Michael Zigmond - US grants

Evidence from clinical observations and experimental investigations indicates that few enduring functional impairments are produced by subtotal damage to monoaminergic neurons, despite considerable evidence for their involvement in a wide variety of physiological and behavioral processes. We have proposed that this apparent paradox results from the capacity for "synaptic homeostasis" that exists in these systems and that leads to compensatory events occurring after partial injury. We propose to use a multidisciplinary approach to describe at the cellular level the initial deficits, recovery of function and residual deficits produced by subtotal destruction of such a monoaminergic system and then to explore the biological bases of those phenomena. Our work will focus on the dopaminergic projection of the nigrostriatal bundle. Where appropriate, other monoaminergic systems will be used for these studies, including the sympathoadrenal system and the noradrenergic locus coeruleus-hippocampus projection. First, we will examine the impact of subtotal injury on monoaminergic function as determined by biochemical, physiological, electrophysilogical, and behavioral measurements. Second, we will attempt to determine the biological bases of the sparing of function that occurs after moderate lesions and the recovery of function that occurs after larger lesions. Third, we will test the ability of certain treatments to extend recovery of striatal function. Fourth, we will examine several other states which may be related to lesion induced-hypoinnervation, including development and chronic neuroleptic treatment. Finally, we will examine the influence of the age at which brain injury is sustained on the behavioral and biological consequences. These results should provide insights into the neurobiology of monoaminergic systems under normal conditions and after damage, as well as provide information of relevance to the detection and treatment of subclinical brain damage in disorders involving abnormalities of monoamine-containing systems.

Evidence from clinical observations as well as from experimental investigations indicates that subtotal damage to monoaminergic neurons produces few enduring functional impairments despite considerable evidence for their involvement in a wide variety of physiological and behavioral processes. We have proposed that this apparent paradox results from the capacity for "synaptic homeostasis" which exists in these systems and which leads to compensatory events occurring after partial injury. Our research will focus on describing at the cellular level the initial deficits, recovery of function and residual deficits produced by subtotal destruction of the dopaminergic component of the nigrostriatal bundle and to explore the neurochemical bases of those phenomena. This work will utilize an in vitro model to address four issues. (1) The impact of the injury on dopaminergic function: Striatal slices exposed to field stimulation will be used to examine the influence of dopamine (DA) and of partial injury produced by 6-hydroxydopamine on acetylcholine (ACh), GABA, and cAMP efflux. Studies will be carried out as a function of post-operative time and lesion size. (2) Lesion-induced changes in the synthesis, release, and reuptake of DA: We will measure DA efflux from stimulated slices and determine the relative contribution of altered release and reuptake. We also will begin to examine the genetic regulation of tyrosine hydroxylase synthesis. (3) Lesion-induced changes in responsiveness of striatal targets to DA: We will focus on D-2 receptor mediated events, examining changes in agonist-induced ACh efflux and in D-2 binding sites. We also hope to begin an analysis of the importance of changes in signal transduction distal to the binding site itself. (4) The ability of treatments to extend recovery of striatal function: Possible modes of "therapy" to be examined include DOPA, tyrosine, pterine cofactor supplementation as well as in vivo exposure to stressors such as insulin-induced glucoprivation. These results should provide insights into the neurobiology of moneaminergic systems under normal conditions and after damage, as well as information of relevance to the detection and treatment of subclinical brain damage in those psychiatric and neurological disorders involving abnormalities of monoamine-containing systems.

Evidence from clinical observations as well as from experimental investigations indicates that subtotal damage to monoaminergic neurons produces few enduring functional impairments despite considerable evidence for their involvement in a wide variety of physiological and behavioral processes. We have proposed that this apparent paradox results from the capacity for "synaptic homeostasis" which exists in these systems and which leads to compensatory events occurring after partial injury. Our research will focus on describing at the cellular level the initial deficits, recovery of function and residual deficits produced by subtotal destruction of the dopaminergic component of the nigrostriatal bundle and to explore the neurochemical bases of those phenomena. This work will utilize an in vitro model to address four issues. (1) The impact of the injury on dopaminergic function: Striatal slices exposed to field stimulation will be used to examine the influence of dopamine (DA) and of partial injury produced by 6-hydroxydopamine on acetylcholine (ACh), GABA, and cAMP efflux. Studies will be carried out as a function of post-operative time and lesion size. (2) Lesion-induced changes in the synthesis, release, and reuptake of DA: We will measure DA efflux from stimulated slices and determine the relative contribution of altered release and reuptake. We also will begin to examine the genetic regulation of tyrosine hydroxylase synthesis. (3) Lesion-induced changes in responsiveness of striatal targets to DA: We will focus on D-2 receptor mediated events, examining changes in agonist-induced ACh efflux and in D-2 binding sites. We also hope to begin an analysis of the importance of changes in signal transduction distal to the binding site itself. (4) The ability of treatments to extend recovery of striatal function: Possible modes of "therapy" to be examined include DOPA, tyrosine, pterine cofactor supplementation as well as in vivo exposure to stressors such as insulin-induced glucoprivation. These results should provide insights into the neurobiology of moneaminergic systems under normal conditions and after damage, as well as information of relevance to the detection and treatment of subclinical brain damage in those psychiatric and neurological disorders involving abnormalities of monoamine-containing systems.

