1985 — 1987 |
Choi, Dennis W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glutamate Chemosensitivity of Cortical Neurons
The goal of the proposed experiments is a detailed characterization of the membrane chemosensitivity of mammalian cerebral cortical neurons to glutamate, a probable cortical excitatory neurotransmitter. Previous studies of glutamate action in intact systems have suggested a variety of effects on membrane potential and conductance, but include unknown contributions from several factors, including local increases in extracellular K+ during glutamate action, electrogenic uptake of glutamate into terminals, and electrotonically distorted effects of glutamate on "remote" dendritic sites. It is proposed to study glutamate action in a sparse cortical cell culture system where these factors can be reduced or eliminated. In addition, care will be taken to block glutamate-evoked release of other neurotransmitters from presynaptic terminals, another potential complicating factor. Mouse cortical neurons will be plated in sparse cell culture and impaled with an intracellular electrode for recording or current injection. Glutamate will be applied to the soma or proximal dendrites under direct visual guidance, either by iontophoresis, or at known concentration by pressure ejection from blunt micropipeties. Effects of glutamate on membrane potential and conductance will be defined, with attention to underlying membrane I-V non-linearities, and difficulties associated with finite length constants. K+ sensitive electrodes will be used to measure neighborhood extracellular K+ accumulation, which is expected to be small in sparse culture. Subsequent quantitative experiments will define the glutamate dose-response relationship, and the pharmacology of glutamate antagonists. Glutamate chemosensitivity on individual neurons will be mapped out by focal iontophoresis, looking for evidence of either multiple response types, or "hot spots". Population studies will look for differences in the glutamate chemosensitivity of morphologically defined subclasses of cortical neurons. The proposed experiments will provide information critical to subsequent studies of glutamate neurotransmission, and will also provide a necessary background for more detailed studies of the glutamate channel. The proposed pharmacological characterization of cortical glutamate receptors could have eventual relevance to the therapy of certain human cortical diseases, including degenerative diseases, hepatic encephalopathy, and epilepsy.
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1 |
1988 — 1992 |
Choi, Dennis W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pharmacologic Reduction of Hypoxic Neuronal Injury
Central hypoxic neuronal injury (HNI) contributes to the morbidity and mortality of many neurological diseases, including stroke, drowning, cardiac arrest, and head injury. However, the responsible cellular mechanisms are not currently defined, and no proven therapy is available. The proposed study is viewed broadly as initiating a investigation into these cellular mechanisms, in the hope of eventually developing an effective pharmacological therapy. A recent, rapidly growing body of evidence suggests that HNI may be in large part mediated by the neurotoxicity of endogenous excitatory amino acids (EAAs), accumulating in brain extracellular space under hypoxic conditions. The proposed study will focus specifically on postsynaptic antagonists of EAA neurotoxicity, utilizing an established murine cortical cell culture model system to achieve both rapid screening, and quantitative drug characterization at the neuronal level, free of the systemic effects which can complicate studies in vivo. The study should be synergistically facilitated by an ongoing parallel investigation of the neurotoxicity of exogenously applied EAAs. In addition to identifying and characterizing promising compounds (or combinations of compounds) with protective efficacy against cortical HNI, the study will seek to define several principles governing this pharmacological approach, including comparative efficacy among different classes of EAA antagonists, time constraints (efficacy of "late" administration of antagonists), effect of zinc (likely co-released with EAAs at many central synapses), and differences in the intrinsic vulnerability (or response to various therapies) of specific neuronal subpopulations. Information gathered during this study will likely be helpful in guiding testing in vivo, and furthermore should produce insight into the basic nature of EAA involvement in HNI.
