NIH 1999 Almanac/The Organization/NINDS/       National Institute of Neurological Disorders and Stroke: Major Divisions The institute is organized into a division of extramural research and a division of intramural research. Division of Extramural Research The Division of Extramural Research plans and directs initiatives for grant and contract support for research, research training, and career development to assure maximum utilization of available resources in the attainment of NINDS objectives. Research activities include studies on: fundamental cellular, molecular, and systems neuroscience; developmental neurobiology; developmental disorders; neurogenetics; stroke; traumatic brain and spinal cord injury; neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease; brain tumors; development of artificial prosthetic devices to restore function to the damaged nervous system; convulsive disorders, including epilepsy; infectious disorders of the brain and nervous system, including AIDS; immune disorders of the brain and nervous system, including multiple sclerosis; and disorders related to sleep mechanisms. In addition, the division maintains surveillance over developments in these program areas and assesses the national need for research on the cause, prevention, diagnosis, and treatment of disorders of the brain and nervous system. Program scientists also track technological development, the application of research findings, and research training and career development in these areas. In addition to determining program priorities and recommending funding levels for programs to be supported by grants and contracts, division scientists (a) collaborate with other institutes of the NIH on national research efforts related to these program areas, (b) prepare reports and analyses of national needs to assist NINDS staff and advisory groups in carrying out their responsibilities and in developing new areas of emphasis, and (c) consult with voluntary health organizations and with professional associations in identifying research needs and developing programs to meet these needs. The Division of Extramural Research is organized into work groups known as clusters. The current operational clusters are: Repair and Plasticity; Systems and Cognitive Neuroscience; Channels, Synapses, and Circuits; Neurodevelopment; Neural Environment; and Neurodegeneration. Two additional clusters, one for Clinical Trials and another for Neurogenetics, will become functional soon. Resources and information will move fluidly among the clusters, and new ones will be created and old ones abolished as science, technology, and resources dictate. There are also four administrative branches in the extramural program devoted to support and coordination, an Office of Research Training and Special Programs, and a Training and Special Programs officer. Topics of special interest to each cluster are listed below. Many clinical and basic research problems are addressed collaboratively by members of several clusters. Repair and Plasticity To elucidate mechanisms of synapse formation. To restore function in neurologically disabled individuals. To encourage development of stem cell biology to repair the injured nervous system. Systems and Cognitive Neuroscience To promote understanding of the neural bases of cognition, emotion, and their interaction. To identify risk factors for developmental cognitive disorders. To encourage a broad analysis of the experience of pain. To expand efforts in behavioral genetic studies of complex traits. To develop better methods for assessing behavior and other neurological functions in the mouse as a useful model for human conditions. To encourage research on brain circuits and motor control. To expand research in sleep and rhythmicity. Channels, Synapses, and Circuits To promote further study of ion channel structure and function. To emphasize the molecular bases of synaptic transmission. To encourage new approaches to circuits analysis and focus attention on particular circuits of immediate medical relevance. Neurodevelopment To promote better understanding of the processes of early development of the nervous system. To better understand the influence of developmental stages on the outcome of insult or injury to the brain. To promote understanding of the genetics, risk factors, pathophysiology, and potential therapies for neurological disorders in infancy and childhood, as well those that begin in early life and last into adulthood. Neural Environment To encourage research on normal functions of astrocytes, oligodendroglia and micro glial cells, microvascular endothelia, and cells of the immune system within the nervous system. To promote efforts to understand the blood-brain barrier in health, disease, and drug delivery. To expand ongoing molecular analysis of CNS tumors. To stimulate translational research on phenotype/genotype relations in glial diseases (such as brain tumors and multiple sclerosis). To encourage research on the involvement of infectious agents in the development of neurological diseases (such as chlamydia infection in stroke, JC virus in brain tumor, and campylobacter jejuni in Guillain-Barré syndrome). To promote studies of vascular mechanisms of neurological diseases, e.g., Alzheimer's disease, AIDS, multiple sclerosis, stroke, epilepsy, and trauma of the head, spinal cord, and peripheral nerves. To promote studies of immune disorders of the nervous system and muscle. Neurodegeneration To stimulate research on the mechanisms of neuron death and neurodegeneration underlying a wide range of neurodegenerative disorders, stroke, trauma, and infections. To promote the development of advanced research technologies necessary for achieving new breakthroughs in neurodegeneration research (e.g., array technology for assessment of gene expression and high-throughput assays of biochemical and cellular process modulators). To encourage the development of integrated national registries and population-based epidemiological studies of neurological disorders, in order to elucidate the natural history of neurodegeneration and to identify biomarkers for neurodegenerative disorders. Division of Intramural Research The division conducts basic and clinical research in neurological and related disciplines. Notable achievements have included drug therapies for debilitating neurological diseases such as parkinsonism and new techniques to help scientists better understand how the brain and nervous system function. Major research advances in neurovirology, neurochemistry and neuroimmunology have also come from the division. NINDS scientists continue to explore central nervous system disorders such as Creutzfeldt-Jakob disease that appear to be slow infections caused by transmissible viruslike agents. These agents are unique in some respects, but in others exhibit classical viral properties. Research focuses on delineating the agents' chemical, biological and genetic nature, and on learning the nature of disease pathogenesis. Inherited disorders of lipid metabolism such as Gaucher's, Niemann-Pick, Fabry's, Krabbe's, and Tay-Sachs are studied. This work includes biochemical and diagnostic studies, carrier identification, and genetic counseling. Studies on the molecular basis of the diseases have reached a new frontier; enzyme replacement therapy has been successfully developed for patients with Gaucher's. Gene replacement is also being explored for patients with this and other metabolic disorders. Many research projects in computed tomography advance the clinical applications of the technique as well as provide scientists with a wealth of valuable research data. In other imaging work, studies with the PET scanner have shown a relationship between glucose uptake and brain tumor growth. This scanning technique allows scientists to obtain axial transverse or coronal images of the brain. It also provides dynamic functional data such as rates of glucose consumption in different parts of the brain and measurements of the storage, degradation, and turnover of radioactively tagged metabolites. Functional magnetic resonance imaging is a new technique being used to study brain activity. Biometry