We propose to examine the impact of acute and chronic stress on the functional capacity of central noradrenergic neurons. Our studies may be divided into three groups:. (1) We wish to determine whether changes in tyrosine hydroxylase (TH) activity within locus coeruleus (LC) neurons during chronic cold stress are indicative of alterations in norepinephrine (NE) efflux. Release will be monitored using in vitro slices, in vivo voltammetry, and local perfusion in situ via dialysis tubing. The LC and two of its terminal fields will be examined, cerebellum and hippocampus will be examined. Among the questions to be studied are the following: Does acute, severe stress lead to an initial depletion of releasable NE? Does the activation of TH, which occurs within a few minutes of stimulation, increase the capacity for subsequent release? Does TH induction lead to a still greater capacity for release? (2) We wish to determine the extent to which increases in NE release during stress are due to alterations in the firing rate of LC neurons. We will record from LC neurons and determine the impact of stress on the firing rate of these cells. In addition, we will measure the amount of transmitter released from slices per impulse. (3) We wish to determine the degree to which adaptive changes occurring in central NE neurons in response to chronic stress affect the functioning of post-synaptic cells. We will record from single hippocampal granule cells and cerebellar Purkinje neurons during electrical stimulation of the LC to determine if exposure to chronic stress alters the inhibitory effect of release of endogenous NE on the spontaneous firing rates of these target neurons. In addition, electrical stimulation of glutaminergic, perforant path input to granule cells during electrical stimulation of the LC will be used to determine if exposure to chronic stress alters the modulatory effect of NE on other, non-NE inputs to target neurons. This work will be related to the basic neurobiology of monoaminergic neurons and their targets and to the relation between stress and depression.

This Program Project represents an integrated exploration of key aspects of the neurobiology of the basal ganglia, both in intact animals and in animals sustaining lesions of the dopamine (DA)- containing nigrostriatal projections. In many animals, the neurotoxin 6-hydroxydopamine will be administered intracerebrally to adult rats to produce an animal models of Parkinsonism. We have identified seven interrelated themes, each of which appear in most if not all of the projects; (1) nonsynaptic interactions between neuronal elements in striatum; (2) direct versus indirect output pathways of dorsal striatum (3) the relation between D1 and D2 receptors on medium spiny neurons of straitum; (4) DA-interactions with other neurochemically defined neuronal elements; (5) pathology and pathogenesis related to Parkinsonism; (6) compensations that occur in response to partial lesions of monoaminergic pathways; and (7) the impact of L-DOPA on neural functioning and on behavior. As part of this endeavor, a multi-disciplinary characterization of many aspects of the normal basal ganglia will be required. Include in this characterization will be an anatomical and neurochemical examination of the interactions among dopaminergic, cholinergic, glutamatergic, and GABAergic neurons. An in-depth study of the catecholamine-synthesizing enzyme, tyrosine hydroxylase also will be carried out. Among the clinically-related issues to be examined are (1) the neurobiological basis of the pre-clinical and the clinical phase of Parkinsonism and related disorders, (2) mechanisms of action of L-DOPA in Parkinsonism, and (3) the underlying basis of the selective vulnerability of nigrostriatal DA neurons. It is expected that these studies will provide insights into the relation between neuropathology and symptomatology in a variety of neurodegerative disorders will be of value in developing new modes of therapy for these conditions, and will add important new information concerning the neurobiology of the basal ganglia.

DESCRIPTION (Adapted from applicant's abstract): Short-term exposure to stressful, aversive stimuli elicits a variety of adaptive physiological and behavioral responses, and chronic exposure to stress can evoke useful, adaptive responses as well. However, chronic exposure to stress also has been implicated in the development of many pathological conditions, including psychiatric disorders, and schizophrenia. In order to elucidate the central component of stress-induced abnormalities, the application will examine the impact of stress on central noradrenergic transmission, particularly the NE- containing neurons of the LC. Recently the investigators observed that exposure to chronic cold increased by 2-fold the ability of a subsequent acute stressor to release NE, a phenomenon that has been termed "sensitization" when observed in other systems. However, little is known about the impact of chronic stress (i.e., as a result of its quality, intensity, and duration) or whether a given stress-induced change may be adaptive or maladaptive. Hence, the project proposes to systematically explore the relation between stress and its effects on LC neurons as well as the functional significance of those effects. In addition, it is planned to study the basic neurobiology of LC neurons, in particular, the nature of the heterosynaptic input to LC terminals and the rate of TH transport in these neurons. Four specific aims are proposed: First, the impact of chronic stress on the synthesis and release of NE in response to a subsequent acute stress will be further characterized. Second, the mechanisms underlying these interactions will be examined by focusing on changes in NE synthesis, release, and inactivation, and by examining both autoreceptor and heteroreceptor input. Third, the application plans to explore the synthesis and transport of TH, the rate-limiting enzyme in NE synthesis. Finally, changes in the electrophysiology of LC neurons during acute and chronic stress will be investigated. The longer-term objectives of this project are to provide new insights into the basic neurobiology of LC neurons and the psychopathology that develops from chronic stress.