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1 |
1991 — 1994 |
Choi, Dennis W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glutamate Neurotoxicity in Cortical Cell Culture
The goal of the proposed experiments is a continued characterization of the cellular mechanisms underlying glutamate neurotoxicity, a process which may participate in the neuronal degeneration associated with certain acute or chronic neurological diseases, including Alzheimer's disease. Even is glutamate neurotoxicity is not involved in the pathogenesis of any chronic neurodegenerative diseases, it is proposed that elucidation of its underlying mechanisms could yield valuable insights into some common principles of neuronal cell injury. To achieve cellular and molecular resolution, investigations will take place in an established dissociated murine cortical cell culture model system. Using pharmacological, physiological, and neurochemical approaches, including neurotoxicity assays, histochemical stains, whole cell recording, HPLC, 45Ca2+ accumulation, and fura-2 videomicroscopy, answers to these four questions will be specifically sought: 1) What is the role of the "metabotropic" glutamate receptor in glutamate neurotoxicity? 2) What is the role of calcium in glutamate neurotoxicity? 3) What is the role of lipid peroxidation in mediating glutamate neurotoxicity? and 4) Why are cortical neurons containing high concentrations of NADPH-diaphorase resistant to NMDA receptor- mediated injury? The proposed experiments will provide new information regarding the nature of glutamate neurotoxicity and could lead to the development of therapeutic approaches designed to reduce pathological neuronal cell death in Alzheimer's disease or related disorders.
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0.955 |
1992 |
Choi, Dennis W |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Basic Clinical Neuroscience Training |
0.955 |
1993 — 1996 |
Choi, Dennis W |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Basic Neuroscience Training For Neuronal Injury |
0.955 |
1995 — 1999 |
Choi, Dennis W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cell Cell Interaction and Hypoxic Brain Injury
Grant funds are requested to support a new research program project aimed at the long-term goal of understanding how cell-cell interactions influence the pathogenesis of hypoxic-ischemic brain injury. Glutamatergic neurotransmission in particular has been implicated in the pathogenesis of hypoxic-ischemia neuronal death. While attention in the area has centered historically around N-methyl-D-aspartate (NMDA) receptor-mediated contributions to hypoxic brain damage, recent evidence has indicated that alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionate/kainate (AMPA/KA) receptors may also be important mediators of injury in the hypoxic-ischemic brain. Elucidation of several mechanisms underlying this AMPA/KA receptor mediation is the specific goal of the present application. The proposed experiments enlist the efforts of 8 faculty investigators, collaborating on 4 highly interlinked experimental Projects and 5 supporting Cores. Project 1 will investigate the contribution of AMPA/KA receptors to hypoxic neuronal injury in cortical cell cultures, with special attention to the minority subset of AMPA/KA receptors which gate Ca2+-permeable channels. Project II will harness a recent discovery that certain benzothiadiazides can modify AMPA/KA receptor desensitization, using these drugs as probes to study the role of AMPA/KA receptor desensitization in limiting hypoxic neuronal injury. Project III will use fura-2 videomicroscopy to examine the hypothesis that AMPA/KA receptor overactivation contributes to hypoxic neuronal injury by disrupting neuronal intracellular Ca2+ homeostasis. Project IV will explore the relationship between hypoxic AMPA/KA receptor activation and free radical-mediated cell damage. These experimental sections are supported by an administrative core (A); an animal (in vivo ischemia) core (B); a histology core (C); a computing and image analysis core (D); and a statistics core (E). taken together, proposed experiments will combine molecular, cellular, and whole animal approaches to answer several specific relevant to understanding the pathogenesis of hypoxic- ischemic brain damage. Information gathered from these studies may aid the future development of effective clinical therapies for stroke and cardiac arrest.
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0.955 |
1995 — 1999 |
Choi, Dennis W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Contribution of Ampa/Ka Receptor to Hypoxic Neuronal Injury in Vitro
Recent evidence has suggested that alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionate/kainate (AMPA/KA) type glutamate receptors may contribute importantly to brain damage following hypoxic-ischemic insults in vivo. The proposed experiments will utilize the simplified model system of cortical cell cultures exposed to oxygen-glucose deprivation to address three aspects of this contribution: 1) the contribution of AMPA/KA receptor-mediated injury to overall hypoxic neuronal death, and the relationship between this AMPA/KA receptor-mediated injury and hypoxic NMDA receptor-mediated injury; 2) the role of AMPA/KA receptors in mediating the hypoxia-induced death specifically of a newly=discovered cortical neuronal subpopulation, that likely expresses large numbers of AMPA/KA receptor-gated Ca2+ channels, and hence may be especially vulnerable to AMPA/KA receptor-mediated injury; and 3) the effect of sublethal oxygen-glucose deprivation on the subsequent neuronal expression of GluR-2 mRNA and functional AMPA/KA receptor-gated Ca2+ channels. These studies should yield novel information about the way AMPA/KA receptors may mediate hypoxic neuronal death, which may prove valuable in guiding the developing of effective therapeutic strategies aimed at reducing this form of injury in disease settings such as stroke or cardiac arrest.
