Marc A. Dichter, MD, PhD - US grants

The regulation of inhibition in mammalian cortex is intimately involved with the development and control of epilectic events. An understanding of the factors which modulate inhibition could lead to the development of improved methods for treating epilepsy. This grant proposes to study the physiological and pharmacological processes which underlie normal synaptic inhibition in mammalian cortex, employing dissociated cell cultures of mammalian neocortex and hippocampus as model systems. The currents responsible for inhibition generated by GABA, the inhibitory neurotransmitter, will be analyzed using whole cell patch clamp and single electrode voltage clamp techniques. The Cl channel which GABA activates will be characterized, using gigaseal patch clamp techniques. The mechanism which underlies desensitization of the GABA response will be determined at the level of whole cell currents and single channels. Drugs which interact with the GABA receptor complex and other antiepileptic drugs will be examined for their effects on IPSPs, and their mechanisms of action at the level of channel kinetics will be determined. In addition, the mechanisms by which several cortical neuropeptides (SOM, CCK, VIP, ENK) act to modulate synaptic inhibition will be examined and their effects on channel kinetics will be determined. Other "inhibitory" currents in the cortical neurons, especially Ca activated K and Cl currents, which may play important roles in the control of epileptogenic processes, will be examined. The channels underlying these currents will be characterized. The "plasticity" of these events in relation to repetitive activation and the ability of drugs or neuropeptides to modulate these events will also be determined. It is hoped that an increased understanding of the physiological and pharmacological regulation of synaptic and non-synaptic inhibition in mammalian cortex will contribute toward the development of improved strategies for treating epilepsy.

The goal of this research is to understand the physiological mechanisms by which excitation and inhibition in the hippocampus can be modulated over relatively short time periods based on patterns of activation of the synapse. It is our hypothesis that the differential modulation of excitatory and inhibitory synaptic efficacy is responsible, at least in part, for the transition between relatively stable interictal activity and seizures and for the spread of seizure activity from areas of focal abnormality to normal areas of CNS. By increasing our understanding of these processes, it is hoped that new strategies for preventing or treating epilepsy will be developed. These experiments will be carried out in a new preparation of very low density dissociated cultures of rat hippocampal neurons. Recording will be made with whole cell patch clamp electrodes from single neurons and from isolated pairs of synaptically connected neurons. The synaptic interactions between the neurons will also be analyzed with histological techniques. The properties of miniature synaptic potentials, both mini- EPSCs and mini-IPSCs, will be examined and it will be determined if they behave as predicted by the quantum hypothesis. Changes in mini-psc amplitude, shape or frequency will be used to help determine if frequency dependent changes in synaptic efficacy are due to presynaptic or postsynaptic factors. Once this is determined, the mechanisms involved will be ascertained. The following hypotheses will be directly tested: (1) Excitatory and inhibitory synapses between hippocampal neurons behave differently when activated at moderate or high frequencies. (2) Neurotransmitter release characteristics are substantially different for excitatory and inhibitory synapses under comparable physiological conditions. (3) Synaptic transmission between hippocampal neurons in culture can be described by the quantum hypothesis. (4) Frequency dependent decrement in inhibitory synaptic efficacy is due predominantly to presynaptic mechanisms. (5) Frequency dependent increment in excitatory synaptic efficacy is due partially to presynaptic mechanisms. (6) Changes in postsynaptic receptor properties may contribute to the frequency dependent effects and this is more prominently involved in the increment of excitatory synaptic function. At the end of these experiments, the mechanisms which underlie frequency- dependent potentiation of excitatory synapses and decrement of inhibitory synapses will be better understood. It will then be possible to extrapolate these findings to an appropriate epilepsy model (a slice preparation or animal model) to test the hypothesis that these changes in synaptic efficacy are responsible in whole, or in part, for the transition to seizures in an epileptogenic area and for the spread of seizure activity from a seizure focus to normal areas of cortex. Using selective methods for preventing or reversing these effects, it is hoped that, ultimately, seizures can be prevented or suppressed.