Success in science requires a solid background in a scientific discipline in addition of extensive laboratory experience. However, for scientists to develop into accomplished professionals, a wide range of "survival skills" also are needed. These skills can be divided into three categories: communications (e.g. the ability to communicate both orally and in writing), economic issues (e.g. securing employment), and personal development (e.g. managing time and stress). While higher education focuses almost exclusively on the content of the scientific discipline and on research methodology, little attention is paid to those skills that can make the difference between technical and creative competence and success. This award is to enable (1) development of a comprehensive series of workshops on survival skills; (2) production of materials and relevant literature to assist instructors in higher education to teach their students these skills; and (3) support of workshops to disseminate this information to faculty members from a wide range of institutions so as to enable other educational programs to initiate similar courses. Evaluation will be carried out for the materials and workshops to assess the success of these activities. Special emphasis is being placed to improve access for under-represented groups. Providing a solid foundation in career skills for women and ethnic minorities may play a pivotal role for ensuring their successful entry into the scientific profession. An important benefit of increasing the diversity of scientists while maintaining their quality is the incorporation of a diversity of approaches to scientific problems which can only serve to improve science.

[unreadable] DESCRIPTION (provided by applicant): We request continuing support for postdoctoral training in basic neuroscience related to psychiatric disorders. Our program is closely integrated with several key research programs related to cognition and schizophrenia, developmental disorders, mood and depression, and the dementia associated with Alzheimer's disease, and AIDS. Additional programs in neurodegenerative disease, stroke, and basic neuroscience further enhance the environment. In our program, the major research advisor, an individualized advisory committee, and the training grant steering committee share the task of guiding and monitoring each trainee. The training program (1) focuses on mentored research, but includes (2) a seminar on the neurobiology of psychiatric and neurological disorders, (3) a monthly research discussion, (4) an annual trainee retreat, and (5) a professional development program. In addition, (6) discussion of issues of the responsible conduct of research occurs throughout the program. Moreover, (7) trainees also participate in courses as needed and have opportunities to (8) teach and (9) observe clinical practice. The 31 members of the training faculty are members of the university-wide program in neuroscience with a particular interest in the neurobiology of psychiatric disorders and affiliated with several academic departments and programs. All 20 full members of the faculty have active, funded research programs and training experience research. In addition, we have included 8 more junior faculty with outstanding promise who will be available to serve as co-mentors in conjunction with a more senior faculty member. We are requesting stipends to support an initial complement of 4 postdoctoral trainees, increasing to 6 after 2 years. These stipends will be used to support trainees for up to two years, although all trainees will be required to apply for their own fellowships within 6 mo of beginning our program and this should make available funds for other individuals. We believe that our program will increase the capacity for translational research of relevance to the NIMH mission. [unreadable] [unreadable] [unreadable] [unreadable]

Beside a solid background in their science, scientists need a wide range of `survival skills` to develop into accomplished professionals. They need to communicate effectively orally and in writing, secure employment and funding, manage stress and time, teach, mentor, and behave responsibly. Higher education focuses almost exclusively on disciplinary content and methods. The principal investigators of this proposal have developed a `survival skills curriculum.` This program includes (1) using active scientists rather than individuals from other disciplines as instructors, (2) discussing ethical issues within the context of the skills to which they are relevant, (3) integrating topics of special relevance to women, minorities, the physically challenged and others with special needs, (4) providing information about a broad range of employment opportunities and emphasizing their value, and (5) extensive evaluation. This funding will be used further to develop this model and disseminate it to other institutions via faculty training in this type of instruction. This grant will allow a substantial number of faculty from US institutions to receive the training and materials to establish their own courses, and a substantial number of graduate students and postdoctoral fellows to attend the workshops on survival skills and ethics.

This revised application requests funds to initiate a new predoctoral and postdoctoral training program in basic neuroscience as it relates to neurodegenerative disease. The program will build on existing training programs, an NINDS-funded Program Project on basal ganglia and parkinsonism headed by the program Director of this application. These training and research programs have already helped to develop and integrate diverse elements of the basic neuroscience community at the University of Pittsburgh and to strengthen its ties to clinical neuroscience. If funded, the addition of pre- and postdoctoral positions provided by NINDS would provide the critical mass needed to recruit the best possible pre- and postdoctoral students (including underrepresented minority students) and to offer a training program of the highest quality. Our proposed program focusses primarily on research training. However, predoctoral students will take a series of core courses in neuroscience and other areas of basic biological science, a course in clinical neuroscience, and advanced seminars, and pass through a series of "milestones," including a comprehensive exam. Postdoctoral trainees will attend courses and seminars as needed. In addition, both groups will participate actively in a series of professional development workshops. These workshops provide explicit training in such "survival skills" as written and oral communication, obtaining jobs and grants, teaching and managing a research lab. Training in responsible conduct is an integral part of both the survival skill workshops and the core curriculum itself. Students are encouraged to consider a wide range of employment opportunities within which to exercise their skills in research and seminars are held to permit them to become familiar with employment both within and outside of traditional academic research universities. Members of the training faculty come primarily from the Department of Neuroscience. Other participating departments and centers include the Departments of Neurobiology, Neurology, Pathology, and Pharmacology, Psychiatry, and the Center for Neuroscience. Two groups of faculty are described: a core group of full faculty , consisting of individuals with extensive experience in training and in research, and a small group of "pro- faculty" who have outstanding promise and will be available to serve as co-mentors in conjunction with a more senior faculty member. Data are provided to support our belief that we will be able to obtain outstanding trainees for the proposed program.