and Field Studies Branch Exploring the design, conduct, and analysis of experimental or observational studies of the nervous system is the work of the Biometry and Field Studies Branch. Branch scientists develop new methods to meet the institute's needs for designing experiments and field studies, analyzing data, and devising statistical models of biological processes. The branch also acts as a statistical coordinating center for several continuing or planned clinical trials and for longitudinal field studies involving U.S. and foreign scientists. Developmental and Metabolic Neurology Branch The Developmental and Metabolic Neurology Branch is concerned with inherited disorders of metabolism such as Gaucher's disease, Niemann-Pick disease, Fabry's disease, and Tay-Sachs disease. Investigations include the identification of enzymatic and molecular defects, devising diagnostic and carrier detection methods for genetic counseling, and development of enzyme and gene replacement therapy for patients with these disorders. The branch is also involved in the development of transgenic animals that mimic human metabolic disorders. The pathogenesis of heritable disorders for which the metabolic basis is unknown, such as type C Niemann-Pick disease and mucolipidosis IV, is also under investigation through "reverse genetics" including chromosomal mapping and identification of the mutated genes and the normal gene products. Epilepsy Research Branch The Epilepsy Research Branch investigates the pathophysiology of seizure disorders and cognitive function in individuals with epilepsy, as well as the organization of language and memory function in normal controls, using positron emission tomography studies of cerebral blood flow, metabolism, and neurotransmitters; intracerebral electrode recordings', and magnetic resonance imaging. Animal and cellular models are used to study excitatory and inhibitory mechanisms, the neuropharmacology of antiepileptic drugs, and potential novel therapeutic compounds. Experimental Therapeutics Branch The Experimental Therapeutics Branch seeks to develop improved pharmacotherapies for neurologic disease. At the molecular level, scientists are working to characterize central transmitter receptors and information transduction processes as well as to develop pharmaceutical approaches to the selective regulation of gene expression within the central nervous system. At the systems level, studies focus on basal ganglia function especially in relation to dopamine receptor mechanisms and the effect of drugs that influence motor behavior. At the clinical level, investigators attempt to elucidate pathophysiologic mechanisms and develop novel pharmaceutical interventions for neurodegenerative disorders that impair motor and cognitive function. Medical Neurology Branch The Medical neurology Branch's Human Motor Control Section focuses on how the brain controls voluntary movement and how these processes become deranged with different movement disorders. Recent advances have been made in understanding how the brain makes and learns sequences of movement and the pathophysiology of focal dystonia and ataxia. Human Cortical Physiology Section This section conducts innovative research on plasticity of the human brain following injury, functional relevance and mechanisms of these changes and potential ways to enhance cortical plasticity when it is beneficial and down-regulate it when it is maladaptive. Laryngeal and Speech Section The aim of this section is to determine the factors and mechanisms involved in the epigenesis of idiopathic voice and speech disorders including stuttering and spasmodic dysphonia. Both basic and clinical studies are currently addressing the role of sensory function in laryngeal and speech control disorders. Neuroepidemiology Branch A major goal of research in the Neuroepidemiology Branch is to understanding factors influencing the occurrence of neurological disorders in population groups. Using epidemiological methods, the branch carries out research that may resolve clinical problems related to the cause, prevention, and treatment of nervous system diseases. The branch is currently involved in research on cerebral palsy, pediatric migraine, and progressive supranuclear palsy. Neurogenetics Branch The purpose of the branch is to investigate the causes of hereditary neurological diseases, with the goal of developing effective treatments for these disorders. Particular areas of research interest include the polyglutamine expansion diseases (Huntington's disease, Kennedy's disease, and spinocerebellar ataxia), spinal muscular atrophy, Charcot-Marie-Tooth disease, myotonia congenita, muscular dystrophy, hereditary motor neuron disease, and Friedreich's ataxia. The disease mechanisms are studied in cell culture and other model systems. Gene transfer techniques are being investigated as potential treatment. A related area of investigation is the mechanism of androgen effects on muscle strength and motor neuron survival. A genetic outreach program is intended to identify and characterize patients and families with hereditary neurological diseases. Neuroimmunology Branch In the Neuroimmunology Branch, the role of immunological mechanisms as they may relate to the cause of diseases such as multiple sclerosis is being studied. Immunological and genetic factors are being examined in families with multiple affected members or in twins in which either one or both twins have multiple sclerosis. The role of HTLV-1 and other retroviruses as the cause of demyelinating disease is being assessed. Finally, new approaches to the treatment of multiple sclerosis are being examined and MRI is being used as a tool to study the natural history of the disease and to assess the efficacy of experimental treatments. Stroke Branch The Stroke Branch seeks a molecular understanding of the mechanisms underlying the initiation and progression of brain damage in stroke and the development of preventive and therapeutic measures based on that understanding. Preclinical studies in animal models and tissue culture as well as clinical studies on human tissue samples and stroke patients are performed. The preclinical studies include natural tolerance to hypoxia and ischemia in hibernating ground squirrels, tolerance induced by ligand-receptor interactions involving tumor necrosis factor-a and studies of the intracellular signaling that regulates these tolerant states. In addition, the contribution of inflammatory and immune mediators to the initiation and progression of stroke are studied. Carotid plaques from asymptomatic and symptomatic patients are analyzed for message and protein of inflammatory mediators to determine their roles in the destabilization of the plaque. Acute stroke patients are treated in experimental protocols and the treatments are evaluated by intensive neuroimaging to quantitate the degree to which salvageable tissue is protected by the interventions. Surgical Neurology Branch In the Surgical Neurology Branch, NINDS scientists have undertaken intensive studies of brain tumors, pituitary tumors, neuronal implantation, gene therapy and immunotoxins for brain tumors, and selected aspects of cerebrovascular disease and epilepsy. Clinical Neurochemistry Section The section conducts patient-oriented and preclinical research about neurocardiologic disorders, with an emphasis on catecholaminertic systems. Long-term goals of the section are to (1) elicudate pathophysiological mechanisms and test novel treatments of neurocardiologic disorders; (2) test concepts of integrative medicine, via collaborative studies about the physiology and pathophysiology of catecholaminergic systems; and (3) direct a world-class, CLIA-certified Catecholamine Assay Laboratory, both to support neurocardiology protocols of the section and support collaborative studies about catecholaminergic systems. Cognitive Neuroscience Section This section investigates the brain basis of human cognitive processes such as planning, reasoning, decision-making and memory using traditional cognitive science methods and functional neuroimaging techniques in both patients and normal volunteers. The