Jaideep Kapur - US grants

Epilepsy is a major health care problem afflicting 4% of Americans. Partial epilepsy is refractory to currently available medical therapy in 30% of the patients. Kindling is a useful animal model of partial epilepsy. Epileptogenesis in kindling may result from loss of GABA- mediated inhibition in the hippocampus. GABA-mediated inhibition is diminished in the CA1 region of hippocampus but transiently enhanced in dentate gyrus of the hipppocampus due to kindling. Recent advances in understanding the structure and function of GABA (A) receptor (GABAR) may explain these findings. GABAR is a pentameric protein formed from 14 different subunits of 5 classes: alpha, beta, gamma and rho. There are 6 alpha, 3 beta and 3 gamma subunits described. GABAR protein delta subunit mRNA is expressed in dentate gyrus but not in the CA1 region and GABAR protein alpha5 subunit mRNA is expressed in CA1 region but not in dentate gyrus. The hypotheses to be tested are: 1) the native GABARs in dentate gyrus have distinct physiologic and pharmacologic properties, and structural composition from those in the CA1 region; 2) the BABARs in CA1 pyramidal neurons and dentate granule cells respond differently to kindling stimulus. Whole-cell BABA (A) currents and single-channel GABAR activity from native GABARs from CA1 pyramidal neurons and dentate granule cells and from recombinant GABARs expressed in mammalian L929 cells will be recorded to fulfill the specific aims of this proposal: 1) Is there differential sensitivity of native BABAR in CA1 pyramidal neurons and dentate granule cells to various benzodiazepines? 2) Do deactivation and desensitization properties of whole cell GABA (A) current in CA1 pyramidal neurons and dentate granule cells suggest differential localization of delta subunit? 3) Are the single-channel properties different for GABARs in CA1 pyramidal neurons and dentate granule cells? 4) Can GABARs of known subunit composition be expressed in a heterologous system to replicate the pharmacologic properties and single channel kinetic properties of GABARs in CA1 pyramidal neurons and dentate gyrus? 5) Does kindling differentially alter the properties of whole cell and single channel GABA (A) currents in CA1 pyramidal neurons and dentate granule cells? The proposed experiments will provide a description of heterogeneity of GABA (A) receptors in the central nervous system and may suggest a potential for development of more specific drugs acting on the receptors.

Status epilepticus (SE) is a neurologic emergency characterized by very prolonged, sometimes refractory seizures associated with a 23 percent mortality. SE is a progressive condition where seizures reduce GABA- medicated inhibition in the hippocampus which in turn leads to more seizures. Past and recent studies of patients and experimental animals having SE suggested the hypothesis that gamma-amino butyric acid type A (GABAA) receptor (GABAR) function is altered during SE. This proposal will directly test this hypothesis by using whole cell patch clamp to study the GABARs present on hippocampal neurons isolated from rats undergoing SE and by study of treatment of SE in rats. Experiments are proposed to accomplish following specific aims: 1) In whole animals the potency and efficacy of anticonvulsants acting at the benzodiazepine site and drugs acting at the barbiturate site of the GABARs will be measured after brief seizures and prolonged seizures of SE. 2) Compare the time course of loss of diazepam sensitivity of dentate granule cell GABARs during SE with the time course of loss of efficacy of diazepam in whole animals undergoing SE. 3) Compare the diazepam and pentobarbital sensitivity of CA1 pyramidal neurons acutely isolated from naive rats and rats undergoing 45 minutes of SE will be. 4) Characterize the detailed pharmacological properties of dentate granule cell GABARs following SE.

Status epilepticus (SE) is a neurological emergency that afflicts 120-160,000 Americans each year, which can cause brain damage and contribute to mortality. Approximately one third of patients in SE are refractory to current therapies. We will investigate the role of AMPA receptor plasticity in benzodiazepine resistant SE using genetically modified mice, advanced imaging and electrophysiology; and we will test a novel therapy. In Aim 1, we propose to test whether a single seizure and SE enhance AMPAR mediated synaptic transmission in activated CA1 pyramidal neurons and the entorhinal cortex using patch clamp electrophysiology and biochemical techniques. The experiments in Aim 2 test the role of GluA1 plasticity in seizure spread and duration using global and conditional knockout mice. In Aim 3, we test the efficacy of IEM 1460, a drug that targets modified AMPA receptors, in terminating SE. This project will provide novel insights into the mechanisms of SE initiation by repeated seizures. It will define the role of GluA1 subunit plasticity in seizure spread during benzodiazepine refractory SE; and provide the neurobiological basis for the development and use of drugs targeting calcium permeable AMPA receptors for terminating SE.