This project will focus on the adaptive properties of catecholamine neurons as they relate to neuronal injury. Our principal hypothesis is that dopamine (DA) exerts both a synaptic and a non-synaptic influence on target cells in the dorsal striatum and that after DA-depleting lesions non-synaptic communication and gross motor behavior is spared until DA loss is extensive, whereas synaptic communication and more subtle motoric capabilities are disrupted in rough proportion to the injury. In the proposed experiments we will continue our examination of an animal model in which partial injury of the DA-containing projections of the nigrostriatal pathway is produced in rats by the intracerebral injection of 6-hydroxydopamine (6-OHDA). Four sets of experiments are proposed: First, we will determine the extent to which a dopaminergic influence is preserved after the partial loss of the DA input to dorsal striatum as a function of lesion size, post-operative time, and the availability of L-DOPA. Neurobiological measures of the impact of DA on its targets in intact and lesioned animals will include: acetylcholine (Ach) release (a D2-mediated response), cAMP production (primarily and D1-response), and GABA release (a complex interaction involving both D1 and D2 sites). Second, we will carry out parallel studies using behavioral endpoints, including food and water intake, motor activity, operant reaction time, and a neurological test battery. In the third experimental series we will explore the basis for the discrepancies between terminal loss and functional deficits, focussing on lesion-induced changes in DA synthesis, DA autoreceptor sensitivity, and changes glutamatergic input. Finally, we will examine the changes related to tyrosine hydroxylase (TH) gene expression after partial injury of DA neurons, comparing our results from those obtained in response to damage to noradrenergic neurons. Alterations in the amount of TH supplied to the nerve terminal will be examined, as will changes in the stability of TH within the terminal. The rate of TH synthesis also will be measured, focusing on rate of TH gene transcription and translation. We believe that the findings that will emerge from this research will have implications for understanding the cause and treatment of Parkinsonism, and will also provide important information regarding the neurobiology of dopaminergic neurons and their interactions with neurons in the basal ganglia.

This Core provides neurochemical analyses for members of the Program Project. In particular it analyzes the dopamine (DA) content of tissue taken from 6-OHDA-treated animals as a means of quantifying the impact of the lesion. Other assays available include that for serotonin, tyrosine hydroxylase activity, and protein. Additional analytical procedures will be developed as necessary.

As summarized in the Center Overview, schizophrenia appears to be associated with a reduction in the dopamine (DA) innervation of prefrontal cortex (PFC). However, the functional impact of such a deficit especially on other elements of PFC circuitry that are though to be dysfunctional schizophrenia, has not been well characterized. The proposed experiments will use a rat model and in vivo neurochemical methods to examine four questions relevant to this issue: (1) What is the impact of loss of DA axons in PFC on extracellular DA in this region? (2) What is the impact of this loss on the function of gamma-aminobutyric acid (GABA) interneurons in PFC? (3) Does loss of DA in PFC alter the ability of PFC to regulate the activity of one of its subcortical targets, the mesoaccumbens DA projection? (4) Does loss of DA axons in PFC sustained early in development produce changes in PFC function that are different from those seen in rats lesioned as adults? The neurochemical experiments described in the present project complement the studies of Projects Grace and Barrionuevo, which explore factors regulating the electrophysiological activity of PFC in rat and monkey. In addition, results of the proposed experiments will generate information about neurochemical interactions in the PFC of rat that will be used to guide the interpretation of data collected in behavioral and imaging studies in schizophrenic subjects (Projects-Cohen and Project-Sweeney), as well as to generate hypotheses regarding the neurochemical deficits of schizophrenia that may then be tested in functional imaging studies (Projects-Cohen and Sweeney) or be analysis of postmortem tissue (Project-Lewis).

INT 9904207
Zigmond

This U.S.-Hungary project between Michael Zigmond of the University of Pittsburgh and Laszlo Harsing of the Institute for Drug Research, Budapest, will examine neurotransmitter interactions in the neostriatum of the brain. The researchers intend to conduct in vivo and in vitro research which investigates neocortical glutamate inputs and outputs to midbrain dopamine neurons from GABAergic interneurons in the neostriatum. The researchers hypothesize that these interactions are reciprocal and occur with and without involvement of GABAergic projection neurons and interneurons. Their collaborative effort builds upon established Pittsburgh expertise in neurotransmitter measurement and release, a Budapest-developed special superfusion chamber, and Hungarian experience with a unique method for stimulating a single afferent to the striatum in vitro. Findings are expected to improve our basic understanding of the neurochemistry of the striatum and specifically, the transmitter interactions within the basal ganglia, an essential area in the control of sensory and motor information.