section focuses on the role of the prefrontal cortex in cognition and behavior. Neuromuscular Diseases Section This section focuses on the investigations and new or experimental therapeutic interventions in patients with inflammatory myopthies, peripheral neuropathies especially those related to infections or dysfunction of the immune system, postpolio syndrome and other motor neuron disorders and in patients with the stiff-person syndrome. Laboratory of Adaptive Systems The Laboratory of Adaptive Systems studies the molecular basis of associative memory and the related behavior of living animals to signal processing in neuronal networks and to subcellular molecular cascades. Biophysics and Electrophysiology Section This section within the Laboratory of Adaptive Systems studies the molecular, biophysical, and integrative bases of associative memory in brain networks. Section observations have related learning and memory behavior of living animals to signal processing in neuronal networks and to subcellular molecular cascades. Our data have implicated molecular and biophysical mechanisms that are conserved in molluscan and mammalian species and thus could have relevance for human learning and memory. Cellular analyses of associative memory in the snail Hermissenda (Pavlovian/classical conditioning), the rabbit (classical conditioning), and the rat (spatial maze learning, olfactory discrimination) revealed a cascade of cellular and subcellular events during memory formation. These events include: long-term synaptic transformation of GABAergic inhibition into excitation; elevation of intracellular calcium and DAG; translocation of PKC; PKC-mediated phosphorylation of the Ca2+ and GTP-binding protein, cp20 (also called Calexcitin): inactivation of voltage-dependent K+ channels; learning-specific regulation of gene transcription; and rearrangement of synaptic terminal branches. Behavioral Neuroscience Unit The goal of the Behavioral Neuroscience Unit of the Laboratory of Adaptive Systems is to conduct research designed to understand the behavioral laws and identify the physiological basis of classical conditioning, an associative form of learning and memory. In behavioral experiments, the relationship between the elements of associations and have shown that the properties of each element that enters into an association may alter an organism's behavior is explored. The long term memory for simple associations has been shown to last as long as six or even nine months, and we have identified a number of sites in rabbit and human brain that are implicated in learning and memory. Considerable electrophysiological and human imaging data from this and other laboratories point to the cerebellum as an important site in learning and memory. Laboratory of Central Nervous System Studies This laboratory focuses research efforts on slow, latent, and temperate viral infections associated with chronic degenerative neurological diseases. An important area of study is the pathogenesis of slow infections and mechanisms of viral persistence, Creutzfeldt-Jakob disease. The Laboratory of Developmental Neurogenetics This laboratory identifies and analyzes genes whose products are relevant for nervous system development and function and whose mutations may be associated with neurological disorders. The focus is on genes regulating the development and differentiation of oligodendrocytes whose malfunction may result in dysmyelination or demyelination, and of pigment cells whose malfunction may result in blindness or hearing loss. Genetic mouse models as well as cell cultures and yeast one- and two-hybrid systems are employed as the primary experimental systems. Developmental Genetics Section The Developmental Genetics Section focuses on the analysis of genes regulating the development and differentiation of the myelin-forming oligodendrocytes whose malfunction may result in severe neurological disorders such as Multiple Sclerosis or Pelizaeus-Merzbacher Disease. The key question is how a network of genes functions to bring about the coordinate synthesis of both structural proteins and lipids in myelin. To understand this process in more detail, we have isolated a number of cDNAs for proteins binding specific promoter elements of the proteolipid protein (PLP) gene and other myelin genes. Studies are also aimed at deciphering how extracellular signals from various cytokines are transmitted from tyrosine kinase receptors to key transcription factors in cells of the oligodendrocyte lineage. Mammalian Development This section focuses on the molecular mechanisms that govern the generation of distinct cell types from unspecified precursors during mammalian development. A detailed knowledge of these mechanisms will not only help us understand fundamental principles of normal ontogeny but also explain, and ultimately correct, instances where development has derailed and disease has resulted. Studies are currently focusing on the role of the basic-helix-loop-helix-zipper transcription factor Mitf whose mutations in rodents may lead to small eyes, retinal degeneration, deafness, and abnormalities in skin pigmentation and whose mutations in humans are associated with the hereditary deafness syndrome Waardenburg IIa. An ultimate goal of these studies is to characterize the network of factors involved in the generation and function of melanocytes which are evidently of crucial importance for the development and function of mammalian sensory organs. Laboratory of Functional and Molecular Imaging The Laboratory has two major research interests. First, is to develop non-invasive imaging techniques to assess brain function. This work primarily uses MRI and takes advantage of the latest generation of high field MRI. The second thrust is to combine imaging and molecular biological tools for functional genomic studies. They are interested in understanding mitochondrial function and development and function of the brain. Laboratory of Molecular Biology This laboratory studies the origins of the cellular diversity in the central nervous system. Neuronal precursor cells can be identified after gastrulation by their expression of the nestin gene . By using transgenics and footprinting, 50 bases in the second intron that are sufficient to direct gene expression to precursor cells throughout the embryonic CNS have been identified. Defined tissue culture conditions allow the expansion of primary nestin-positive cells and their differentiation into neurons and glia. Nestin-positive cells from the embryonic CNS can be clonally expanded in vitro. This stem cell can self-renew and differentiate to give neurons, astrocytes and oligodendrocytes. Addition of defined growth factors to the medium directs this differentiation process by instructive mechanisms. In addition we have defined conditions that permit the efficient differentiation of ES cells into synaptically connected neurons. Thus, non-cell-autonomous mechanisms play a major role in directing the differentiation of CNS stem cells to appropriate regional fates. Our current data supports a model where a common stem cell is present in different brain regions and that this cell comes under the influence of extracellular signals to generate locally appropriate neuronal types. Laboratory of Molecular and Cellular Neurobiology This Laboratory is composed of two sections which share common interests in the structure and function of membrane proteins and lipids, transmembrane signaling and cell surface glycoconjugates. Techniques of biochemistry, molecular biology, cell biology and immunology are utilized to investigate basic and clinically-related questions in neurobiology. Myelin and Brain Development Section The primary goals of research in the Myelin and Brain Development Section are to elucidate the physiological and pathological roles of glycoproteins and glycolipids of myelin and myelin-forming cells. The Section does both basic and clinically-related research with the primary objectives of elucidating physiological and pathological roles of glycoproteins and glycolipids of myelin and myelin-forming cells. One aspect of the basic research focuses on glycoproteins