Gary L. Westbrook - US grants

The central role of excitatory amino acids as neurotransmitters, neuromodulators and as mediators of neuropathological processes is now widely accepted. Specifically, receptors and ion channels activated by L-glutamate and related endogenous excitatory amino acids may play a role in synaptic plasticity in the hippocampus and integration of both sensory and motor information in the spinal cord; current hypotheses of the pathogenesis of neuronal cell death in stroke, Huntington's disease, Alzheimer's, spinocerebellar degenerations and seizure- related brain damage also rely in part of 'excessive' activation of these same receptors. The broad objective of this project is to explore the cellular and molecular mechanisms by which excitatory amino acids exert their effects with a particular focus on the role of the N-methyl- D-aspartate (NMDA) receptor subtype in synaptic transmission. This unique agonist-gated channel has two features - voltage- dependence and calcium permeability - which make this channel an excellent candidate as a modulator of neuronal excitability. The general strategy will be to explore, using electrophysiological methods, the properties of L-glutamate activated ion channels which are likely to contribute to excitatory synaptic efficacy. In particular the contribution of second messenger activation, desensitization and agonist-gated transmembrane calcium influx on agonist-evoked responses and on single excitatory synapses will be examined. The relevance to synaptic transmission and drug action of three modulatory binding sites (Mg, Zn and glycine) on the NMDA receptor channel will also be examined. These three potent endogenous modulators have the capability to regulate ion flux through NMDA-receptor channels, and underscore the potential for pharmacological action at NMDA receptors. The experimental approach will use voltage and patch clamp methods on primary dissociated neurons in cultures prepared from rodent central nervous system, primarily hippocampus. Significant effort will be directed to preparations allowing study of identified cells types, and microisland cultures to allow study of single excitatory synapses.

The role of glutamate as a transmitter at central excitatory synapses is now well established. Although these pathways are of obvious importance for the normal transfer of information throughout the brain, it has come as somewhat of a surprise that signalling at glutamate synapses is much more complex than predicted from classical studies of the neuromuscular junction. This is nowhere more apparent than in the limbic system where such diverse phenomena as learning and memory, phencyclidine (PCP)- evoked psychosis and 'excitotoxic' brain injury have all been closely linked to the activity of glutamate receptors. Thus the activity of glutamate receptors is likely to contribute to the symptoms in schizophrenia, dementing illness such as Huntington's and Alzheimer's, and to brain damage caused by prolonged seizures or stroke. In some cases, abnormalities of glutamate receptors may be causal. Transmitter released from presynaptic terminals activates two classes of postsynaptic glutamate-activated channels, selectively activated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) and NMDA (N- methyl-D-aspartate). Despite intense scientific interest in glutamate receptors, their regulation remains poorly characterized. The purpose of this project is to examine two aspects of glutamate receptor regulation in hippocampal neurons. I. Increases in intracellular calcium can lead to slow 'rundown' of the NMDA channel, and high energy phosphates counteract rundown by a mechanism that does not appear to require direct receptor phosphorylation. This downregulatory mechanism may limit calcium influx into dendritic spines and thus modulate cellular responses to synaptic stimulation. Aims 1-3 will examine the action of intracellular calcium and ATP on NMDA receptor/channels. II. Phosphorylation of postsynaptic glutamate receptors is postulated to be an important regulator of synaptic transmission. However, the rapid action of phosphatases and phosphodiesterases suggest that kinases may need to be located near a membrane substrate such as a receptor in order to be effective. In Aim 4 the hypothesis that kinase localization by specific anchoring proteins is required for phosphorylation of AMPA receptors in the postsynaptic density will be tested. Peptide inhibitors of anchoring proteins for the regulatory subunit (RII) of cAMP-dependent protein kinase will be introduced into hippocampal neurons. Whole-cell patch clamp recording and measurements of intracellular calcium in single cultured hippocampal neurons will be used. The composition of the cell cytoplasm will be controlled using intracellular perfusion and flash photolysis of "caged" compounds. Single glutamate channels will be studied in the cell-attached and inside-out configuration. Molecular methods including expression of receptor subunits in cell lines will be used to probe the regulator sites on specific receptor subunits. The results of these studies are expected to lead to more effective therapeutic strategies for altering synaptic transmission in neuropsychiatric disorders.