This project in neuroendocrinology fulfills the program objective of advancing scientific knowledge by enabling experts in the United States and Central Europe to combine complementary talents and share research resources in areas of strong mutual interest and competence.

DESCRIPTION (provided by applicant) We request continuing support for this postdoctoral training program in basic neuroscience as it relates to neurodegenerative disease and stroke. The program is closely integrated with several key research programs related to Parkinson's disease, Alzheimer's disease, ALS, and stroke. Additional programs in psychiatric disorders and in neuroAIDS further enhance the environment. The grant will permit us to maintain the critical mass needed to recruit the best possible postdoctoral trainees, including under represented minority trainees, and to offer a training program of the highest quality. Our training program focuses primarily on basic and translational laboratory research that can readily be related to neurodegenerative disease and stroke. The major research advisor and an individualized research advisory committee, a professional development committee, and the training grant steering committee share the task of guiding and monitoring each trainee. All trainees participate in three activities which supplement their research experience: (1) Two seminars on the neurobiology of clinical disorders, (2) a monthly research discussion, and (3) a series of professional development workshops. Issues of responsible conduct are integrated into the entire program. Trainees also participate in courses as needed and have opportunities to teach and to observe clinical practice. The 39 members of the training faculty are members of the university-wide program in neuroscience with a particular interest in neurodegenerative disorders and stroke. Most of the faculty members have extensive experience in training and in research and have significant grant support. In addition, we have included a small group of junior faculty who have outstanding promise and will be available to serve as co-mentors in conjunction with a more senior faculty member. At present our training faculty supervise more than 90 postdoctoral trainees (in addition to a large number of predoctoral students), of whom about 20 are eligible for NRSA support. We are requesting stipends to support an initial complement of 4 postdoctoral trainees, increasing gradually to 8 over a 5-year period. We believe that our training program provides an opportunity for outstanding postdoctoral trainees to prepare for a career of direct relevance to issues of neurodegenerative disease and stroke.

Parkinson's disease (PD) poses a serious threat to the health of a large segment of our society. Having focused on the compensatory changes that underlie the preclinical phase of PD, we are now concentrating our efforts on the issue of neuroprotection. The present project is one component of this effort and focuses on strategies for inducing endogenous neuroprotective mechanisms in animal models of PD. The work derives from recent evidence from our labs indicating that the contralateral motor neglect normally following unilateral damage to the nigrostriatal dopamine (DA) projection can be ameliorated by forced use of the contralateral limb. Moreover, we find that this behavioral sparing is accompanied by a dramatic reduction in the loss of DA. We hypothesize that forced execution of a motor act that is otherwise compromised by PD is neuroprotective, and that this results from an interaction between the motor act, injury, and concomitant increase in the availability of one or more trophic factors (e.g., GDNF) and hormones (e.g. estrogen). Our project utilizes 6-hydroxydopamine (6-OHDA)-treated rodent models of PD and has five specific aims: (1) We will determine the impact of forced use/disuse on the anatomical and functional state of DA neurons, including the use of microdialysis to measure in vivo DA efflux. (2) We will determine the relationship between use-dependent neuroprotection and increased trophic factor expression by using antisense oligonucleotides to reduce expression, fusion proteins to serve as decoys for the factors, and animals deficient in a protein key to trophic factor signaling. (3) We will examine the ability of estrogen to exert a neuroprotective influence and the possible role of that influence on the impact of exercise. (4) We will use microarrays together with more classical techniques to examine changes in other trophic factors and their receptors in striatum of animals subjected to lesions and/or casting. Later we will also look at substantia nigra and at regions where no neuroprotection is seen. (5) Finally, we will examine the impact of age on neuroprotection induced by trophic factors by first determining whether neuroprotection induced in young adult rats continues to be effective as animals reach old age, and then ask whether neuroprotective strategies that work in young adults can also be used to reduce the impact of 6-OHDA in aged rats.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) poses a serious threat to the health of a large segment of our society. This is an extensively revised renewal application for a Program Project Grant now in its 18th year. During much of the history of the PPG, we have focused on the compensatory changes that underlie the preclinical phase of PD. That line of investigation will continue, while at the same time we will also add two new foci: first, the development of neuroprotective strategies and, second, the detection of PD it its preclinical phase. Neuroprotection: This will now provide the principal long-term focus of the entire PPG. Our approach derives from recent evidence from our labs indicating that the contralateral motor neglect and loss of DA normally following unilateral damage to the nigrostriatal DA projection can be ameliorated by forced use of the contralateral limb. We hypothesize that forced execution of a motor act that is otherwise compromised by PD is neuroprotective, and that this results from an interaction between the motor act, injury, and concomitant increase in the availability of one or more trophic. We will explore this hypothesis using our 6-hydroxydopamine (6-OHDA) rat model. Our work will involve studies of the role of trophic factors (e.g., GDNF, BDNF, and FGF2), estrogen, and aging, as well as anatomical studies to differentiate between protection, rescue and sprouting (Project 1: M. Zigmond, PI). We also use multineuron recording in awake animals to examine the effect of forced use on the functioning of the basal ganglia more broadly (Project 2, D. Woodward, PI). Compensation: In the past, our studies of compensation have focused our studies on adaptations within the nigrostriatal dopamine (DA) system. Our multineuron recordings will now allow us to explore adjustments within other components of the basal ganglia (Project 2: D. Woodward, PI). Early detection: For neuroprotective strategies to be most effective, it is likely that they must be applied as early in the course of the disease as possible. In this respect, the compensatory changes noted above represent a problem to be overcome through the development of diagnostic tests that can detect PD before the emergence of gross neurological deficits. To do so we will develop a multi-dimensional clinical test battery, using PET imaging as the ultimate criteria for nigrostriatal damage (Project 3, N. Bohnen, PI). We believe that by combining a variety of basic, translational, and clinical approaches we will make significant progress toward the development of a therapeutic approach to PD.