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0.955 |
1995 — 1999 |
Choi, Dennis W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glutamate Neurotoxicity in Vitro |
0.955 |
1997 |
Choi, Dennis W |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
21st Princeton Conference On Cerebrovascular Disease
We seek support for the 21st Princeton Conference on Cerebrovascular Disease. For several decades, this biannual conference has brought junior and senior investigators, both basic and clinical together to reflect upon the current state of stroke research, and to formulate future collaborations and research projects aimed at furthering understanding of disease pathogenesis and the development of new treatments. The conference will be held from Thursday, May 7, 1998 to Sunday, May 10, 1998 in St Louis, Missouri, and will be hosted by Washington University Medical School. The conference will be limited to 130 participants with special interest in cerebrovascular disease, or special expertise relevant to cerebrovascular disease. Invitees will include clinical or bench scientists currently active in stroke research, as well as key scientists from other related disciplines. A special effort will be made to invite young scientists, as well as scientists from under-represented minorities. The NIH funding sought here will be dedicated to enabling 60 young investigators to attend the Conference; the organizers plan to raise other funds to help with the expenses of senior investigators. Topics for intense scrutiny will be selected key areas of basic and clinical cerebrovascular disease research with impact on clinical issues in stroke management. The proceedings will be published. The overall goal of the conference is to generate a vision of future directions in stroke research.
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0.955 |
1997 — 2000 |
Choi, Dennis W |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Nervous System Development and Injury |
0.955 |
2000 |
Choi, Dennis W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cell-Cell Interaction and Hypoxic Brain Injury
This is a revised submission to request support for fimds to support continuation of a productive Program Project aimed in the long-term at understanding how cell-cell interactions influence the pathogencsis of hypoxic-ischemic brain injury. The current grant period focused on alpha- amino-3-hydroxy-5-methyl-4-isoxazolcpropionate (AMPA) / kainate receptor-mediated injury, whereas the present proposal follows up on several newer themes that emerged during the current period (2/02/95 - 1/31/00, P01 NS32636). The proposed experiments enlist the efforts of 10 faculty investigators, collaborating on 3 interlinked experimental Projects and 4 supporting Cores. Project 1 will test the hypothesis that toxic levels of Zn2+ entry may contribute to neuronal death after transient global ischemia and mild transient focal ischemia, utilizing both in vitro and in vivo injury models. Project II and III move past the main second messenger of injury, entry of Ca2+ or Zn2+, to examine later events. Project II will examine downstream intracellular processes, specifically examining hypoxia- induced changes in astrocyte mitochondrial free radical production, and testing a novel hypothesis that this increase in astrocyte free radial production contributes to neuronal death. Finally, Project III will look beyond the injury horizon and examine the recovery of dendritic structures and fimctional synapses post injury, specifically testing the hypothesis that synapses lost due to mild hypoxic insults can reform and regain firnction. These experimental sections are supported by an Administrative, Computing, and Statistics Core (A); an Animal Core (in vivo ischemia) (B); a Microscopy and Imaging Core (C); and a Stereology Core (D). Besides sharing a central investigative theme of cell-cell interactions, the three proposed Projects will collaborate on experiments involving specific signaling processes mediated by Zn2+ or astrocytes. Taken together, proposed experiments will combine molecular, cellular, and whole animal approaches to answer several specific questions relevant to understanding the pathogenesis of hypoxic-ischemic brain damage. Information gathered from these studies may aid the future development of effective clinical therapies for stroke and cardiac arrest.