The central role of excitatory amino acids as neurotransmitters, neuromodulators and as mediators of neuropathological processes is now widely accepted. Specifically, receptors and ion channels activated by L-glutamate and related endogenous excitatory amino acids may play a role in synaptic plasticity in the hippocampus and integration of both sensory and motor information in the spinal cord; current hypotheses of the pathogenesis of neuronal cell death in stroke, Huntington's disease, Alzheimer's, spinocerebellar degenerations and seizure- related brain damage also rely in part of 'excessive' activation of these same receptors. The broad objective of this project is to explore the cellular and molecular mechanisms by which excitatory amino acids exert their effects with a particular focus on the role of the N-methyl- D-aspartate (NMDA) receptor subtype in synaptic transmission. This unique agonist-gated channel has two features - voltage- dependence and calcium permeability - which make this channel an excellent candidate as a modulator of neuronal excitability. The general strategy will be to explore, using electrophysiological methods, the properties of L-glutamate activated ion channels which are likely to contribute to excitatory synaptic efficacy. In particular the contribution of second messenger activation, desensitization and agonist-gated transmembrane calcium influx on agonist-evoked responses and on single excitatory synapses will be examined. The relevance to synaptic transmission and drug action of three modulatory binding sites (Mg, Zn and glycine) on the NMDA receptor channel will also be examined. These three potent endogenous modulators have the capability to regulate ion flux through NMDA-receptor channels, and underscore the potential for pharmacological action at NMDA receptors. The experimental approach will use voltage and patch clamp methods on primary dissociated neurons in cultures prepared from rodent central nervous system, primarily hippocampus. Significant effort will be directed to preparations allowing study of identified cells types, and microisland cultures to allow study of single excitatory synapses.

The action of glutamate-activated ion channels determines the flow of information via excitatory synapses throughout the mammalian brain. As a result, the normal function and regulation of glutamate channels (of the AMPA, kainate and NMDA subtypes) are involved in virtually all brain functions. In the past 10-15 years, fundamental studies of N- methyl-D-aspartate (NMDA) receptors provide one of the clearest rationales for the relevance of basic research to clinical problems. These studies have provided new insights into normal brain functions such as synaptic plasticity, the formation of memories, and the action of psychomimetic drugs such as phencyclidine (PCP) on human behavior. Excessive stimulation of glutamate receptors can cause neuronal cell death in seizures and stroke, and may play an important role other neuropsychiatric illnesses. An amazing complexity of regulatory mechanisms influence glutamate receptors. For example, NMDA receptors are regulated by allosteric mechanisms, multiple kinases, phosphatases and soluble second messengers. Although such complexity may seem fitting given the central role of excitatory synapses, the question of what determines the specificity of such interactions is unexplored. Calcium influx into neurons through open NMDA channels at synapses initiates several of these regulatory mechanisms, thus we have focused on the regulation of hippocampal NMDA receptors by intracellular calcium. Our results suggest that compartmentalization and local interactions between glutamate receptors, regulatory proteins and cytoskeletal elements in the postsynaptic density (PSD) are keys to this puzzle. These interactions are likely to affect the activity of synaptic NMDA channels as well as the formation and receptor composition of hippocampal synapses. We will test two aspects of this general hypothesis in this proposal. First, we will examine the domains of the NMDA receptor responsible for calcium regulation (Aim 1-2) and desensitization (Aim 3). Preliminary results demonstrate that calcium regulation is NR2A specific and chimeric/deletion constructs suggest regions of NR1 and NR2A that are involved, perhaps by a ball-and-chain mechanism. We will also examine the possible inductive role of NMDA receptors, and the NR2B subunit in particular, in the function and localization of individual synapses on hippocampal neurons (Aim 4). These studies will make use of transgenic mice lacking the NR2B subunit. The proposed studies will use recombinant NMDA receptors expressed in 293 cells and Xenopus oocytes as well as native receptors in cultured hippocampal neurons. Novel methods we developed for studies of synaptic NMDA receptors and function of individual synaptic sites will be used.