DESCRIPTION (provided by applicant): Overall hypothesis: Exposure of developing animals to severe stress causes an increase in the vulnerability of the brains of those animals to neuronal death, which persists into adulthood, leading to an increase in the susceptibility to neurological and psychiatric disease. Possible routes of rehabilitation include environmental enrichment, exercise, and specialized diets. We are seeking funds to further develop the tools necessary for collaborative studies on the impact of developmental stress and the vulnerability of the adult brain to neurotoxin exposure. Over the next two years, we have three major Specific Aims: Aim 1: To develop models of developmental stress in laboratory rats. Three models will be explored: (a) maternal deprivation, (b) nutritional deprivation, and (c) an inflammatory challenge. Aim 2: To obtain pilot data to see if developmental stress increases the vulnerability of the brain to neurotoxins. We will determine whether developmental stress increases the damage done by application of such neurotoxins as quisqualic acid and 6-hydroxydopamine. Special attention will be given to dopamine neurons in the brain, although other types of neurons also will be examined. Both behavioral and neurobiological indices of damage will be monitored. Aim 3: To further develop the research capacity of the University of Cape Town and the University of Stellenbosch through (a) attendance by faculty and trainees at international meetings, (b) attendance of key individuals at specific courses on research methodology, and (c) provision of instruction to faculty and trainees other professional skills such as oral and written communication, applying for research funds, and responsible conduct of research. Having accomplished these aims in 2003-2005 (FY 04 and FY05), we will submit a full proposal for research to begin in July 2005 (FY 06) that utilizes these tools to explore the molecular and cellular basis of DA neuron cell death and protection. Moreover, the methods and reagents that we develop will be made available to others in the field.

[unreadable] DESCRIPTION (provided by applicant): Researchers representing the University of Pittsburgh and two institutions in South Africa, the University of Cape Town and the University of Stellenbosch are seeking funds as part of an overall effort between our institutions to examine a specific hypothesis pertaining to the impact of environment on brain and behavior and also to help create within the academic community in Cape Town a regional center of excellence in neuroscience and in professional skill training. Their hypothesis is that exposure of developing animals to severe stress will have a significant detrimental effect on brain and increase its vulnerability to neuronal death, but that this can be offset through an exercise intervention program. There are five specific aims: four are research aims and one is a set of capacity building objectives. Research objectives: Aim 1 - To examine the impact of a developmental stressor (maternal separation) on several variables related to the vulnerability of the brain, including (a) the hypothalamic-pituitary-adrenal (HPA) axis, (b) mitochondrial function, and (c) alterations in a specific class of proteins (neurotrophic factors) by using a proteomic screen. Aim 2 - In addition, the state of central dopamine neurons will be carefully examined. Aim 3 - To examine the effects of exercise (voluntary running) on these same variables, and then to determine whether the effect of maternal separation can be offset by later exposure to exercise. Aim 4 -To examine the effects of maternal separation on the vulnerability of dopaminergic neurons to oxidative stress using 6- hydroxydopamine as the stressor, then determine whether any increase in vulnerability caused by the maternal separation can be offset by exercise. Aim 5 - Capacity building objectives: To further develop the research capacity of the Universities of Cape Town and Stellenbosch as well as other institutions in the Western Cape through attendance by faculty and trainees at international meetings, participation in specific courses on research methodology, and provision of instruction in other professional skills, such as oral and written communication, applying for research funds, and responsible conduct of research. The research team believes that their research project will provide important insights into epigenetic influences on behavior and brain function more generally, and also serve as a context for promoting the research capacity of two key institutions in southern Africa. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): We request continuing support for our postdoctoral training program in basic neuroscience as it relates to neurodegenerative disease and stroke. The program is closely integrated with several key research programs related to Parkinson's disease, Alzheimer's disease, ALS, and stroke, and to the Pittsburgh Institute for Neurodegenerative Diseases, now housed in contiguous space within a newly constructed state-of-the-art facility. Additional programs in psychiatric disorders and in neuro-AIDS further enhance the environment. The grant will permit us to maintain the critical mass needed to recruit the best possible postdoctoral trainees, including underrepresented minority trainees, and to offer a training program of the highest quality. Our training program focuses primarily on basic and translational laboratory research that can readily be related to neurodegenerative disease and stroke. The major research advisor and an individualized research advisory committee and the training grant steering committee share the task of guiding and monitoring each trainee. All trainees participate in four activities that supplement their research experience: 1) A seminars on the neurobiology of clinical disorders, 2) a monthly research discussion, 3) a series of professional development workshops, and 4) at least one outreach activity. Issues of responsible conduct are integrated into the entire program. Trainees also participate in courses as needed and have opportunities to teach and to observe clinical practice. The 33 members of the training faculty are members of the university-wide program in neuroscience with a particular interest in neurodegenerative disorders and stroke. Most of the faculty members have extensive experience in training and in research and have significant grant support. In addition, we have included a group of 9 junior faculty who have outstanding promise and will be available to serve as co-mentors in conjunction with a more senior faculty member. At present our training faculty supervise more than 90 postdoctoral trainees (in addition to a large number of pre-doctoral students), of whom about 20 are eligible for NRSA support. We are requesting stipends to support an initial complement of 4 postdoctoral trainees, increasing gradually to 6 over a 5-year period. We believe that our training program provides an opportunity for outstanding postdoctoral trainees to prepare for a career of direct relevance to issues of neurodegenerative disease and stroke. [unreadable]

