George V. Rebec - US grants

Ascorbate, a water.soluble vitamin found in high concentrations in the mammalian forebrain, appears to modulate several different neural functions. This neuromodulatory role is especially evident in the neostriatum where physiological amounts of absorbate have been shown to alter neuronal activity and to interfere with the behavioral response to drugs that act in part via neostriatal mechanisms. To determine the neurochemical systems and processes underlying these actions of ascorbate a series of experiments will focus on a ppossible interactions between ascorbate and some well.known neurotransmitters. Single.unit recording techniques, including electrochemically.quantified iontophoresis, will be used to examine the effects of ascorbate on the neuronal response to iontophoretically.applied dopamine and glutamate and to glutamate released endogenously from cortico.neostriatal terminals. Intraneostriatal infusions in freely.moving rats will be used to examine the role of the neostriatum in the behavioral effects of ascorbate. Parellel experiments will examine the mechanisms that control extracellular levels of ascorbate, especially the dramatic rise produced by amphetamine. Carbon.fiber microvoltammetic and electrochemically.modified electrodes will be used to obtain unequivocal information on extracellular ascorbate levels in the neostriatum. Follow.up work will extend these investigations to other forebrain regions known to contain a high endogenous level of ascorbate. These lines of research will provide basic information about the mechanisms by which ascorbate regulates and controls some important neural and behavioral processes.

The alarming abuse potential of amphetamine and its profound clinical effects have stimulated considerable research on the neuronal systems and processes underlying the actions of this drug. The neostriatum has been implicated in many of the behavioral effects of amphetamine including the focused, repetitive behaviors (stereotypies) that are enhanced during long-term treatment. Little information is available, however, concerning the neurochemical systems that modulate the neuronal effects of amphetamine in the neostriatum and the relationship of these effects to the amphetamine behavioral response. In this application, experiments are proposed that will shed new light on these issues. Single-unit recording techniques, including electrochemically-quantified iontophoresis and studies of freely-moving animals, will be used to identify changes in the sensitivity of pre- and postsynaptic dopamine receptors that may occur during long-term amphetamine treatment. These techniques also will be used to investigate corresponding changes in serotonergic neurons that recently have been found to modulate the neuronal effects of amphetamine in specific neostriatal regions. A growing body of evidence also suggests that neostriatal neurons are responsive to ascorbic acid and that this effect may have important implications for understanding the actions of amphetamine in this site. In fact, ascorbic acid has been shown to attenuate amphetamine-induced stereotyped behaviors. Thus, a parallel series of experiments are proposed to elucidate the role of ascorbic acid in regulating the neuronal and behavioral actions of amphetamine.

DESCRIPTION (provided by applicant): Repeated use of amphetamine, cocaine, and related psychomotor stimulants can lead to high rates of relapse. In fact, relapse to drug use even after prolonged periods of abstinence is a common characteristic of experienced stimulant users. Relapse appears to be triggered by three major events: exposure to acute stress, presentation of cues previously associated with the drug, and exposure to the drug itself. Studies of reinstatement of drug seeking in experimental animals, primarily rodents, suggest that, as in humans, information related to these events converge in prefrontal cortex (PFC) to drive the relapse response. Although relating rodent cortical areas to those in primates requires caution, ample evidence now indicates that the interface between anterior cingulate and pre-limbic cortex (Cg1-PLC) is crucial for the reinstatement of drug seeking triggered by drug-related cues and by re-exposure to the drug. In this application, research is aimed at identifying how neurons in this region process cocaine and cocaine-related cues to reinstate cocaine-seeking behavior in rats. We contend that activation of dopamine (DA) neurons in the ventral tegmental area (VTA), which innervates a large expanse of PFC, plays a critical role in the activation of Cg1- PLC neurons underlying cocaine relapse. Three specific aims will address this hypothesis. First, we will characterize the dynamics of neuronal activity during cocaine self-administration and during reinstatement of cocaine seeking induced by the cocaine-related cue and by cocaine priming. Cg1-PLC activity will be monitored by chronically implanted micro-wire electrodes. The goal is to understand how these neurons process information related to cocaine seeking. Consistent with our hypothesis, pilot data suggests that Cg1-PLC neuronal activation is a critical component of this behavior. Second, to determine the role of VTA in cocaine relapse, VTA activation will be blocked by local infusions of kynurenate, an antagonist of ionotropic glutamate receptors. Early findings confirm the importance of VTA activation, and subsequent work will examine the extent to which VTA activation drives relapse-related responding of cortical neurons. Third, to confirm reinstatement-related release of DA in Cg1-PLC, we will use fast-scan cyclic voltammetry to monitor catecholamine transients induced by cocaine-related cues. The extent of the DA contribution will be examined in follow-up studies that focus on the VTA as the source of the catecholamine signal. Collectively, our results will provide important, new information on the involvement of PFC neurons and their DA input in relapse to cocaine seeking.