of myelin sheaths with particular emphasis on the myelin-associated glycoprotein (MAG). MAG is a member of the immunoglobulin (Ig) superfamily that is localized in periaxonal glial membranes of myelinated fibers and is thought to function in transmitting signals in both directions between axons and myelin-forming cells. The clinically-related research in the Section is divided into two broad categories. The first has to do with immune-mediated neuropathies associated with serum antibodies to glycoconjugates. Membrane Biochemistry Section This section focuses on the regulation of cellular signaling with emphasis on beta-adrenergic receptors, the adenylyl cyclase system and heterotrimeric G proteins. Our research is focused on cellular signaling and its regulation using the adenylyl cyclase system as a model. The latter consists of receptors that bind hormones and neurotransmitters, G proteins that transduce the signal, and a catalyst that generates cyclic AMP, the intracellular second messenger. One the Section's major goals is to understand how cells modulate their response to stimuli. The mechanisms are complex and include desensitization, internalization, up- and down-regulation, and cross-talk between different signaling systems. We are currently investigating the differences in regulation of the three ß-adrenergic receptor subtypes, ß2, ß1, and ß3. Thus, human ß1 receptors internalize less then ß2 receptors in response to an agonist; but, we recently found that over expression of ß-arrestin eliminates the difference. ß-arrestin also binds to clathrin and is believed to act as an adapter to recruit receptors into the coated-pit endocytic pathway. Our results suggest that ß-arrestin has a lower affinity for ß1 than ß2 receptors. We also have identified multiple mechanisms of down-regulation at both receptor mRNA and protein levels. One involves repression of rat ß1 gene transcription by the inducible cAMP early repressor (ICER). A second involves activation by protein kinase C of a transcriptional repressor that binds to a novel response element in the rat ß1 promoter. In a third, human ß1 mRNA is destabilized by a cAMP-induced mRNA binding protein. Finally, the human ß1 receptor protein can undergo both inactivation in response to cAMP and degradation in response to agonist. Another aim is to characterize the physical interactions that occur between the components of the adenylyl cyclase system during signal transduction, especially the subunit interactions of the heterotrimeric G proteins. Laboratory of Molecular Medicine and Neuroscience The goals of the research in the The Laboratory of Molecular Medicine and Neuroscience primarily involve understanding the pathogenesis of human neurotropic virus infections during the clinical course of disease in situ and establishing biological models using neural cell cultures in vivo. The neurotropic viruses studied are the human polyomavirus, JCV, which causes a demyelinating disease in immune compromised individuals, termed Progressive Multifocal Leukoencephalopathy (PML) and the human lentivirus, HIV-1, which causes an encephalopathy/encephalitis as a significant complication in AIDS. A second goal is to determine the response of neural cells to viral gene expression at the molecular, transcriptional level to define not only the susceptibility to infection but also to gain insight into normal cell functions. And a third goal is to apply the data and expertise gained from studies utilizing human neural cells and viral gene expression to the development of cell and viral vectors to delivery of therapeutic molecules to the CNS. Molecular Medicine and Virology Section This section conducts experiments on the cellular events involved during the course of viral infections in the central nervous system and understanding virus-cell interactions at the molecular level. Two human neurotropic viruses are the focus of study, JC Virus and HIV-1, which are linked together for study in several ways. Both viruses are neuroinvasive, involve white matter diseases in the brain, and infect neuroglial cells as well as immune cells. In the human brain, JCV lytically infects the oligodendrocyte causing a fatal demyelinating disease, Progressive Multifocal Leukoencephalopathy, PML. PML occurs almost exclusively in immune compromised individuals particularly in AIDS cases. Since the AIDS epidemic began, the incidence of PML has increased dramatically with thousands of cases reported worldwide. By current estimates, AIDS is the underlying immunosuppressive disorder in 90% of PML cases. Of all AIDS cases, 4-6% will develop PML and, not infrequently, it will be the defining illness . The goals and experimental approaches used in our studies of JCV and HIV-1 have been similar because of the neurovirulence of both viruses and their involvement with PML and AIDS. In cell cultures derived from human fetal brain, JCV multiplies well in glial cells, predominantly the GFAP positive astrocyte, the most highly represented cell in these cultures. Because of the efficiency of infection in glial cells, JCV is considered highly neurotropic . Like JCV, HIV-1 also infects astrocytes in both in vitro and in vivo, but they are not the primary focus of its infection. HIV-1 predominantly infects macrophages and microglial cells ultimately causing an encephalitis and/or encephalopathy . HIV-1 infection in the brain also leads to severe cognitive impairments in approximately 25% of AIDS cases formerly described as the AIDS dementia complex. Specific neurotropic strains of HIV-1 however have not been identified. The genetic and physical maps of both JCV and HIV-1 genomes have been well characterized. Both viruses show genetic variations which effect host range and influence their neurovirulence. Studies of these two viruses are the major focus of the Section's investigations. However, most experiments for JCV or HIV-1 are conducted using cells derived from human fetal brain. Consequently a third project has evolved in the lab dedicated to the characterization of these cells during development. Molecular Therapeutics Section The section studies therapeutic options for the treatment of neurological disorders. Given the unique biology of the brain and spinal cord, non-traditional and novel methods are being developed to treat neurological diseases. The experimental approach in the section is two fold: Somatic cell therapy based upon development and transplantation of neuroglial cell lines into the CNS and viral vectors for sustained delivery of therapeutic genes. Laboratory of Neural Control The primary goal of scientists in the Laboratory of Neural Control is to understand the systems of central nervous system neurons that produce and control movement in human beings and other vertebrate animals. Much of our work is done on systems of neurons in the spinal cord and brain stem, where the input elements (sensory afferent supraspinal descending fibers) and output elements (the motoneurons that control specific muscle groups) have clear functional identities. Investigators in LNLC are also pursuing related mathematical and computational studies of the properties of individual nerve cells and of systems of interconnected neurons. Neural Mechanisms Section Research in the Neural Mechanisms Section in the Laboratory of Neural Control includes work on the organization of spinal cord interneurons, computational and mathematical studies of the structure and function of neuronal dendrites. One major focus is to working out circuit organization of excitatory last-order interneurons in the spinal cord of adult cats. The Section also uses computational studies of the structure and function of neuronal dendrites Dendrites are critical to understanding the way in which synaptic information is processed to produce action potentials that relay that information to the cell's targets. Two major problems in this area are: 1) defining the morphology of dendrites and the synaptic endings from identified afferent systems that terminate on them; and 2) defining the functional properties of the synapes and their postsynaptic receptors, as well as the electrical properties of the dendritic