DESCRIPTION (provided by applicant): Responsible conduct in research (RCR) is an essential part of good science. Traditionally, it was assumed that explicit training in RCR was either unnecessary or was provided through the normal process of preparing individuals for a scientific career. However, experience over the last several decades suggests that a more explicit training program is necessary. Furthermore, a strong case exists for combining training in RCR with training in other aspects of professionalism, including the ability to communicate scientific results orally and in peer-reviewed manuscripts, teaching, and mentoring. This is because many of the key aspects of ethical behavior are embedded in those skills and may not be included in conventional RCR courses. The NIH has recently issued requirements for RCR training that recommend a minimum of eight hours of training, provided in person by research faculty. Unfortunately, many institutions are not prepared to provide such instruction, particularly given the large number of individuals that are now required to receive it and the likelihood that this number will continue to expand. Over the past 25 years the PIs have developed an educational program that provides graduate students, postdocs, and junior faculty with explicit training in RCR within the context of instruction in professional skills. In addition, for the past 16 years they have offered an annual trainer-of-trainers conference in which faculty obtain the instruction and materials necessary to establish such programs at their own institutions. In this application, funding is requested to extend the PIs' efforts at disseminating that educational model. There are five specific aims. Aim 1: Continue to develop training modules in RCR and in professional skills that will enable faculty to establish courses at their home institutions in RCR and professional skills. These modules will be designed to fulfill and expand on NIH requirements. Aim 2: To bring together an instructional team that will work together to provide that training. Over the years the PIs have invited a great many individuals to provide lectures on RCR and professional skills; from that group they have identified several outstanding individuals, each of whom has the knowledge, the professional standing, and the teaching skills to convey the necessary information. This team, which forms the Organizational Committee, is well balanced with respect to gender and ethnicity. Aim 3: To recruit 40 qualified participants per year who are committed to implementing training at their home institution. Individuals will be selected based on an application that includes a description of their experience, their rationale for attending the conference, and their plans to utilize the training. Care will be taken to recruit a diverse group of attendees with regard to gender, ethnicity, ability, and the types of individuals they plan to train. Aim 4: To carefully evaluate the effectiveness of this project, through on-site anonymous feedback from conference participants and annual follow-up surveys of past participants. Data will be used to improve subsequent activities. Aim 5: To further disseminate this educational model and our results at meetings, in articles published in relevant journals, and on the Internet.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) affects at least 1 million individuals in the US alone. Although much is known about its pathophysiology and information is emerging about its cause, there are no pharmacological treatments shown to have a significant, sustained impact on the prevention of PD or on attenuation of its progress. However, clinical evidence suggests that physical exercise is such a treatment, and this is supported by studies of animal models of the dopamine (DA) deficiency associated with the motor symptoms of PD. Moreover, exercise is a practical and sustainable therapeutic intervention likely to act simultaneously on most if not all of the cellular events capable of protecting DA neurons and restoring DA function. This proposal is designed test the hypothesis that exercise increases DA function and protects DA neurons against toxic insult due in part to increased neurotrophic factor (NTF) signaling, protection of mitochondrial respiration, and stimulation of angiogenesis. An MPTP mouse model will be used to test this hypothesis. Aim 1. To determine the effects of exercise on the impact of MPTP on dopaminergic function (1a) the optimal temporal relationship between exercise and toxin exposure will first be established, thus exploring both protection (exercise before toxin) and rescue (exercise after toxin). (1b) The impact of the optimal exercise paradigm on DA cell loss and on changes in pre- and post-synaptic DA receptors will then be assessed. (1c) Levels of DA and metabolites in striatal tissue and extracellular fluid will be measured in MPTP animals treated with exercise as dictated by Aim 1a. (1d) Mitochondrial respiration will be assessed after MPTP exercise. Aim 2. To assess the role of NTFs in exercise-induced protection members of four distinct NTF families known to protect DA neurons will be examined: GDNF, BDNF, MANF, and VEGF. (2a) The temporal and anatomical profile of NTF changes will be determined after exercise, MPTP, and the optimal combination of MPTP and exercise. (b) The ability of exogenous NTF to mimic the effects of exercise identified in Aim 1 will then be assessed. (2c) The NTFs and/or their receptors suggested by Aim 2a and 2b will be conditionally knocked out to determine if this increases the toxin impact of MPTP and reduces the protective impact of exercise, thereby strengthening the hypothesis of a causal relationship between NTF and exercise-induced protection. Aim 3. To examine the role of angiogenesis in exercise- and NTF-induced neuroprotection, (a) vasculature be assessed in substantia nigra, striatum, and cortex using BrdU to measure new cells and PECAM-1 (CD31), to mark blood vessels. (3b) FITC-conjugated tomato lectin will be used to detect patent blood vessels. (3c) A 3-dimensional analysis of vascular density will be performed. (3d) Finally, expression of angiogenesis-promoting proteins, including angiopoetins 1 and 2, will be measured. These results will provide the basis for the next iteration of a research program focusing on additional mechanisms underlying exercise-induced protection, provide stronger evidence for exercise-induced protection in PD, and establish targets for pharmacological treatment of the condition. PUBLIC HEALTH RELEVANCE: Parkinson's disease affects well over a million individuals in the US alone. Although we know a good deal about how the brain is affected by the disease, there are no pharmacological treatments shown to have a significant and sustained impact on the prevention of the disease or on the attenuation of its progress. Such treatments are urgently needed. Clinical evidence suggests that physical exercise is just such a treatment, and studies of animal models of the dopamine loss associated with the motor symptoms of the condition further support this hypothesis. This study is designed to test that hypothesis. Such a demonstration would not only strengthen the case for advising individuals to engage in exercise, but may also provide insights into drug development for the condition.