DESCRIPTION (provided by applicant): The past 20 years has seen a remarkable increase in our knowledge of cellular and molecular properties of the nervous system, but how these properties are combined into functional circuits remains a daunting challenge. Small synaptic circuits represent the functional modules that underlie information processing in the brain. Disruption of circuits is central to diseases ranging from mental retardation, autism and schizophrenia to traumatic brain injury, epilepsy and Alzheimer s disease. These modules share many common features in different brain systems, yet our knowledge of their functional organization at the cellular level remains rudimentary. Sensory systems provide an attractive experimental system for such questions because their modular organization is amenable to direct study, e.g. barrels in the somatosensory cortex. In this project, we will use the olfactory bulb as a model to address how circuits process information. The olfactory system is attractive for this purpose because of the elegant spatial mapping of sensory input and its relatively simple circuitry. While theories of olfactory function abound, based largely on macroscopic recording or molecular maps, information is lacking at the cellular level to match functions, e.g. signal amplification or discrimination, with elements in the chain from receptor neuron to olfactory bulb to olfactory cortex. This is a problem of synaptic integration, involving the strength and duration of individual synaptic responses;the connectivity of synapses within the network;and the excitable properties of neurons. We hypothesize that fast and slow synchronization of cellular activity in the glomerular layer are required for high fidelity coding of sensory information while interneuronal networks within the bulb (periglomerular and granule cells) play a role in discrimination, but not in the traditional sense of lateral inhibition. The principal cells, mitral cells, receive input from only one glomerulus, thus comparison of mitral cells within the same or different glomeruli provide a powerful in vitro approach to circuit analysis. We will use paired recording from cells in rodent brain slices, guided by use of cell-specific genetically-labeled mice, to manipulate assess the cellular properties and organization modules that determine information processing in this circuit. Our preliminary data provide strong clues to the role of rapid signaling by gap junctions as well as slow synaptic signaling mechanisms. These data also provide hypotheses about behavior that we will directly test. These studies are expected to provide important insights into synaptic integration in the olfactory bulb as well as for other brain circuits.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] In response to RFA-MH-05-011 (Course Development in the Neurobiology of Disease), we propose the development of an integrated Course in the Neurobiology of Disease at Oregon Health and Science [unreadable] University with the follow general goals: [unreadable] 1. To provide a foundation in the underlying mechanisms of neurological and psychiatric disease. Our premise is that a Theme-oriented approach to fundamental cellular, molecular as well as organismal mechanisms, rather than a disease-specific approach, is best able to engage students who are interested in basic aspects of brain function. We expect that we may not only recruit students into disease-oriented research, but we will make them "ready observers" so that possible disease-related issues in their own research are more quickly noted. [unreadable] 2. To provide a Toolbox of topical methods and issues in the neurobiology of disease. These toolbox sessions will also probe the links between basic mechanisms and behavioral manifestations of disease. 3. To provide a sampling of neuropsychiatric disorders that serve as training examples for the themes discussed in Objective 1. These examples will be based on the expertise of the faculty at OHSU, but will change with each course presentation so that the course remains vibrant and relevant to students, as well as other trainees and faculty [unreadable] 4. To provide hands-on exposure to clinical situations through live patient presentations, multimedia presentations, and visits to clinics, hospital wards, and other clinical settings. These Clinical Demonstrations will stress hands-on interactive experience so that graduate students experience first-hand the impact of neurological and psychiatric disease on brain function, and on the social fabric of the patient's life, their families and their community. [unreadable] Three NIH training grants qualify OHSU for this RFA: T32NS007466 (Gary Banker, PI; T32AA007468 [unreadable] (Christopher Cunningham, PI), and T32AT002688 (Barry Oken, PI). Three other training grants are currently funded and would qualify except that they will be subject to competitive renewal before September 2006. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Synaptic plasticity is widely regarded as the basis for learning, memory, and our ability to adapt to our surroundings. At the level of single neurons or at the level of human behavior, it is clear that plasticity is more robust when we are young. However there is untapped potential for plasticity, repair and regeneration in the adult mammalian brain, as evidenced by the birth of new neurons in several brain regions. These issues are central to the understanding and potential treatment of neurodevelopmental disorders, autism and mental retardation, as well as conditions that result in neural loss or degeneration such as stroke, epilepsy, brain and spinal cord trauma, Parkinson's disease, and Alzheimer's disease. The long-term goal of this project is to understand the mechanisms of circuit formation at the level of single synapses in the hippocampus. Much experimental effort has been directed at the very early period of neurogenesis, whereas much less is known about the integration of adult-generated neurons into functional networks in the adult brain. As for neural development, understanding the functional integration of new neurons in the adult is a daunting task because of the presumed contribution of hundreds of molecules in a spatially and temporally precise sequence. How does one approach this immense problem in the intact animal, yet gain access to single cells and synapses? This project makes use of a novel transgenic mouse to track the development of dendrites and synapses as new neurons leave their niche, and integrate into the adult circuitry of the dentate gyrus. Preliminary data suggests that this process occurs in distinct stages with a period of limited dendrite outgrowth and exclusively GABAergic synapses, followed weeks later by additional dendritic growth and new excitatory synapses. The project will use electrophysiology and cell imaging to chart the inputs and outputs as new neurons integrate into the adult network, both in normal conditions and following perturbations such as exercise and epileptic seizures. The role of molecules that regulate this stage-specific development will be tested using selective marking with specific promoters in transgenic mice, and viral-mediated gene manipulation in vivo. Assays will use biochemical and molecular methods as well as brain slice physiology. The ability to track these distinct stages will also be used to test the role of cell adhesion molecules in synapse maturation.Project Narrative [unreadable] Many neuropsychiatric illnesses cause loss of nerve cells and/or disruption of connections between nerve cells (synapses). This project takes advantage of a unique mouse model to examine the integration of adult- generated (new) neurons into synaptic networks in the hippocampus, thus providing access at the single nerve cell level to mechanisms of synapse formation, repair and regeneration in the brain. These issues are central to the understanding and potential treatment of neurodevelopmental disorders, autism, mental retardation and mood disorders, as well as conditions that result in neural loss or degeneration such as stroke, epilepsy, brain and spinal cord trauma, Parkinson's disease, and Alzheimer's disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal requests R13 support for a longstanding, well-attended, and well-received Gordon Research Conference (GRC) on Excitatory Synapses and Brain Function. Perhaps no other structure is more fundamental to our understanding of the brain than the synapse. In the central nervous system, excitatory synapses represent the primary source of information transfer between regions as well as for local interactions within circuits. Synapses also serve as the site of action for many commonly prescribed medications and their disruption contributes to many neurological and psychiatric disorders. These include schizophrenia, autism, depression, substance abuse and addiction, Parkinson's Disease, Alzheimer's disease, traumatic brain injury, stroke and epilepsy. In some cases, synaptic dysfunction is causal in disease, whereas in other cases it represents the downstream sequelae of one or more underlying molecular defects. In either case, a fundamental understanding of the formation, structure, molecular organization, signaling function, and plasticity of synapses is essential to progress in lessening the burden of human neurological disease and for predicting and improving mental health. This conference is unique in its focus on the excitatory synapse, and in its multidisciplinary group of participants including structural biologists, molecular and developmental biologists, cell biologists, biochemists, cell/molecular imagers, biophysicists and physiologists. The conference is intended to relate fundamental insights in excitatory synaptic function to the impairments in synaptic function that occur in disease, as well as the maladaptive plasticity that occurs in substance abuse. The goal of the conference is not to survey abnormalities in each disease, but rather to probe new fundamental insights into synaptic function and dysfunction from a thematic approach. The program has been designed to highlight cutting edge approaches and to stimulate new concepts, methods and technologies within a sound biological framework of fundamental neuroscience. The conference will bring together expert scientists worldwide in an environment that is conducive to discussion and exchange of ideas. The exchange of ideas at this conference has been a driving force for the field. We expect the 2009 GRC on Excitatory Synapses and Brain Function will shape future scientific directions, and provide critical support for the mission of multiple institutes at NIH including NIMH, NINDS, NIDA and NIA. The synapse is the fundamental unit of information processing in our brain. Synaptic dysfunction is responsible for many neurological and psychiatric diseases. This conference brings together experts on excitatory synapses to update progress and stimulate new approaches to improve mental health and reduce the burden of neurological disease.