GABAA-mediated synaptic inhibition plays an important role in many normal physiological processes in the CNS and appears to be critically important in the physiology of epilepsy. The development of epileptic activity, the onset of seizures in an epileptogenic area, and the spread of seizures to normal brain are all either due to, or are associated with, a decrease in inhibitory efficacy. The factors which regulae GABAA mediated inhibition at the level of the postsynaptic receptor are only partially understood and the Cl channels which underlie the GABA response are only partially characterized. Our previous work has been devoted to characterizing GABAA-mediated inhibition in cultured neocortical and hippocampal neurons, at the cellular and molecular level, using intracellular, whole cell patch clamp and single channel recording techniques. We will continue these studies, focusing initially on the molecular mechanisms responsible for desensitization and resensitization of the GABAA receptor at both the cellular and single channel level. We will determine if these processes are related to changes in Cai or cAMP, activation or inhibition of protein kinases or phosphatases, or modulation of G proteins or phosphoinositol pathways. We will describe the properties of the GABA-activated Cl channel, including the size of the main conducting state, the presence of other substates, the kinetics of channel openings and closing, including tendency to open in bursts, and other physiological influences on the channel. We will also determine if desensitization is due to a change in single channel conductance, a change in probability of openings or in burst versus single opening, a change in open times or inactivation of one subtype of Cl channel (as we have recently demonstrated for desensitization at the quisqualate receptor.) The mechanisms of action of several important drugs which work via the GABA receptor complex, including the benzodiazepines, barbiturates, beta-carbolines, and progesterone metabolites will be analyzed at the channel level and we will determine if neuropeptides which co-exist with GABA in cortical neurons have direct modulatory effects on the GABA receptor and Cl channel. We will also determine whether activation of glutamate receptor subtypes affects GABA responses and whether GABA activation influences the changes in CAi induced by excitatory neurotransmitter (NT) action. Finally, if GABA responses are affected by second messenger pathways, we will determine how these effects may be mediated by other NTs which affect these systems, especially NE, DA, SOM, VIP and CCK. It is hoped that by understanding the regulation of GABAA-mediated inhibition at the molecular level we will be able to devise new strategies for preventing the loss of inhibition which appears to promote the development and spread of seizures.

The Society for Neuroscience is the major professional organization for scientists who study the nervous system. An important goal of this organization is to encourage scientists in training to undertake research related to diseases of the nervous system. The objective of this grant application is to support teaching workshops that introduce young neuroscientists to current concepts about the etiology and pathogenesis of disorders of the nervous system. For each workshop, about 12 faculty are chosen by the organizing committee. Clinical presentations provide enrollees with an experience of the human dimension of particular diseases. Lectures cover both clinical research and relevant laboratory work. In addition to lectures, enrollees are given a choice of small group workshops that emphasize either specific conceptual or methodological issues. Since its inception, eleven workshops have been held, usually on the day prior to the start of the Society for Neuroscience Meeting. Topics have included: Stroke, AIDS in the nervous system, Epilepsy, Huntington's and Parkinson's disease, Muscular Dystrophy, Multiple Sclerosis, Prion diseases, Drug Addiction, Pain and Affective Disorders, Stroke and Excitotoxicity. Enrollment generally runs between 100 and 200 attendees. Most enrollees are graduate students or postdoctoral fellows. Current plans are to cover the following topics in the near future: Recovery of function after neural injury, Genetic Diseases of the Nervous System and Disorders of the hypothalamus and circadian rhythms. Other topics will be chosen depending on their potential interest to young neuroscientists, their impact on society and the quality of recent research related to that disease area.

The Society for Neuroscience is the major professional organization for scientists who study the nervous system. An important goal of this organization is to encourage scientists in training to undertake research related to diseases of the nervous system. The objective of this grant application is to support teaching workshops that introduce young neuroscientists to current concepts about the etiology and pathogenesis of disorders of the nervous system. For each workshop, about 12 faculty are chosen by the organizing committee. Clinical presentations provide enrollees with an experience of the human dimension of particular diseases. Lectures cover both clinical research and relevant laboratory work. In addition to lectures, enrollees are given a choice of small group workshops that emphasize either specific conceptual or methodological issues. Since its inception, sixteen workshops have been held, usually on the day prior to the start of the Society for Neuroscience Meeting. Topics have included: Stroke, AIDS in the nervous system, Epilepsy, Huntington's and Parkinson's disease, Muscular Dystrophy, Multiple Sclerosis, Prion diseases, Drug Addiction, Pain and Affective Disorders, Stroke and Excitotoxicity, Neuromuscular diseases, Amyotrophic Lateral Sclerosis, Schizophrenia, Migraine, Mental Retardation and developmental disorders. Enrollment generally runs between 100 and 200 attendees. Most enrollees are graduate students or postdoctoral fellows. Current plans are to cover the following topics in the near future: Tourette's Syndrome and Obsessive-Compulsive Disorder, the neurobiology of brain tumors, AIDS Dementia, Peripheral Neuropathy, Pain, Language Disorders, and Affective Disorders. Other topics will be chosen depending on their potential interest to young neuroscientists, their impact on society, and the quality of recent research related to that disease area. We are especially interested in covering diseases of the nervous system that are important clinically, but which are in need of enhanced basic cellular and molecular understanding.