The University of Pittsburgh in conjunction with the American Association for the Advancement of Scien(AAAS) will design a Train the Trainers of Trainers (T3) model based on some of the leading research of Everett Rogers and others about how .innovations are diffused into existing organizations. T3 will ramp up the training of faculty to train others who train graduate students in responsible conduct of research, conduct research and interviews to gather knowledge about what has worked to date, plan the workshop for training the trainers, develop workshop materials that address issues of pedagogy, program implementation including, the development of support for ethics training, and design new curricula units that relate to professional development issues that arise unexpectedly, but are critical to the professional and ethical success of the graduate students.
The proposal addresses a critically important need across all STEM fields?the training in responsible conduct of research and ethical behavior throughout a STEM career. The model will rapidly multiply the number of people capable of teaching ethics to STEM graduate students with new workshops, curricula and websites.
The collaboration with AAAS and other professional societies augurs well for a fast and useful ramp-up of materials and usage. While the project focuses on graduate students in NSF related fields, it is anticipated that the curriculum materials and training will be useful for teaching ethics to undergraduates, postdoctoral fellows, and faculty, and staff in fields beyond those that are NSF related.

DESCRIPTION (provided by applicant): This project will build capacity in international research ethics in China. The primary focus will be a 2-year program of Master's level training focused on international research ethics to at least 30 Fellows from China, with the objective of preparing them to establish or significantly enhance research ethics training programs, Institutional Review Boards (IRBs), and/or compliance offices at their local institution. In this way, the impact of the training of these 30 Fellows will be greatly amplified in the years to come. The instructional program provided to the fellows will be tailored to each individual needs, based on their prior knowledge and experience, their specific interests, and their specific local environment. The curriculum will include (a) instruction in research ethics provided through workshops, seminars, and coursework taken primarily in China and at the University of Pittsburgh, (b) training in professional development (e.g., publishing research articles, preparing proposals, project management, and evaluation) and the aspects of responsible conduct associated with those skills, (c) instruction and involvement in the design, development, and management of research programs, (d) attendance at selected meetings of IRBs at the University of Pittsburgh, as well as discussions with the administrators of those units that will focus on preparing the participants to establish and direct such programs in China, (e) visits to IRBs at additional institutions, (f) a visit the NIH, the American Association for the Advancement of Science (AAAS), the Office of Research Integrity (ORI), and Johns Hopkins University to discuss critical issues in research ethics and science policy, and (g) instruction in pedagogy and distance learning provided in China by the PIs and other US- and China-based experts in the field of education. In the second year, the Fellows will be expected in initiate or significantly improve a course on research ethics with input from a mentoring committee consisting of one of the PIs and US- and China-based consultants. Fellows will also be encouraged to become engaged on relevant research and start up grants will be available on a competitive basis. In addition, we will develop a locally sustainable conference on research ethics for researchers, administrators, and policy makers in China to be held in the fifth year of the project. The PIs and their co-investigators and consultants will be involved in the planning and implementation of the first conference. However, the major responsibility will be that of the Fellows themselves, and a mechanism will be put in place to ensure that future conferences of this sort will be held regularly and be organized entirely by program alumni and those their trainees. A rigorous evaluation of the program will occur at all phases, and both the curriculum and the outcome of the program will be disseminated at professional meetings, on the web, and in peer-reviewed publications. The program will be overseen by an External Advisory Committee made up of individuals with relevant expertise, most of whom will not be directly affiliated with the training program except that they will assist in participant selection and the evaluation of the curriculum and its effectiveness.