DESCRIPTION (provided by applicant): We request continued support for the Multidisciplinary Training Program in Neuroscience at Oregon Health & Science University (OHSU). The program, which is about to enter its fifteenth year, offers broad-based, early stage training for students in OHSU's two PhD-granting neuroscience programs, the Neuroscience Graduate Program and the Behavioral Neuroscience Graduate Program. Together these programs enroll about 70 students. The program of training we propose is based on a strong core curriculum that provides a common knowledge base for subsequent training in the classroom and at the bench, together with extensive and varied opportunities for research, beginning as soon as students enter the program. Our training faculty of 75 scientists offer research opportunities that range from fundamental studies of nerve cell function to translational research programs that target nervous system disorders. Through workshops and individual instruction, the program also provides instruction in scientific communication skills and career development. The spirit of collegiality that has developed at OHSU combined with the diversity of its many research institutes and the close proximity of basic and clinical research facilities provide a unique opportunity for predoctoral students to form cross-disciplinary collaborations during their trainin and to gain an appreciation for the growing importance of neuroscience research in the treatment of neural disease.

Summary/Abstract This project would continue support for the Multidisciplinary Training Program in Neuroscience at the Oregon Health and Science University (OHSU) in Portland. The program, about to enter its 20th year, provides broad, early stage training for graduate students entering the Neuroscience Graduate Program (NGP). The program is based at the Vollum Institute but includes faculty and students in many centers, department and institutes on the OHSU campus. The program includes 55 students (ca. 10 per year). Our training faculty of 55 scientists offers thesis research opportunities that include all levels of modern neuroscience research from state-of-the-art cryoEM studies of membrane proteins to systems neuroscience to disease-oriented and translational neuroscience. The program has undergone a number of innovations and changes since the last renewal as fully outlined in the proposal, highlighted by the recruitment of new leaders and new faculty in the neuroscience community at OHSU. The new faculty members bring new areas of research strength that supplement the ongoing research strengths in the NGP. The innovations include a unique core curriculum structure that begins with a week-long Boot Camp of followed by a 12-week intensive course that provides a broad foundation in neuroscience for all students in the program. The curriculum format, now in its 3rd year, allows first year student to engage in fulltime laboratory rotations within 4 months of entry into graduate school. The core curriculum is supplemented by workshops and individual instruction in specific techniques as well as professional skills, ethics and career planning. Additional emphasis is placed on fostering skills in experimental design, programming, and quantitative approaches to address NIH mandates in rigor, reproducibility and transparency. The spirit of at OHSU among the large neuroscience community, numbering approximately 150 affiliated scientists, combined with the diversity of its many research institutes, and the close proximity of basic and clinical research facilities provide a unique opportunity for early stage pre-doctoral students to establish and benefit from cross-disciplinary collaborations. This foundation will foster the skills necessary for graduates to be successful in a variety of science-related careers needed to fully understand and treat complex neuropsychiatric diseases.