biomedical equipment resource; biomedical equipment purchase;

DESCRIPTION: (Adapted From The Applicant's Abstract) Amphetamine and related drugs of abuse elicit species-specific motor responses characterized by repetitive or stereotyped patterns. Research on animals, typically rodents, as models of the human response, has implicated the striatum and related basal ganglia circuitry in the motor-activating effects of these drugs. Critical elements of this circuitry include both dopamine- and glutamate-containing fibers that contact neurons in dorsal striatum. During amphetamine-induced motor activation, these neurons establish a pattern of discharge activity mediated, at least in part, by a complex interaction between dopamine and glutamate inputs. Research in this application extends this line of work on behaving animals in two directions. One involves characterization of the neuronal response pattern to amphetamine in substantia nigra pars reticulata, a major target of striatal neurons and an important output nucleus of the basal ganglia. After basic neurobehavioral correlations are established, further studies will examine the extent to which amphetamine-induced changes in reticulata neurons are mediated by the striatum via descending GABA-containing projections. The aim is to determine how amphetamine-induced neuronal response patterns established in striatum are represented in reticulata neurons. A second focus of the proposed research is to examine at the single-neuron level how the major transmitters altered by amphetamine-- dopamine and glutamate--interact with each other and with GABA to influence the activity of striatal neurons in an intact, normally functioning animal. Dopamine, glutamate, and GABA will be applied directly by iontophoresis to electrophysiologically isolated single units in awake, unrestrained rats. Attention will center on the mechanisms by which synaptic dopamine modulates glutamate- and GABA- mediated responses. A major component of this work also involved iontophoresis of amphetamine and other indirect dopamine agonists in striatum to determine how local changes in dopamine transmission modulate striatal activity and to reveal the synaptic action of these drugs unaccompanied by concomitant activation of other neuronal pathways. Collectively, these lines of research will provide important new information on the neurochemical systems and processes by which amphetamine alters neuronal function and motor behavior.

[unreadable] DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominant condition characterized by cognitive impairment and progressive loss of motor control. Despite identification of the underlying genetic defect and its mutant protein, huntingtin, effective treatment remains elusive. Although the ultimate pathology in HD involves the death of neurons primarily in the striatum and selected components of the corticostriatal pathway, the onset and progression of the behavioral phenotype is likely to reflect deficits in how the striatum receives and processes information. This line of research has been enhanced by the recent development of transgenic and knock-in mouse models of HD. In work proposed for this application, we will use these models to build on our long-standing program of research on ascorbate, a water-soluble vitamin critically involved in striatal function. Our overall objective is to assess how a behavior-related deficit in striatal ascorbate release in HD mouse models contributes to motor symptoms. We will test the hypothesis that low ascorbate is linked to alterations in glutamate, an excitatory amino acid released by the corticostriatal pathway. Three parallel approaches are planned. In one, we will use on-line microdialysis to quantify basal extracellular glutamate in behaving HD and wild-type controls. Follow-up experiments will monitor cortically evoked glutamate changes in striatal extracellular fluid and examine mechanisms of extracellular glutamate clearance. Consistent with our prediction, preliminary data indicate that an ascorbate deficit in behaving HD mice indicates elevated striatal glutamate owing to an impaired uptake mechanism. A second approach will examine dysfunctions in striatal neuronal processing by recording single-unit activity in the striatum of HD mouse models during spontaneous movement and sensorimotor stimulation. Follow-up experiments will test hypothesized changes in glutamate receptor sensitivity on abnormally active neurons recorded from awake HD mice and modulation of these changes by ascorbate. A third approach will examine whether reversal of the striatal ascorbate deficit by repeated, intermittent injections of ascorbate leads to a corresponding improvement in the behavioral phenotype. Preliminary data not only support this hypothesis but indicate that ascorbate treatment also reverses abnormalities in striatal neuronal processing. Collectively, these approaches are designed to assess how fluctuations in striatal ascorbate and glutamate contribute to the neuronal malfunctions underlying the motor phenotype of HD. [unreadable] [unreadable]

