Steven M Rothman - US grants

The major goals for the coming year are to further define excitatory synaptic transmission in hippocampal cultures and to study the role of excitatory neurotransmission in mediating anoxic neuronal injury in the hippocampal slice. Specific aims include: 1. Determining whether newly developed blockers of excitatory amino acids, such as kynurenic acid, antagonize EPSP's in culture. 2. Determining the role of the kainate and N-methyl-D-aspartate receptors in neurotransmission, long term potentiation, and burst generation. 3. Studying pairs of synaptically connected neurons to determine whether any form of synaptic potentiation occurs in culture. 4. Injecting presynaptic neurons with various transmitter candidates to see whether the post-synaptic potentials can be increased. 5. Determining whether blockade of excitatory neurotransmission in hippocampal slices protects them from anoxic injury. These experiments will use cultures of dissociated rat hippocampal neurons and slices of rat hippocampus. Physiological recording will be done with standard electrodes and amplifiers, as well as patch clamp electrodes in special cases. The long term objectives of this project are to obtain a better understanding of the physiology and pharmacology of excitatory neurotransmission and relate this to human neurological diseases. There is convincing evidence that excitatory transmitters, probably amino acids, are responsible for hypoxic/ischemic neuronal injury, epileptic brain damage, and some hereditary degenerative neurological illnesses. Clearly, more information about excitatory transmitters will be required if the treatment of these human diseases is to proceed on a rational basis.

The experiments described in this grant application are designed to provide information about the participation of excitatory amino acids, especially glutamate, in neuronal death, development, and communication. They will utilize dispersed cultures of rat hippocampus and explant cultures to explore three aspects of the neurobiology of glutamate. These experiments should enhance our knowledge of events likely to take place during cerebral anoxin and ischemia, which are extremely important clinical problems. Major questions to be considered include: 1. What is the mechanism of glutamate neurotoxicity and how does it relate to anoxic neuronal injury? a. Is elevated intracellular calcium directly related to cell death? b. Is decreased intracellular pH directly related to cell death? c. Are perturbations of second messengers/modulators/intracellular enzymes causally linked to neuronal death? d. Are there circumstances when glutamate alters neurons without killing them? e. What is the sequence of morphological changes which neurons undergo prior to glutamate-induced death? 2. Does ongoing release of glutamate alter the structure or function of developing neurons? a. Will chronic blockage of glutamate receptors modify the dendritic structure of hippocampal neurons? b. Is the level of intracellular calcium in cultured neurons partially regulated by ongoing glutamate release and synaptic activity? 3. What factors control the release of synaptic glutamate in vitro? a. What is the peak concentration of glutamate released-into the synaptic cleft? b. Is calcium always required for glutamate release?

Over the last decade there has been an enormous increase in basic neuroscience research in a variety of areas including systems neuroscience, developmental neuroscience, molecular neuroscience, and cellular neurophysiology/neuropharmacology. While clinical neuroscientists have managed to keep pace with the information and technology that most directly impacts upon the daily practice of medicine, they have been unable to appreciate the full implications of much of this basic research. Moreover, they have not been able to actively participate in the actual acquisition of new neurobiological data because the research technology is beyond the scope of medical school and neurology residency education. The NSADA institutional award represents a creative new mechanism to bring potential young academic pediatric neurologists into fundamental neuroscience research. The five year award allows motivated individuals to get sufficient didactic and laboratory experience to begin sophisticated independent investigation in the neurosciences. We expect to admit pediatric neurology residents into the NSADA program for two research years prior to the start of their core clinical neurology residency. After completion of clinical neurology training, they will have three additional years of intense laboratory exposure. This is the equivalent of two standard postdoctoral fellowships and should provide the NSADA fellows with a substantial research background. The Division of Pediatric Neurology and Program in Neurosciences at Washington University have extensive intellectual and physical resources that will be available to NSADA trainees. The 18 mentors identified for this grant have been selected from over 75 full-time faculty members with neuro-science labs. They represent a group with superb teaching skills and proven interest in the research development of clinicians. They should insure our ability to educate these pediatric neuroscientists for the next millennium. This research immersion should translate into better care for children with neurological disease.

This is an application to establish an Experimental Neonatal Brain Disorders Center focused on the vulnerability of the developing brain to hypoxia and ischemia. The application is divided into two Preclinical Projects and three Clinical Projects, all supported by three Cores. Taken as a whole, the project will provide valuable diagnostic and therapeutic information relevant to newborn brain injury. Project 1, The Neuropharmacology of Cortical Injury, contains experiments that explore the role of excitatory amino acids in neuronal injury in fetal and newborn rats and in brain slices obtained from focal cortical resections in humans. These should help elucidate the cellular pathophysiology of neuronal injury. Project 2, Intraventricular Hemorrhage and Cerebrovascular Reactivity, will investigate the role of vasospasm and altered vascular reactivity in newborn brain injury associated with intracranial bleeding. This information may suggest alternate therapies for newborns with cerebral hemorrhage. Project 3, Doppler Evaluation of Fetal Neurological Integrity, will determine whether quantitative ultrasound examinations show characteristic patterns in neurologically normal and abnormal fetuses. These examinations may help identify high risk newborns in utero. Project 4, Cerebral Blood Flow and Metabolism in Newborn Infants, will use positron emission tomography to measure glucose metabolism in newborns and also validate near infrared spectroscopy as a noninvasive indicator of cerebral blood flow. These results could alter the fluid and respiratory management of critically ill neonates. Project 5, Nuclear Magnetic Resonance Imaging of Diffusion Coefficient and Blood Flow, will determine whether NMR techniques can noninvasively identify early brain damage in sick newborns and whether they can localize abnormalities of cerebral blood flow. This information could alter management of these infants.