membrane. Developmental Neurobiology Section Research in the Developmental Neurobiology Section is concerned with the development and operation of circuits in the spinal cord. Most of the work is done on the isolated spinal cord preparation of the chick embryo, although some work is done on other species. Of particular interest is the understanding of how spontaneous rhythmic activity is generated by developing spinal networks. To address this question classical electrophysiological techniques coupled with optical methods for understanding the behavior of neuronal populations are used. Our current thinking is that the spontaneous activity is a self-organizing property of developing spinal networks. In particular it arises from the coupling of widespread excitatory connections within the network with a set of activity-dependent depressors of network excitability. One of the important depressors appears to be synaptic depression which follows an episode of activity. Cellular and Systems Neurobiology Section Research in the Cellular and Systems Neurobiology Section is directed toward understanding brain mechanisms underlying the generation and control of innate motor behavior in the mammalian CNS. One of the fundamental challenges in contemporary neuroscience is to explain the generation of behavior and complex regulatory functions of the higher vertebrate brain in terms of cellular and network properties of neural systems. Neural networks generating movement, particularly brainstem or spinal circuits producing innate motor behaviors such as breathing and locomotion, provide important model systems to address these problems in the mammalian CNS. It has long been postulated that these rhythmic motor acts are produced by central pattern generation networks (CPGs) that are intrinsically organized to generate the underlying complex spatiotemporal patterns of oscillatory neural activity. Our long-range goal is to explain the ontogeny and neurogenesis of respiratory rhythm and pattern in terms of the molecular, biophysical, synaptic, and network properties of functionally identified CNS respiratory neurons. Current studies focus mainly on identifying the cellular and network mechanisms operating in the respiratory rhythm generator (oscillator)- one of the main components of the respiratory CPG. One of our seminal discoveries is that we identified neurons in the pre-Bötzinger complex, the main locus of rhythm-generating neurons, with pacemaker properties that are the current candidates for the rhythm-generating cells. Structural Cell Biology Section The Section performs Video and confocal microscopy, image processing, biochemistry, and advanced electron microscopy and these are used are used in conjunction with isolated or cultured nerve cells, in vitro models, and reconstituted systems to address a range important questions in cellular neurobiology. The squid giant axon has been an important preparation for much of this work. Current areas of interest are functions of the various kinesins and kinesin related proteins in the axon; the motors and mechanisms of slow axonal transport; role of myosins in axonal transport and in other axonal functions; role of endoplasmic reticulum and other calcium buffers in synapses, dendrites and spines; mechanisms of mRNA migration in dendrites; and relationships of the endoplasmic reticulum to the axonal transport; structure of the postsynaptic density. Analytical Cell Biology Section This section investigates the fundamental cell biology of neurons, with emphasis on the organization and function of the specialized membranes and intracellular transport pathways which underlie synaptic transmission. The Section focuses on calcium regulation of postsynaptic responses to synaptic activity. The Section uses many of the principal microscopical techniques of modern structural biology, while continuing its commitment to the development of new methods designed to advance the quantitative and molecular aspects of electron microscopy. The following is a brief description of major areas of research: (1)Calcium regulation in dendrites and dendritic spines. These studies aim to evaluate the role of various calcium uptake, release, sequestration and extrusion mechanisms in the generation and termination of free Ca2+ transients in spines and dendrites of hippocampal pyramidal cells and cerebellar Purkinje cells. It also aims to characterize in detail the relationship between intraneuronal free Ca2+ and total Ca. One important goal is to characterize the interplay between mitochondria, endoplasmic reticulum, and cytoplasmic Ca2+ buffers in the spatio-temporal buffering of free cytosolic Ca2+. (2)Molecular organization of protein assemblies underlying intracellular transport. Scanning transmission electron microscopy (STEM) techniques are being used to characterize, at the nanometer level, the molecular shape and organization of several important proteins or cellular assemblies which underlie intracellular transport and/or synaptic transmission. Cellular Organization Unit Studies the sorting of membrane proteins in excitable cells (muscle and nerve). Sorting of membrane proteins is an essential component of the subcellular organization of all cells . But it is especially challenging for cells of nerves and muscles because of their large size. One alternative to the transport of proteins from their site of synthesis to a distant site of action is to synthesize the proteins close to the very sites where they are needed. This process requires sorting of mRNAs and of the subcellular organelles involved in protein synthesis, the endoplasmic reticulum (ER) and the Golgi complex. Some studies are on on the mechanism by which mRNAs are localized in cultured myotubes and in cultured rat hippocampal neurons, using non-isotopic in situ hybridizations. The present focus is on the mechanism by which the Golgi complex is reorganized during differentiation of muscle and nerve cells. Laboratory of Neurochemistry This laboratory is concerned with the development, structure, and functional organization of the nervous system, with a special focus on molecular and physiological mechanisms that are involved in establishing and maintaining the neuronal phenotype. A wide variety of techniques ranging from the anatomic to the molecular genetic, and invertebrate as well as mammalian model biological systems, are employed to study various processes of neurogenesis, migration, differentiation and specification of the neuronal phenotype during development, and the maintenance of diversity and plasticity in the mature nervous system. Cellular and Developmental Neurobiology Section The section focuses on the development and regulation of Luteinizing hormone releasing hormone (LHRH) neurons. As integral components of the hypothalamic-pituitary-gonadal axis, LHRH neurons are exquisitely regulated, exhibiting pulses of LHRH secretion in reproductively mature animals. Developmentally, LHRH neurons originate outside the CNS, in the olfactory placode, and thereafter migrate into the brain. Alterations in normal development or regulation of this system results in reproductive dysfunctions. Work in this Section exploits in vitro models in which it is possible to systematically perturb LHRH migration, gene expression and/or secretion. Major projects are: Mechanisms Underlying Phenotypic Choice and Neurophilic Migration Regulation of Neuropeptide Expression in Neuroendocrine Cells Molecular Neuroscience Section The principal research goal of the Molecular Neuroscience Section is to understand the molecular mechanisms which are involved in the adaptive, and homeostatic regulation of cell-specific gene expression, biosynthesis, and secretion of neuropeptides in the nervous system. Over the past twenty years, neuropeptides have become increasingly prominent as intercellular messengers in the peripheral and central nervous system, acting as neurohormones, neuromodulators, neurotrophic factors, and/or neurotransmitters. The research projects in the Section are concerned with the elucidation of the peptidergic phenotype, and specifically focus on the cell biological processes that underlie