DESCRIPTION (Adapted From The Abstract Provided By Applicant): This proposal requests support to begin a unique, integrative research training program at Indiana University in the study of sensorimotor neuroplasticity. Remarkable advances at the neurobehavioral level have made it clear that neuronal processing of information for motor output involves highly flexible mechanisms evident at all levels of the nervous system. Further development of the field requires a supply of creative, methodologically competent neuroscientists dedicated to research careers. The proposed program draws upon the strengths of a multidisciplinary faculty in neuroscience and psychology to meet that goal. Training is built on a foundation of intensive research experiences, interactive group meetings, and formal course work. Throughout the course of the program, trainees are immersed in collaborative research with one or more of the core faculty who use a range of neurobehavioral approaches to study many different mammalian species. Faculty research directions include: the neurobiology of motor lerning, cortical and subcortical reorganization of sensorimotor systems after peripheral nerve injury, motor related changes in neuronal processing and their underlying neurochemical substrates, remodeling the morphology of adult neurons in a spinal motor system, assessing the functional plasticity of aging neuromuscular systems, and neuroethology of vocal communication. At the group level, training activities involve regular laboratory meetings, research seminars, specialized colloquia, and participation at scientific and professional meetings. Formal course work is designed to provide a firm grounding in the organization and operation of the nervous system as well as in behavioral issues ranging from learning to motor control. Funding is requested for 2 predoctoral students and 2 postdoctoral fellows over a 5year period with individual trainees typically receiving support for two years. Pre-doctoral trainees will be drawn from two Ph.D. programs, the Program in Neural Science and the Department of Psychology, on the basis of their academic and research credentials. Post-doctoral trainees will be recruited nationwide by a variety of mechanisms and selected on the basis of their suitability for the training program. The longterm goal is to provide specialized, interdisciplinary research training in sensorimotor neuroplasticity that will spawn novel and creative approaches to basic research problems in motor control and movement.

A grant has been awarded to Dr. Paul A. Garris at Illinois State University
to develop a new instrument for wireless monitoring of neural activity in
the brain of awake, unrestrained animals. The new instrument, called
real-time animal telemetry (RAT), will combine two powerful technologies;
microsensors for spatially and temporally resolved measurements, and digital
telemetry for remote data transmission and system control with high speed
and high fidelity. The primary advantage of RAT will be sub-second
characterization of brain function with minimal perturbation of behavior.
The proposed RAT instrument holds great promise for advancing the study
of brain-behavior relationships.

RAT will be a modular and multifunctional instrument, a design that advances
development and affords flexibility to its application. Several types of
measurement techniques will be incorporated into RAT: Voltammetry which
monitors the chemistry of the brain and electrophysiology which
measures brain bioelectrical activity. Combined, the techniques assess the
release of a neurotransmitter and its postsynaptic effect to obtain a more
integrative view of brain function. Although great strides have recently been made
applying real-time voltammetry and electrophysiology to awake animals,
the connection between sensor and recording equipment is made by a cable tether. Unfortunately, the hard connection affects behavior and hinders or even prevents
investigation of important paradigms such as those involving social interactions
and complex environments. To overcome this problem, the new instrument will
use a wireless link. Moreover, because real-time voltammetric and electrophysiological measurements are very susceptible to transmission artifacts, RAT will use high fidelity digital telemetry.

Ultimately, RAT should be commercially viable instrumentation in the support of
biological research. The incorporation of well-established techniques will
make the proposed RAT instrument attractive to large number of users working
in the neuroscience fields. With further development, there is enormous
potential for RAT to support other existing real-time microsensors. RAT can
also evolve to accommodate sensor technologies that emerge in the future.

DESCRIPTION (provided by applicant): The purpose of this application is to establish an integrative predoctoral training program in the neuroscience of drug abuse at Indiana University. Research aimed at the problem of drug abuse has seen remarkable advances in recent years, driven in large part by a rapidly expanding arsenal of technological innovations at all levels of analysis from ion channel, proteins to human brain mapping. But as the science becomes increasingly complex and specialized, there is a growing danger that opportunities for integration and interaction across disciplines will be lost. Narrowly focused researchers cannot produce the well-rounded scientists the field needs - scientists who have the flexibility to respond to changing techniques and paradigms and who can understand and appreciate the multi-faceted problem of drug abuse. If the expanding research knowledge base is to lead to effective prevention and treatment strategies, the time has come to put a translational perspective on research training. To meet this need, we have assembled a dedicated group of faculty trainers drawn from the Program in Neuroscience and the Department of Psychological and Brain Sciences. This group, which has, a strong and longstanding tradition of collaboration on issues directly relevant to drug abuse, includes senior and junior investigators, molecular neurobiologists and cognitive neuroscientists, and animal behaviorists and clinical scientists. Working together in a state-or-the-art research environment, this group has access to a pool of highly talented trainees motivated to become the next generation of drug abuse researchers. The proposed training program involves trainees in three key components: integrative course work, translational research training, and professional skills development. Course work covers basic neuro- and psychopharmacology, provides an integrative view of biobehavioral processes in substance use disorders, and concludes with an overall perspective on the translation of theoretical and empirical knowledge as it applies to different experimental approaches. Research is guided by a mentor in cellular, systems, cognitive, or clinical neuroscience interacting with a co-mentor representing a different but complimentary level of analysis. This integrative approach is reinforced through discussion groups, attendance at colloquia, and participation at national meetings. Trainees also learn to develop skills in grant writing, manuscript preparation, and teaching. In short, a combination of course work and research training aimed at integrating and translating bench and bedside approaches will create scientists well prepared for the next decade of research on the addicted brain.