Abstract Women constitute a majority of the patients with epilepsy, and many of them experience a cyclical exacerbation of seizures related to periodic changes in serum progesterone and estrogen levels during the menstrual cycle. This condition is called catamenial epilepsy. Currently, there are no scientifically tested effective treatments for catamenial exacerbation. In a multi-center trial of progesterone therapy for catamenial epilepsy failed to show efficacy. We have developed a model of chronic temporal lobe epilepsy in female rats and will use this model to study the effect of prolonged progesterone exposure on excitatory and inhibitory synaptic transmission and on seizure frequency and intensity. Specifically, we will test our hypothesis that a chronic elevation of progesterone in female animals diminishes the anticonvulsant action of progesterone by enhancing AMPA receptor-mediated glutamatergic synaptic transmission and suppressing GABAA receptor mediated synaptic transmission. In Aim 1, we will determine the impact of progesterone treatment on glutamatergic synaptic transmission on hippocampal principal neurons in na¿ve and epileptic female rats using a combination of patch clamp electrophysiology and analysis of the expression of the AMPA receptor subunits via both biochemical and immunohistochemical techniques. In Aim 2, we will determine the impact of progesterone treatment on GABAergic synaptic transmission on hippocampal principal neurons of na¿ve and epileptic female rats with a similar combination of techniques as in aim 1. In Aim 3, we will determine the role of progesterone receptors in progesterone-induced tolerance during progesterone treatment and withdrawal. These studies will provide novel insights into the mechanisms of catamenial epilepsy. These studies could explain the mechanism of failure progesterone therapy for treatment of catamenial epilepsy and potentially identify novel therapeutic targets for the treatment of catamenial exacerbation of seizures.

DESCRIPTION (provided by applicant): Epilepsy is characterized by recurrent unprovoked seizures. Biological rhythms and external stimuli can modulate seizure frequency partly through circulating steroid hormones. Neurosteroids like allopregnanolone are synthesized in the brain from circulating steroids and have powerful anticonvulsant effects. AIIopregnanolone exerts its actions in the central nervous system by acting on GABAA receptors. Physiological concentrations of allopregnanolone (10 -30 nM) potently enhanced whole cell GABA-evoked currents in hippocampal dentate granule cells acutely isolated from naive rats. In contrast, in dentate granule cells acutely isolated from epileptic rats these physiological concentrations of allopregnanolone failed to enhance whole cell GABAA receptor currents. Whole cell GABA-A receptor currents are generated by activation synaptic and extrasynaptic GABAA receptors. In dentate granule cells of naive rats, a1 and g2 subunits are expressed at synapses while a4 and d subunits are extrasynaptic. The extrasynaptic receptors mediate tonic inhibition while synaptic receptors mediate phasic (synaptic) inhibition. The tonic inhibition is insensitive to allopregnanolone, diazepam, and sensitive to Zn2+ while synaptic inhibition is sensitive to allopregnanolone, diazepam, and insensitive to Zn2+. These preliminary findings support the hypothesis that in the hippocampal dentate granule cells of epileptic rats, there is diminished allopregnanolone modulation of GABAergic inhibition. Decreased allopregnanolone modulation is due to increased expression of alpha4 and delta subunit-containing receptors, which in epileptic rats are present extrasynaptically and in subsynaptic membrane. We will test the predictions of our hypotheses by accomplishing the following specific aims: 1) To characterize allopregnanolone, diazepam, zinc, and furosemide modulation of GABA mediated miniature inhibitory synaptic currents (mlPSCs), and of GABAA receptor mediated background tonic inhibition of hippocampal dentate granule cells of epileptic and control rats. 2) To characterize the amount and site (synaptic versus extrasynaptic) of expression of specific GABAA receptor subunits in dentate granule cells in epileptic and control rats by immunohistochemistry and immunoblot studies combined with confocal laser microscopy. Significance: These experiments are of particular relevance to understanding epilepsy-induced alterations in GABA-A receptors and their modulation by neurosteroids.

[unreadable] DESCRIPTION (provided by applicant): This proposal proposes to develop novel, mechanism-based therapies for the treatment of organophosphate (OP) nerve agent-induced seizures. OP nerve agents, such as sarin, soman and VX act by central and peripheral cholinesterase inhibition and enhance cholinergic transmission. In experimental animals, all known nerve agents, produce convulsive seizures and status epilepticus within minutes of exposure. Seizures were also observed in humans exposed to nerve agent poisoning during the Iran-Iraq war, and in the Tokyo subway attacks where sarin and VX were used. The mechanism of OP-induced seizures remains uncertain. Preliminary studies in our laboratory have used the OP, paraoxon, as a surrogate agent to study OP-induced seizures. We demonstrate that low doses of paraoxon infused into the hippocampus cause prolonged seizures. Furthermore, we demonstrate that the concentrations of paraoxon that cause seizures in hippocampal slices can enhance excitatory neurotransmission by stimulating glutamate release from presynaptic terminals. These findings lead to the formulation of the central hypothesis guiding this proposal: convulsant concentrations of OP nerve agents enhance glutamatergic neurotransmission. This proposal seeks to confirm preliminary findings regarding the effect of OP agent on excitatory transmission and to test candidate compounds that diminish glutamate release from presynaptic terminals as potential therapy against seizures induced by cholinergic agents. Experiments are proposed within three aims, each group has specific milestones toward therapeutic interventions. We present quantitative outcomes criteria that represent "go-no go" decision points. Experiments outlined in Aim 1 seek to confirm and extend the observation that OP cholinesterase inhibitors enhance glutamate release from presynaptic terminals in the hippocampus, using patch clamp electrophysiology and FM dye technique. Experiments proposed in Aim 2 seek to test two classes of compounds, somatostatin and its analogs, and adenosine and its analogs, for their ability to diminish glutamate release during control resting conditions and following OP stimulation, using patch clamp technique and FM dye technique. Two classes of compounds, galanin and neuropeptide Y, are held in reserve and will be tested in case either one of the two proposed agents fail to reduce glutamate release by 30%. Aim 3: To test anticonvulsant action of two classes of compounds in controlling lithium/pilocarpine and OP paraoxon-induced status epilepticus. Two classes of compounds, neuropeptide Y and galanin are held in reserve and will be tested in case either one of the two proposed agents fail to control diazepam refractory seizures in 50% of experimental animals. By studying the mechanisms of OP-induced seizures, we are proposing to identify novel therapeutic targets for the treatment of OP-induced seizures. Second, we propose to develop therapies based on novel therapeutic targets. Furthermore, we develop a model of OP-induced seizures that can be used in civilian [unreadable] [unreadable] [unreadable]