[unreadable] DESCRIPTION (provided by applicant): After the first four years of our Bioengineering Research Partnership, implantable devices for epilepsy are now a reality. This is due, in part, to translation of technology developed by our group to industry. Data from multi-center clinical trials of first generation responsive antiepileptic devices indicate that this new technology is safe, and that there is promise of significant benefit to patients. They also demonstrate that 1st-generation devices rarely make patients seizure free. This is because we do not yet understand when, where and how to deliver electrical stimulation to pre-empt seizures, or the mechanisms underlying seizure generation in epileptic networks. These challenges, and translating them into more effective second-generation devices, are the focus of this proposal. Specifically, our aims are: (1) To understand mechanisms underlying seizure generation in two well characterized, spontaneously seizing animal models of epilepsy with documented similarities to refractory human epilepsy, (2) To map seizure generation in the epileptic network to determine where to place sensing electrodes and when to stimulate to maximize seizure suppression and minimize side effects. (3) To develop more effective closed loop stimulation algorithms for controlling seizures. These Aims will be accomplished through a series of projects led by established collaborators in neurology, neuroscience, bioengineering and industry, at Penn, CHOP, Georgia Tech, and BioQuantix, Inc. Teams will focus on improving upon results from first-generation human devices through detailed animal experiments on multiple temporal and spatial scales. These include: (1) the cellular level, through broad-band unit recording and biophysically accurate computational modeling; (2) the network level, with in vitro experiments on hippocampal slices using voltage sensitive dyes and multi-electrode arrays; and (3) the whole brain level, through simultaneous micro and macroelectrode field recordings and responsive brain stimulation in vivo. These experiments will build upon the substantial progress made during the first cycle of our Bioengineering Research Partnership grant. The unique composition of our group, its track record of successful technology transfer, and our ability to learn from and immediately convey our discoveries to existing programmable devices, provide an unprecedented opportunity to perform cutting-edge neuroscience and bioengineering research and immediately translate it into better treatment for patients. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This is a proposal to develop a comprehensive Neurobiology of Disease curriculum, consisting of an overview course and a group of 12-16 new, half-semester Neurobiology of Disease course "Modules" offered over a three year cycle. Modules will provide graduate students with in-depth training in the pathobiology and clinical aspects of a wide range of neurological and psychiatric diseases/ disorders/ conditions. Some Modules will be organized around biological themes common across many nervous system disorders; others will be organized around specific diseases/ conditions. The curriculum for all Modules will include: clinical presentation of the disease/condition, including a patient presentation; pathobiology; disease mechanisms in common with other disorders; how basic science has translated into clinical trials in the given area; basics of clinical trial design and conduct; and ethical issues in patient-oriented research for the particular patient population. Course formats will include lectures by basic and clinical faculty, student presentations, reading assignments from the current literature, small group discussions. The Neurobiology of Disease overview course that exists currently (and has, at Penn, for more than 20 years) and will continue to be offered. All Modules will be developed and co-taught by both clinical and basic science faculty from multiple departments. Graduate students in any University program may enroll; most will come from one the eight areas of Biomedical Graduate Studies, or from the Departments of Psychology, Biology, Biomedical Engineering, or from the Schools of Veterinary Medicine and Nursing. Post doctoral research fellows, medical residents and medical students will be encouraged to enroll, audit and/or make individual use of the materials developed. There are more than 600 graduate students in relevant basic science program at Penn at any given time who likely would be interested in these courses. All materials developed for the overview course and Modules, including slides from course lectures and detailed lecture notes, will be tailored for web education and be made freely available on the web. The T32 training grant that confers eligibility is 5T32GM007517-26 Nusbaum, Michael, "Graduate Training in Systems and Integrative Biology." Penn also has two other neuroscience training grants that would have conferred eligibility. Relevance to public health: This project will develop and implement a new and comprehensive curriculum to teach neuroscience graduate students (i.e., PhD students) about various neurological diseases and injuries and psychiatric conditions. Students will learn how patients' lives are affected, and about the biological mechanisms that malfunction to cause the clinical problem. Students will discuss how treatments have been developed in the past, and how their research in a laboratory can help to lead to new treatment in the future. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] We propose an integrative training program in translational research in neural injury and neurodegeneration for MDs or MD-PhDs who finish clinical training in Neurology, Neurosurgery, Psychiatry, Ophthalmology, Anesthesia, Emergency Medicine or any other clinical neuroscience specialty, and desire academic careers in laboratory-based translational research in the Neurobiology of Disease. Clinician-scientists have a unique ability to ask new scientific questions inspired by patient observations, and are singularly situated to communicate and collaborate with both PhD scientists and health care providers. The program is built around mentoring relationships in which two experienced faculty trainers guide a trainee's research development. Mentors will be chosen to offer complementary experience to the trainee. Trainees will have access to a minimum of 22 laboratories within the "core" group of Penn faculty, but could train in other laboratories as well. Trainee research must be collaborative, asking questions relevant to at least two groups. The 2-year program will focus on neuroprotection for acute and chronic neurodegenerative diseases and conditions, including stroke; brain and spinal cord injury; Alzheimer's, Parkinson's and motor neuron diseases; epilepsy; retinal degeneration; depression; behavioral disorders; autism. Training will focus heavily on laboratory research, with trainee projects directed toward understanding the genetic, molecular and cellular pathophysiology and the development of strategies for prevention, treatment, or cure. Didactic training will include: coursework in the University's comprehensive Neurobiology of Disease curriculum and workshops in clinical trial methodology, grant preparation, statistics, bioethics, patient-oriented research, and new drug development. The program would begin with 2 trainees, growing to 5 within 2 years. A unique feature of the proposed Program is that prospective trainees will know of their acceptance into the Program well before their clinical training ends. This will allow trainees to plan for this stage of their professional development by taking advantage of the time during residency to gain research, and research-related experience, with the final effect of expanding the total amount of training they will have acquired before writing their K-award proposal. It is expected that trainees who complete this program will be very competitive for independent research grant funding, will become faculty members at academic medical centers, and will pursue careers in translational research in the neurobiology of disease. Relevance: We propose to teach physicians to conduct laboratory research into causes of neurological diseases. A better understanding will lead to the development of new treatments and cures. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Clinician-scientists are uniquely positioned to ask new and insightful scientific questions inspired by patient observations, yet, they often lack the expertise to be able to translate their observations into carefully designed basic scientific and translational experiments. There are likely many reasons for this, but the most cited barriers are lack of specific training, mentoring, funding, and time. If these barriers could be removed, more highly motivated clinician-scientists could pursue careers in laboratory-based translational research, thereby helping to reverse the current state of affairs in many neurological disorders, in which basic research is proceeding at an increasingly rapid pace but translational research is lagging, and most patients with neurological disorders are left without effective treatments or cures. Here we propose a research training program for MD-PhDs or MDs who have finished their clinical training in a neuroscience-related specialty and are highly motivated to pursue careers as physician-scientists in innovative laboratory-based translational research in neurological diseases. The program consists of intense research training under the close mentoring of two faculty mentors, chosen to offer complementary experience. Trainees design and conduct independent multidisciplinary research projects that they can take with them when they transition to independent support, and upon which they will base their NIH K-award application. Research projects are directed toward the translation of the genetic, molecular and cellular pathophysiology of neurological diseases into strategies for prevention, treatment or cure. Trainees will be encouraged to pursue projects that are collaborative and cross-disciplinary, as collaboration fosters their research development, and linking disciplines helps generate ideas that are novel and innovative. Trainees will have access to 22 laboratories within the core faculty, but could collaborate with other groups as well. Trainees will work with PhD researchers; participate in journal clubs, lab meeting and basic science seminars. The curriculum includes a course on Neurobiology of Disease, a certificate program in patient-oriented research, which also covers experimental design and biostatistics, and structured discussions on critical topics in translational research. Trainees will also be exposed to the processes required to bring a therapy from the lab to the bedside, including instruction in clinica trial methodology and relevant regulatory issues. Trainees participate in workshops on developing a K- award proposal, grant and scientific writing, and laboratory and project management, plus mandatory training in responsible conduct of research. A unique feature of this Program is that prospective trainees can know of their acceptance before their clinical training ends, allowing them to schedule research into their remaining clinical time, thereby expanding the total amount of research experience they will have before writing a K- award proposal. The main expected short-term outcome for this program is application for an NIH K award.