Over the last decade there has been an enormous increase in basic neuroscience research in a variety of areas including systems neuroscience, developmental neuroscience, molecular neuroscience, and cellular neurophysiology/neuropharmacology. While clinical neuroscientists have managed to keep pace with the information and technology that most directly impacts upon the daily practice of medicine, they have been unable to appreciate the full implications of much of this basic research. Moreover, they have not been able to actively participate in the actual acquisition of new neurobiological data because the research technology is beyond the scope of medical school and neurology residency education. The NSADA institutional award represents a creative new mechanism to bring potential young academic pediatric neurologists into fundamental neuroscience research. The five year award allows motivated individuals to get sufficient didactic and laboratory experience to begin sophisticated independent investigation in the neurosciences. We expect to admit pediatric neurology residents into the NSADA program for two research years prior to the start of their core clinical neurology residency. After completion of clinical neurology training, they will have three additional years of intense laboratory exposure. This is the equivalent of two standard postdoctoral fellowships and should provide the NSADA fellows with a substantial research background. The Division of Pediatric Neurology and Program in Neurosciences at Washington University have extensive intellectual and physical resources that will be available to NSADA trainees. The 18 mentors identified for this grant have been selected from over 75 full-time faculty members with neuro-science labs. They represent a group with superb teaching skills and proven interest in the research development of clinicians. They should insure our ability to educate these pediatric neuroscientists for the next millennium. This research immersion should translate into better care for children with neurological disease.

DESCRIPTION Over the past two decades biologists have recognized that there are two broad forms of cell death--necrosis and apoptosis. The former is seen after many severe, acute injuries and is associated with rapid cellular destruction. The latter can be triggered by a variety of acute and chronic insults and may take hours to weeks to fully manifest. There is now compelling evidence that a major component of the neuronal death seen in a variety of acute and chronic neurological diseases, including stroke, is apoptotic, and that this type of death is mediated by several endogenous cysteine proteases. The focus of this application is to determine the physiological consequences of aborting apoptotic neuronal death and to determine whether the triggers for apoptotic injury are present in human neocortex deprived of oxygen and glucose. Specifically: (1) Examine the function of voltage-gated and ligand-gated channels in autonomic ganglion neurons and cerebellar granule neurons that have been deprived of trophic factors but rescued from cell death by inhibition of proteases. (2) Determine the time course of protease activation in brain slices from rodents, and then humans (obtained from neocortical biopsy), and then identify interventions that inhibit protease activation. These experiments will utilize standard methods for intracellular recording and voltage clamping of cultured neurons and quantitative confocal imaging of brain slices. They will provide the first direct information about the physiological properties of neurons that have had an apoptotic death program interrupted. They should allow us to determine the functional potential of neurons when apoptosis has either been stabilized or actually reversed. Most important, they will provide direct evidence for the recruitment of apoptotic pathways in ischemic human brain.

[unreadable] DESCRIPTION (provided by applicant): The treatment of many human epileptic syndromes remains unsatisfactory. While anticonvulsant medications allow about 75% of epileptics to achieve excellent seizure control, the remaining 25% of patients suffer from a combination of continued seizures and medication toxicity. It is unlikely that a single medical breakthrough will provide a cure for all of these refractory patients. Focal neocortical epilepsies have proven particularly difficult to manage. While some respond to anticonvulsants, a large fraction remains intractable to medical therapy. This group can respond to cortical resection, but surgical management is problematic. Exact identification of the epileptogenic focus can be complicated and there is a risk of unanticipated, irreversible neurological deficits after resection. Focal cortical cooling has the potential to improve the evaluation and treatment of this epileptic subgroup. The aims of the experiments described in this application are to investigate the potential of focal cooling with thermoelectric (Peltier) chips to rapidly terminate chronic seizure discharges, determine the degree of cooling required to stop these seizures, determine whether cooling can prevent seizures, and develop computer programs that recognize and anticipate seizures in "real time". In addition, the potential pathological consequences of cortical cooling will be determined. These experiments represent a necessary first step toward utilizing these techniques for the therapy of human epilepsy. These experiments will utilize models of acute and chronic rodent neocortical seizures and small Peltier devices developed for the microelectronics industry. If Peltier devices can control focal seizures in our models, they will be refined for future experiments to investigate their potential role in mapping and controlling epileptogenic neocortex in man. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Over the past two decades basic and clinical neuroscientists have become interested in the anatomic and physiological changes in the central nervous system induced by neuronal activity. These changes, categorized as "neural plasticity" are believed to explain many normal phenomena, such as learning and memory. However, changes in brain structure and function may also be induced by pathological neuronal activity. There is an emerging literature suggesting that the excessive neuronal discharges associated with epilepsy can alter the structure of neurons without leading to neuronal death, but no one has determined the extent or the reversibility of these changes in single, identified central neurons. There are now optical techniques and transgenic mice expressing the green fluorescent protein (GFP) and variants that allow "online" tracking of the morphological changes in single neurons during and after in vivo and in vitro seizures. The experiments proposed in this application will begin to test the hypothesis that prolonged seizure discharges can alter the morphology of spines and dendrites in single neurons imaged in real time and that these changes may play a role in the termination of some types of seizures. These are important issues to resolve, because there is an ongoing controversy about the structural and functional consequences of different types of seizure disorders. More conventional histological analysis, which readily identifies neuronal death, lacks the power to resolve questions about subtle anatomic alterations at the level of single neurons and processes. This work is still in an exploratory stage, because the exact parameters required for long term imaging of in vivo and in vitro seizures have not been determined. When the exploaratory work described in this application has been carried out, it should be feasible to begin isolating the individual variables that account for seizure-induced neuronal alterations. This information will be essential in informing debate about more aggressive therapy for specific types of epilepsy.