peptide neurosecretion . In order to systematically examine these issues, the magnocellular neurons of the mammalian hypothalamo-neurohypophysial system (HNS), which synthesize and secrete the nonapeptides, oxytocin (OT) and vasopressin (VP) are studied. These neurons are representatives of a specialized class of peptidergic neurons called Neurosecretory cells (a k a Neuroendocrine cells or Endocrine neurons). Specific research goals with respect to the HNS are: 1) to determine whether the cellular phenotypes in the HNS, usually defined as being either OT- or VP-synthesizing magnocellular neurons, contain other differentially expressed molecular species, 2) to evaluate the molecular genetic bases of cell-specific expression of the OT and VP genes, i.e., identification of the relevant promoter and enhancer sequences in these genes, and ultimately the putative cell-specific transcription factors that may be regulating their cell specific expression, 3) to determine the sorting signals and mechanisms that direct the packaging of the OT and VP peptide precursors into LDCVs, and 4) to determine which calcium channel subtypes and SNARE protein isoforms are involved in the calcium-dependent neurosecretion of the OT and VP peptides. Neuronal Cytoskeletal Protein Regulation Section The section focuses on the molecular basis of neuronal morphology, i.e., its highly asymmetric shape with cell body, elongated axon and branching dendrites, each cellular compartment defined by a unique cytoskeleton. Among the many cytoskeletal molecules in nerve cells responsible for this morphology are the neurofilaments (NFs), which are exclusively neuronal and serve as phenotypic markers. NFs, neuron specific Class IV intermediate filaments, are the major cytoskeletal element of large axons and together with microtubules, they determine the size and shape of the neuron. The NF-subunit proteins, NF-L, NF-M and NF-H, are extensively phosphorylated, with up to 100 or more potential phosphorylation sites in different domains. During NF processing from cell body to axon terminal, phosphorylation is topographically regulated by a dynamic equilbrium between the activities of kinases and phosphatases within cellular compartments. Though all kinases, phosphatases, regulators and substrates are synthesized in cell bodies, the extensive stable phosphorylation of NFP tail domains occurs primarily in the axon during axonal transport. Inasmuch as NFs require multi-site phosphorylation, it is possible that during NF processing, a sequence of multi-site phosphorylations and dephosphorylations are required before the KSP repeat sites become accessible for axonal phosphorylation. We are concentrating on the questions: What are the mechanisms of action of these kinases (primarily the proline-directed kinases) and phosphatases and how are they topographically regulated during NF processing, from synthesis in the cell body to assembly into the axonal cytoskeleton? Neurogenetics Unit The primary goal of the Neurogenetics Unit is to enhance our understanding of the mechanisms that generate neuronal diversity during CNS development. Unique cellular phenotypes in the nervous system are generated in part by the combinatorial actions of neural-identity regulators. Members of the evolutionary conserved POU transcription factor family function as cell-identity regulators during CNS development by establishing/maintaining unique cell fate programs in neural stem cell lineages. Recent studies performed in my laboratory have identified a temporal branch-point in the regulatory hierarchy controlling these genes during Drosophila CNS development. We have discovered that the pdm-1 and pdm-2 POU genes are tightly regulated by two Zn-finger transcription factors, Hunchback (Hb) and Castor (Cas) (Kambadur et al., 1998). Functional analysis of these Zn-finger proteins has revealed that they act in a cooperative, nonoverlapping manner to restrict pdm expression during neuroblast sublineage development. By silencing pdm expression in early and in late developing sublineages, Hb and Cas establish three pan-CNS expression domains whose cellular constituents are marked by the expression of either Hb (early), the Pdms (intermediate), or Cas (late). Phenotypic analysis of loss of function mutations in hb, pdm or cas, by us and others, has shown that each of these regulators carryout distinct roles during sublineage development. Protein-DNA binding studies have identified multiple Cas/Hb docking sites within the pdm-1 neurogenic regulatory DNA, suggesting that these proteins function as direct repressors of pdm gene expression. Our studies have also demonstrated that cas function is required for proper expression of all known Drosophila POU genes, and the effects of cas are both negative, repressing pdm expression, and positive, as drifter and I-POU require cas for full-expression. This differential control over neural-identity genes suggests that Cas may have dual regulatory roles, functioning as an activator to ensure the expression of determinants that control cell fates in late forming neuroblast sublineages and as a repressor to insulate their identity programs from factors that dictate earlier fates. Current efforts are aimed at understanding the molecular mechanisms that establish and maintain this regulatory "fork in the road." In order to learn more about this cell fate machinery and ultimately better understand its biological significance we are pursuing two central questions that address how and why these pan-CNS expression domains are formed. First, what are the regulators that control the sequential hb -> pdm -> cas neuroblast expression? As a step toward identifying the upstream regulators, a genetic screen designed to identify factors required for correct hb and/or cas expression is underway. This P-element induced recessive lethal screen has already yielded several mutations which alter cas lineage-specific expression. Our second goal focuses on understanding the functional significance of these layered expression domains. Due to the shared expression of these transcription factors, cells within a given domain may have overlapping repertoires of downstream target gene expression. To test this hypothesis, we will prepare cDNAs from isolated Hb or Cas expressing neuroblasts. Using differential screening and in situ hybridization techniques we will search for genes whose expression is restricted to specific layers. By identifying additional target genes whose expression is restricted, we hope to not only expand our understanding of this regulatory network but also to connect these genetic circuits to genes those encoded proteins impart distinct neuronal phenotypes. Laboratory of Neurophysiology The laboratory focuses on the cellular and molecular mechanisms underlying the physiological transformation of embryonic mammalian central nervous system (CNS) tissue from its initial proliferative state into fast-transmitting circuits of cells and signals. In vivo, proliferation occurs among cells that couple in a variable manner in an electrical syncytium lining the central core of the developing CNS (the ventricle), which, during embryogenesis, is filled with cerebrospinal fluid containing high concentrations of the same proteins that circulate in the blood. During embryogenesis pre-mitotic cells divide repeatedly to populate CNS regions with millions of cells. In the cortex, waves of post-mitotic neurons migrate from the ventricular zone both radially along, and tangentially to glial elements that span the forming tissue. Migrating neurons disperse in the differentiating region, forming local and long distance circuits and networks. Complementary strategies to access the physiology of this complex process, which involves dramatic changes in tissue form and cell function are used. Conventional electrical techniques are used to record individual cells in real time either in situ or in culture. After ~15 years of research and development this laboratory has developed and optimized laser-based flow cytometry for use in conjunction with fluorescence indicator dyes to quantify physiological properties expressed by individual cells at