Summary/Abstract This project would continue support for the Multidisciplinary Training Program in Neuroscience at the Oregon Health and Science University (OHSU) in Portland. The program, about to enter its 20th year, provides broad, early stage training for graduate students entering the Neuroscience Graduate Program (NGP). The program is based at the Vollum Institute but includes faculty and students in many centers, department and institutes on the OHSU campus. The program includes 55 students (ca. 10 per year). Our training faculty of 55 scientists offers thesis research opportunities that include all levels of modern neuroscience research from state-of-the-art cryoEM studies of membrane proteins to systems neuroscience to disease-oriented and translational neuroscience. The program has undergone a number of innovations and changes since the last renewal as fully outlined in the proposal, highlighted by the recruitment of new leaders and new faculty in the neuroscience community at OHSU. The new faculty members bring new areas of research strength that supplement the ongoing research strengths in the NGP. The innovations include a unique core curriculum structure that begins with a week-long Boot Camp of followed by a 12-week intensive course that provides a broad foundation in neuroscience for all students in the program. The curriculum format, now in its 3rd year, allows first year student to engage in fulltime laboratory rotations within 4 months of entry into graduate school. The core curriculum is supplemented by workshops and individual instruction in specific techniques as well as professional skills, ethics and career planning. Additional emphasis is placed on fostering skills in experimental design, programming, and quantitative approaches to address NIH mandates in rigor, reproducibility and transparency. The spirit of at OHSU among the large neuroscience community, numbering approximately 150 affiliated scientists, combined with the diversity of its many research institutes, and the close proximity of basic and clinical research facilities provide a unique opportunity for early stage pre-doctoral students to establish and benefit from cross-disciplinary collaborations. This foundation will foster the skills necessary for graduates to be successful in a variety of science-related careers needed to fully understand and treat complex neuropsychiatric diseases. 7

Neurons have the remarkable ability to process and respond to complex stimuli such as physical exercise and changes in an organism?s external environment. The value of exercise for brain health cannot be underestimated as its effects impact mood, learning and memory as well as prevention and rehabilitation and recovery from neurological illness. However, experimental effort largely has been focused on the effects of sustained exercise over periods of weeks or months (1-3), which can involve direct effects on the CNS as well as indirect effects through alterations in multiple organ systems. Likewise, most attention in exercise-induced hippocampal plasticity has been directed at newborn granule cells (1, 3-7), but plasticity occurs in the far more numerous mature granule cells as well (8). Aside from its sustained benefits, acute exercise has also been linked to short term increases in learning and memory (9, 10) that are likely mediated by the hippocampus (11-13). How this occurs at the molecular level is not clear. Thus we decided to examine how a single episode of exercise affects neural activity and impacts brain function. We developed a novel approach for in vivo analysis of dentate granule cells activated by a single episode of voluntary exercise. Our approach, akin to an impulse function in engineering terms, allowed us to examine exercise-induced synaptic and molecular changes over a period of days post-exercise. Mature dentate granule cells, activated by voluntary exercise during a two-hour window, were permanently marked using Fos-TRAP mice (14, 15), in which the immediate early gene promoter linked to a fluorescent reporter, permanently marks activated granule cells. The single episode of exercise resulted in selective increases in synaptic function and dendritic spine density in the outer molecular layer of the dentate gyrus, the lamina receiving contextual information from entorhinal cortex. The top upregulated gene in RNAseq of exercised-activated cells was Mtss1L, a previously understudied gene coding for an I-BAR-domain protein. As BAR domains sense and induce membrane curvature, we hypothesize that Mtss1L is an early effector of dendritic spine and synapse formation following stimuli such as exercise. Our preliminary data lead to a number of interesting questions that will be addressed in this proposal. Namely: 1. Where is Mtss1L localized and why are the effects on synapses limited to a specific lamina in the dentate gyrus?; What are the effects of other I-BAR family members as several are expressed at synapses but only Mtss1L is activity-dependent?; and 3. Do exercise-induced synaptic changes prime specific synapses for learning and memory by salient stimuli? Our approach provides the cellular- and temporal-specificity to link physiologically- and clinically-relevant stimuli in vivo (exercise) to individual synapses and expression of specific genes contributing to structural plasticity in the hippocampus.