PROJECT ABSTRACT The University of Virginia has a strong history of training physician scientists in neurological research. This application demonstrates an ideal environment and program for initial exposure of resident trainees to early research that will lead to K awards and successful careers as physician scientists. Dr. Karen Johnston, Professor and Chair, Department of Neurology and Dr. Jaideep Kapur, Professor and Vice Chair for Research in the Department of Neurology will function as co-PIs on this application. They have demonstrated a long commitment to developing research scientists. The application includes 19 outstanding mentors and a specific plan for the first resident research candidate.

Benzodiazepine-refractory status epilepticus (Established Status Epilepticus, ESE) is a relatively common emergency condition with several widely used treatments. There are no controlled, randomized, blinded clinical trials to compare the efficacy and tolerability of currently available treatments of ESE. This and the accompanying Statistical and Data Management Center (SDMC) application describe the ESE treatment trial (ESETT), which is designed to determine the most effective and/or the least effective treatment of ESE among patients older than two years by comparing three arms: fosphenytoin (FOS), levetiracetam (LEV), and valproic acid (VPA). This is a multicenter, randomized, double-blind, Bayesian adaptive, Phase III comparative effectiveness trial. Up to 795 patients will be randomized initially 1:1:1 and response-adaptive randomization will occur after 300 patients have been recruited. Randomization will be stratified by three age groups, 2-18, 19-65, and 66 years and older. The primary outcome measure is cessation of clinical seizure activity and improving mental status, without serious adverse effects or further intervention at 60 min after administration of study drug. Each subject will be followed until discharge or 30 days from enrollment. This trial will include interim analyses for early success and futility. This trial will be considered a success if the probability that a treatment is the most effective is greater than 0.975 or the probability that a treatment is the least effective is greater than 0.975 for any treatment. This will be the first phase III clinical trial of ESE in children and adults.

We propose to map focal motor to bilateral tonic-clonic seizures (FMBSs), which are the most dangerous epileptic seizures. These seizures increase the risk of sudden unexpected death in epilepsy (SUDEP) and lead to fractures and dislocations due to violent falls. SUDEP is the most common cause of death in patients with epilepsy. We propose that the canonical circuit published in Kandel's Principles of Neural Science (2013), which posits that focal seizures engage diencephalic thalamocortical circuits, which leads to secondarily generalized tonic-clonic seizures is too simplistic. It is not consistent with known neuroanatomy of the motor cortex, and modulation of seizures by subcortical structures. We propose that FMBSs originating in the frontal cortex spread through the striatum to the globus pallidus, substantia nigra and thalamus via the indirect pathway, in addition to spreading directly to the thalamus .. We test this hypothesis in three aims. Aim 1: to map FMBS spread at the mesoscale and compare it to anatomical connections of the seizure focus in TRAP mice using tissue clearing and 3D imaging combined with tract tracing and electrophysiological techniques). Aim 2 to map FMBS spread at the microscopic scale through the cortex and direct and indirect basal ganglia circuits in TRAP mice using immunohistochemistry. In aim 3, we will study dopamine type 2 receptor modulation of seizures at the mesoscale and microcircuit levels using a combination of techniques. We incorporated tools and techniques developed by the BRAIN initiative in our laboratory to move seizure circuit mapping research forward. We have used TRAP mice, the CLARITY technique, high resolution, high-throughput imaging, and 3D reconstruction of images to visualize activated neuronal pathways. We have constructed a highly collaborative team with expertise in anatomy, electrophysiology and computer science of imaging, which allows us to generate and analyze large volumes of data and build on each other's creativity. We have acquired sufficient equipment to perform these studies. These studies will generate new targets for the modulation of seizures by deep brain stimulation. Currently, this method is used for anterior thalamic stimulation and responsive neurostimulation, but in the future, multiple subcortical structures could sites for neuromodulation. Receptors and ion channels known to modulate basal ganglia circuits may emerge as novel targets for anticonvulsant development. If our studies confirm seizure passage through the striatum, then ii would be important to understand the underlying cellular mechanisms.