membrane and cytoplasmic levels. The ultra-high throughput (100,000 cells/minute) and the random nature of the recording strategy provide, quick unbiased and complete accounts of properties emerging in proliferative and differentiating cells. The reproducibility in 10,000-cell profiles of physiological properties both under steady-state and stimulating conditions attests both to the high fidelity and precision of the FACS strategy and the stereotypical changes occurring during cortical development. The sort capability of the FACS allows unparalleled purification of specific subpopulations for in vitro investigation. Lamp-based digital videomicroscopy is also used to record at near real time rates two optically-detectable properties simultaneously on ~100 cells in culture. Each of these recording strategies complements the others. Immunocytochemistry has been employed to identify relevant epitopes and specific components distributed in sections and suspensions. Recent results include: 1) immunoidentification of ~98% of all embryonic CNS cells by their surface ganglioside expression, 2) characterization of GABAergic cell components, steady-state physiological properties and pharmacological responses of all immunoidentified CNS cells using indicator dyes and flow cytometry, 3) direct 1:1 correlation between ganglioside expression and surface GABA level over a 100-fold range of signal intensity, 4) co-localization of ganglioside and GABA in patches on cultured neurons, 5) rapid and reversible loss of GABAergic signals in cultured neurons from a surface-accessible compartment by gentle streams of saline, 6) rapid transformation in tonic and transient GABAergic signaling at GABAA receptor/Cl- channels by ruthenium red, which binds sialic acid residues of gangliosides and other membrane compounds, 7) fM and uM GABA-induced, Ca2+-dependent chemotaxis and chemokinesis of cortical neuroblasts from the ventricular zone and cortical plate, respectively via pertussis-sensitive mechanisms, and 6) astrocyte-mediated facilitation of hippocampal and spinal neuron differentiation and synaptogenesis in vitro through Cac2+-dependent mechanisms involving neuronal GABA and glutamate receptors. Ion Channel Biophysics Unit The unit is broadly focused on the role of ion channels, potassium ion channels in particular, in nerve cell function. The Unit interested in the relationship between specific gating properties of ion channels and nerve cell electrical activity, the distribution of ion channels in nerve tissue and in particular nerve cell types, and the trafficking of ion channels between nerve cell bodies, where ion channels are synthesized, and various parts of nerve cells, in particular nerve cell axons. Currently two preparations to address these issues; the squid giant axon and Cajal-Retzius cells in the late embryonic and early post natal period of development in the rat brain are used. Cajal-Retzius cells represent a unique system for developmental neurobiology owing to their relatively short life span and to reliable anatomical markers. We have recently discovered a development loss of a rapidly inactivating potassium ion current during the first two weeks of post-natal development even though the fast activating, tetrodotoxin-sensitive, sodium ion current is up-regulated during this same period. Neural Circuits Unit The unit studies retinal structure & function.Visual information is transduced by photoreceptors and processed by numerous retinal interneurons before transmission through optic nerve fibers to brain visual centers. Exploration of integrative pathways in retina, including special systems of neurons devoted to processing signals either from rods or from cones, or to generating cellular pathways excited either by brightness increments, or brightness decrements (ON-center and OFF center pathways), form the principal focus of this investigation. There are several approaches. One is structure-function studies in retinal slice or whole mount. Photic or drug induced responses are compared to morphology and synaptic connectivity of retinal neurons. Acutely dissociated retinal neurons provide another approach. Dissociated horizontal and bipolar cells can be recognized morphologically, and pharmacological reactions studied with fluourescent probes. The goal of such studies is to generate circuitry models which aid in the understanding of information flow and the specialized roles of individual neuronal types in the processing of visual signals. Molecular Plasticity Section The Section studies neuroplasticity in the striatum. Under normal circumstances, plasticity within the striatum plays a key role, in the formation of important types of implicit memory-including reward-associated learning. Under pathologic circumstances, plasticity within striatal neurons is involved in the pathogenesis of addictive disorders and in limiting the effectiveness of dopamine replacement therapies in Parkinson's disease. Similar processes may be involved in producing some of the symptoms of psychotic disorders. The lab is interested in how neurotransmitters and pharmacologic agents produce long-term changes in the functional properties of striatal neurons by regulating gene expression. The initial synaptic effects of cocaine and amphetamine in the intact striatum (increasing not only synaptic dopamine, but also serotonin and glutamate), and of dopamine agonists in the denervated striatum, leads to receptor-mediated activation of second messenger cascades and protein kinases that can signal to the nucleus. Thus, in addition to their rapid behavioral effects, neurotransmitters and drugs produce slower post-receptor adaptations, including regulated gene expression, that ultimately alter neuronal function and therefore the behavior of neuronal circuits. We have been focused on the intracellular signaling pathways that convert stimulation of dopamine, glutamate, and serotonin receptors into activation of the CREB and AP-1 families of transcription factors in striatal neurons, and have investigated the molecular mechanisms by which such events lead to activation of "target genes" such as the prodynorphin and proenkephalin genes. We continue to investigate the complex interactions of neurotransmitter-activated cyclic AMP and Ca2+-signaling cascades in striatal neurons using the tools of patch clamp physiology and molecular biology. In addition, we are in the process of identifying multiple target genes activated by dopamine in striatal neurons, investigating the pathways by which they are activated, and in trying to understand their function. Neural Development Section The principal research goal of the Neural Development Section is to understand how functionally appropriate synapses form during development of the nervous system. Focusing on a simple synapse in the peripheral nervous system, the sympathetic innervation of an autonomic target tissue, she and her colleagues have provided compelling evidence for unexpected plasticity in the expression of neurotransmitters and neuropeptides in the developing nervous system. Specifically, the sympathetic neurons that innervate sweat glands initially express a noradrenergic phenotype but are instructed via a soluble retrograde signal provided by the target tissue to acquire cholinergic and peptidergic properties. While the change in transmitter properties observed in vivo can be mimicked by several neuropoietic cytokines, including leukemia inhibitory factor, ciliary neurotrophic factor and cardiotrophin-1, none of these corresponds to the differentiation factor derived from sweat glands. Rather, the factor, which requires activation of receptor subunits utilized by the neuropoietic cytokines and shares common downstream signaling pathways with them, appears to be a novel family member. A similar change in neurotransmitter properties occurs in the innervation of a second sympathetic target tissue, the periosteum or connective tissue covering of the bone. Synapse formation in this system requires not only retrograde signaling but also anterograde. Production of the cholinergic differentiation factor by the target tissue requires noradrenergic sympathetic