor-mediated EPSCs in striatal efferent neurons in DLS. AMPA/NMDA ratios will also be determined. Completion of these aims will provide the first insights into whether the transition to habitual control over cocaine-seeking behavior occurs concomitant with changes in astrocyte function and structural connectivity in DLS and the effects of cocaine self-administration history on astrocyte modulation of excitatory afferents to striatal efferent neurons. Such insights will allow for targeting of approaches to diminish relapse in stimulant-addicted individuals.

ABSTRACT The organizers of the Park City Epilepsy Meeting will host a symposium entitled ?Cutting Edge Approaches to Transform Epilepsy Therapy.? This symposium will be held from October 6-8th, 2019, in Park City, UT and will be open to the public. Despite FDA approval of over 16 antiseizure drugs in the last two decades, nearly 1/3 of all patients with epilepsy fail to achieve full seizure control. In addition, it is currently not possible to prevent the development of epilepsy in patients at high risk following a CNS insult. Therefore, there is an acute need for more effective therapies for the patient with intractable epilepsy and for persons at risk. The goal of the symposium is to explore how innovative breakthroughs in neuroscience can be leveraged to rapidly develop new treatments for the patient with epilepsy. A creative conference structure, ample discussion time, and inclusion of junior investigators in all aspects of the meeting will insure that new knowledge, new collaborations, and new insight into state of the art therapy development will be significant outcomes of the symposium. A white paper describing the outcome of the meeting will be submitted for publication by the organizers. This application requests funds to partially support travel for the invited faculty and junior investigators and to provide funds for conference services, printing, and audio-visual technologies.

PROJECT SUMMARY: Viral infections of the central nervous system (CNS) are associated with an increased risk for seizures, status epilepticus (SE), and the development of chronic epilepsy. Our collaborative group has developed the first animal model of viral-induced epilepsy. Mice (C57BL/6) who receive intra-cerebral injections of Theiler's Murine Encephalomyelitis Virus (TMEV) display acute spontaneous seizures several days after infection, survive the initial infection and go on to develop spontaneous recurrent seizures. Furthermore, our data from the last award period using C57BL/6 mice with various cytokines or cytokine receptors knocked-out have shown that manipulations in the TNF? system can significantly alter the pathologic sequelae observed following TMEV injection. Therefore, we propose to test our overall hypothesis that following TMEV infection, calcium dependent increases in production and release of TNF? from microglia activates the neuronal TNF?R1 pathway, contributing to seizure generation. The proposed experiments will lead to a greater understanding of the role of viral and immune contributions to acute seizures, altered neuronal and microglial function, and epileptogenesis. We will use a multidisciplinary approach to test our hypothesis, including, state of the art in vivo and in vitro 2 photon microscopy, calcium imaging, novel transgenic mouse models, chronic video-EEG monitoring and brain slice electrophysiology to: 1) Determine the time course and extent of physical changes, motility, and the development of spontaneous calcium transients in microglia in TMEV infected mice during the acute infection period using GCAMP5G selectively expressed in microglia; 2) Determine the mechanisms underlying calcium transients in activated microglia during the acute infection period and the role of calcium transients in cytokine production; and 3) Determine if signaling through the neuronal TNF?R1 pathway underlies hippocampal excitability and seizure activity following TMEV infection. We anticipate that these experiments will provide important new insight into the role of TNF? and it's receptor, TNF?R1 in cell death, synaptic transmission and epileptogenesis and set the stage for the development of novel therapeutic interventions for the prevention of infection induced epilepsy.

Neuroimmunology is a rapidly growing field of research that touches a number of critical human diseases including multiple sclerosis, epilepsy, spinal cord injury, and viral encephalopathies. Traditionally, researchers have been trained in either neuroscience or immunology whereas the Neuroimmunology Training Program at the University of Utah seeks to develop and train the next generation of researchers in both fields with a cross- disciplinary approach focused on the interplay between the immune and nervous systems during disease. This application is the result of ongoing collaborations between high caliber faculty on campus who recognized the need to develop a training program specific to the challenges of neuroimmunology. This application requests support for 4 pre-doctoral trainees who will be selected from an outstanding pool of candidates within our relevant graduate programs. The Neuroimmunology Training Program at the University of Utah will formally bring together 17 faculty members from across the neuroimmunology research spectrum to participate in mentorship, program-wide meetings, workshops, and boot camps to share expertise and further develop the neuroimmunology field. The training program will be overseen by two faculty directors and a steering committee focused on selecting and supporting trainees who are highly likely to exhibit continued success within the diverse field of neuroimmunology research. Selected trainees will be expected to participate in specialized training in quantitative literacy, statistical analysis, and scientific rigor and reproducibility within the context of neuroimmunology research. Given the exceptional training track record of our faculty, available and unique resources to support research and robust institutional support, the Neuroimmunology Training Program will provide and outstanding opportunity for trainees to develop intellectually, advance and optimize their thesis research projects, create a valuable network of colleagues, and prepare for a highly successful research career focused on the crossroads of immunology and neuroscience.