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0.955 |
2000 — 2001 |
Choi, Dennis W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Es Cell Transplantation After Spinal Cord Injury
Grant funds are requested to support a new research Program Project aimed at initiating an exploration of the potential of embryonic stem (ES) cell transplantation for restoring lost functions after spinal cord injury. Members of the proposed Project team have already developed experience working with mouse ES cells differentiated down neural lineages, methods for reducing cell death in the nervous system, and rodent models of traumatic spinal cord injury. In the experiments that form the specific background for the present proposal, Project team members have obtained evidence that mouse ES-cell derived neural lineage cells (ESNLCs) can be successfully transplanted into the syrinx/cyst that forms nine days after impact injury to the rat spinal cord . These transplanted ESNLCs survived for at least many weeks, partially filling the syrinx and migrating into host tissue, and expressed markers for differentiated oligodendrocytes, astrocytes, and neurons. The requested Program Project funding would enable Project team members to build on these early experiments in a systematic and hypothesis-driven fashion. Based on direct observations that a majority of transplanted ESNLCs die over the first 48 hours following transplantation and that neurons in particular continue to die over subsequent days, the emphasis of the experiments proposed here will be on improving the survival of transplanted ESNLCs through pharmacological and genetic engineering approaches aimed at blocking apoptosis and excitotoxicity. It is anticipated that work performed in subsequent years may be directed at optimizing the ESNLC transplantation procedure in other ways.
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0.955 |
2000 — 2002 |
Choi, Dennis W |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Excitotoxic Cell Death of Es Derived Neural Lineage Cells
stem cell transplantation; embryonic stem cell; cytotoxicity; nervous system transplantation; spinal cord injury; cell death; cell differentiation; neurotoxicology; NMDA receptors; cell population study; neurotrophic factors; oligodendroglia; neurons; antioxidants; glutamate receptor; excitatory aminoacid; cell line; voltage /patch clamp; calcium indicator; tissue /cell culture;
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0.955 |
2001 |
Choi, Dennis W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Zinc Neurotoxicity
DESCRIPTION (From the Applicant's Abstract): Glutamate receptor- and Ca2+-mediated neurotoxicity was the focus of study during past grant periods. Recently, we have begun to examine a related form of neurotoxicity, also enhanced by glutamate receptor activation but mediated by Zn2+ rather than Ca2+. Zn2+-mediated neurotoxicity likely contributes to central neuronal death after certain insults, such as transient global ischemia. Our Central Hypothesis is that extracellular Zn2+ can kill neurons by: 1) entering across the plasma membrane, largely through voltage-gated Ca2+ channels (VGCCs) in depolarized neurons; 2) increasing intracellular free Zn2+ ([Zn2+]i); 3) interfering with glycolysis, causing ATP levels to fall; 4) triggering apoptosis (at lower Zn2+ levels). The proposed experiments will test aspects of this central hypothesis in cultured murine cortical neurons, delineating mechanisms underlying Zn2+-induced neuronal death to advance efforts to develop therapeutic countermeasures that might be used to reduce brain damage after cardiac arrest. Cultured neurons will be exposed to varying concentrations of extracellular zinc for brief ("fast toxicity") or prolonged ("slow toxicity") time periods. We plan to define the relationships linking transmembrane Zn2+ influx (measured with patch-clamp and radio-isotope flux techniques), [Zn2+]I (measured with dye videomacroscopy), cellular Zn2+ content (measured with atomic absorption spectroscopy or inductively-coupled plasma spectroscopy), and cellular apoptosis (v.s. necrosis). We will also measure resultant neuronal levels of ATP, NAD+, NADH and glycolytic intermediates, mitochondrial transmembrane potential, and cytoplasmic reactive oxygen species (measured with dihydroethidium dye). Finally, we will test genetic perturbations of cellular Zn2+ homeostasis, specifically increased or decreased expression of the key plasma membrane Zn2+ transporter, ZnT-1, or the major neuronal intracellular Zn2+ binding protein, metallothionein-III, will produce the changes in vulnerability to Zn2+ neurotoxicity predicted by the central hypothesis.
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0.955 |