innervation. Similarly, in the absence of cholinergic innervation, sweat glands fail to acquire secretory responsiveness during development and fail to maintain it in adulthood. Thus, synapse formation requires reciprocal cell-cell interactions and the neuronal effects on the differentiation status of the target tissue are mediated by classical small molecule transmitters. Neurotoxicology Section Studies in the Neurotoxicology Section involve the viral agent (JC virus) of a fatal demyelinating disease of the CNS known as progressive multifocal leukoencephalopathy (PML). PML occurs in immunocompromised individuals, including about 5% of AIDS patients. Demyelination results from productive infection by JCV of oligodendrocytes, the myelin forming cells. JCV is one of two human polyomaviruses and is closely related to SV40. PML provides a viral model for the demyelinating disease of unknown etiology, multiple sclerosis. Our studies have focused on two levels of JCV variation. First, the viral regulatory region, which is stable in the kidney (archetypal), is rearranged by deletion and duplication in the PML brain (PML type). Each rearranged regulatory region is unique. The process generating these neurotropic viral genomes is unknown. It may occur in the brain, or in lymphocytes which sometimes also have a low level infection. Second, the viral coding region and intergenic regions show mutations which characterize 3 basic genotypes differing in sequence by 1-2%. Type 1 is European, Type 2 is Asian, and Type 3 is African. In addition, a fourth major genotype is derived from a recombinant between Types 1 and 3. Type 4 makes up about 15-20% of the viral genotypes in the U.S.A. Current research is aimed at (a) further definition of the worldwide molecular epidemiology of these JCV genotypes, (b) providing evidence for biological differences between these genotypes, and (c) defining the mechanisms of these biological differences at a molecular level. Neurotrophic Factors Section This section studies how Signaling in the brain occurs among both neurons and glia, and how some of this information flow is mediated by trophic factors. Expression of neurotrophic factors is high developmentally and can be elevated significantly in response to various types of injury to the adult brain. Not only neurons but also astrocytes respond to all types of brain injury and some of the increase in neurotrophic factors occurs in reactive astrocytes. The increased expression of neurotrophic factors may represent part of the brain's attempt to respond to the damage, with the factors involved in the resulting plasticity and restoration efforts. One major objective of the Neurotrophic Factors Section is to determine the phenotype of astrocytes during development and following injury, especially in terms of regulation of the expression of neurotrophic factors and neuropeptides, in order to understand the role that astrocytes may play in the brain's response to injury. We are using both in vivo animal models and tissue culture to address these questions. Synaptic Mechanisms Section The section examines how neurotransmitters are released from presynaptic nerve terminals at fast-transmitting synapses. Much of this work is based on a preparation developed in the lab from the chick ciliary ganglion that allows a direct recording of ion currents in a vertebrate presynaptic nerve terminal. Studies on this nerve terminal have resulted in a number of 'firsts' including: the recording and characterization of a calcium current in a vertebrate presynaptic nerve terminal; the recording of single calcium channels at a transmitter release site; the direct correlation of single calcium channel activity with the quantal release of neurotransmitter; the structural localization of calcium channels at nm resolution on the transmitter release face; and, the direct demonstration and biophysical characterization of a presynaptic ligand-gated receptor. In addition, we have recently developed a formal model of transmitter release based on our experimental findings in the squid and the chick. Current work examines how proteins that are involved in the transmitter release process may modulate the behavior of presynaptic calcium channels. Molecular Physiology and Biophysics Unit This unit is to study the molecular structure and function of ion channel proteins. Ion channels are integral membrane proteins that are present in all cells of the human body and are important for a vast array of physiological processes. These include the generation and processing of electrical signals in the nervous system, regulation of heart contraction, secretion of hormones that regulate blood sugar, and control of water and salt balance in the kidney. The two channels that we focus on in particular are the voltage-gated potassium and calcium channels. We are interested in how these homologous ion channels accomplish various biophysical tasks. For example, how does a given channel select for specific ions or how is the opening and closing of the ion conduction pore coupled to changes in membrane voltage? To address these questions we combine biochemical, molecular biological, electrophysiological, and biophysical techniques to alter and study the behavior of the channel. A key element of our approach is to use various peptide toxins as molecular probes to study specific regions of the channel, such as the ion conduction pore or the voltage-sensing machinery. The laboratory also has a strong interest in understanding how the molecular design of voltage-gated potassium and calcium channels allows them to fulfill specific physiological roles. The structure/function studies help to elucidate the mechanisms underlying the unique functional behavior of a channel and also provide molecular probes that can be used pharmacologically to isolate and study specific channels in complex cellular environments. Synapse Formation and Function Unit This unit studies the functional properties of developing neural networks. There are several examples throughout the developing vertebrate nervous system, including the retina, spinal cord, hippocampus and neocortex, where immature neural circuits generate activity patterns that are distinct from the functioning adult circuitry. It has been proposed that these transitional circuits may provide the activity patterns necessary for normal development of adult neural systems. For example, immature retinal neurons spontaneously generate correlated activity in the form of waves of action potentials that sweep across the retinal ganglion cell layer, providing a signal for organization of downstream visual centers, and perhaps the retina itself. We use a combination of electrophysiology, imaging, anatomical and modeling techniques to study how the biophysical properties of synaptic transmission affect neuronal firing patterns throughout retinal development. Synaptic Function Unit The principal research goal of the Synaptic Function Unit is to understand the molecular mechanisms involved in the neurotransmitter release process and modulation. Our previous studies show that N- and P/Q-type calcium channels bind directly to the synaptic fusion-core complex. Peptides containing the synaptic protein interaction site (Synprint) cause dissociation of N-type calcium channels from the synaptic fusion-core complex and reversibly inhibit synaptic transmission when introduced into presynaptic superior cervical ganglion neurons. These studies provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses. Our present major ongoing project is cloning novel components of neurotransmitter release machinery using the yeast two hybrid cloning system combined with biochemical (in vitro) binding assays with purified or recombinant proteins, immunoprecipitation), molecular genetic (gene knockout), cell biological (co-transfection and immunocytochemistry), and electrophysiological (recording neurotransmission on cultured neurons) techniques. We have recently discovered a SNAP-25-binding protein, called Snapin. Snapin which is an important component in the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin.

National Institutes of Health