DESCRIPTION (provided by applicant): With an ever-growing life expectancy and senior population, costs for healthcare and assisted living for the elderly will place an unsustainable burden on the US economy as the elderly lose their functional mobility. The cost of assisted living for seniors in the US is projected to reach $248 billion by 2012. At the core of the loss of functional mobility are declines in cognitive and motor function, represented by increased intra-individual variability known as greater behavioral inconsistency. Increased neural noise is the higher occurrence of random brain activity causing greater behavioral inconsistency in the elderly. As such, scientists are continuing to seek means of attenuating age-related increases in behavioral inconsistency. Currently, the factors that attenuate these declines remain unknown, presenting a barrier to the development of targeted interventions for the clinic and community. The advantage of animal models become in studies of aging is their shorter lifespan that allows the scientist to obtain outcome data more rapidly. Yet, behavioral inconsistency has not been studied in animal models. Recent theorizations propose that animals housed in enriched experimental environments exhibit smaller age-related increases in neural noise during the aging process. "Enriched" animals that are housed in groups have the opportunity for social interaction and an environment that is varied over the experimental period with toys and food locations being changed frequently. However, the specific source of the benefits of the environment is still being debated, as enriched environments positively affect aging in a manner that cannot be replicated by socialization or exercise alone. It is thus a logical question to ask whether the increased opportunity to engage in different movement patterns provides the positive effects of enriched environments. Our specific aim is to test the hypothesis that greater variance in the movement patterns that one engages in is a primary source of the benefits of environmental enrichment. We will connect the effects of aging on variability in neural and motor activity with that of variety of motor activities afforded by the environment. This study will be conducted by an interdisciplinary research team from Kinesiology, Physics, and Neuroscience to examine the complex scientific problem of aging. Our study has the potential to achieve a significant scientific breakthrough due to its many innovations. The results would show that a key component to healthy aging is to continually acquire new movement skills throughout the lifespan rather than physical activity alone. Interventions that seek to slow age-related declines in cognition and action have focused primarily on maintaining existing function. Successful completion of this study would provide the evidence-base for continually introducing new movement skills to seniors, a process that could be easily implemented through community programs. PUBLIC HEALTH RELEVANCE: With people over 65 years becoming proportionally the largest age bracket in America, this study is relevant to nearly an eighth of the entire nation. Successful completion of this study will show that continually engaging in a greater variety of different movement skills has a protective effect against neural and motor declines in aging, a result that can be directly translated to clinic and community. Maintaining cognitive and movement capabilities are especially relevant to public health, as they are essential components of functional mobility, which lies at the core of quality of life in the elderly.

DESCRIPTION (provided by applicant): This proposal requests support for the first-time renewal of a highly successful integrative pre-doctoral training program in the neuroscience of drug abuse at Indiana University. Despite substantial advances in understanding drug addiction within specific levels of analysis (e.g., behavioral, clinical, and molecular), the problem of drug abuse cannot be solved by focusing on a singular experimental approach. If the next generation of researchers is to make meaningful progress, they must be well-rounded scientists who possess both the flexibility to respond to rapidly changing technologies and the training background to appreciate that drug abuse is, in fact, a multi-faceted problem. To prepare trainees for success in the next decade and beyond, our program emphasizes a team-driven, inter-disciplinary approach based on the translational model. Our program is successful because it brings together 10 core faculty members who are committed to integrative training and have a history of collaboration on issues directly relevant to drug abuse research. They include senior and junior investigators, molecular neurobiologists, cognitive neuroscientists, and clinical scientists. They have joint appointments in the campus wide Program in Neuroscience and the Department of Psychological and Brain Sciences. Working together in state-of-the-art facilities, this group has access to a pool of highly talented trainees motivated to launch a caree in drug abuse research. Our training program develops trainees by emphasizing three key components: integrative course work, translational research training, and professional skills development. Course work covers basic neuro- and psychopharmacology, provides an integrative view of biobehavioral processes in substance use disorders, and brings a translational perspective to theoretical and empirical knowledge. Research is guided by a mentor in molecular, systems, cognitive, or clinical neuroscience interacting with a co-mentor representing a different but complimentary level of analysis. This integrative approach is reinforced through discussion groups, attendance at colloquia, and participation at national meetings. Trainees, moreover, earn joint degrees in Neuroscience and Psychology. Instruction in ethical scientific behavior includes formal course work and campus workshops as well as specialized instruction led by a core faculty member who has many years of experience leading seminars on ethical issues unique to substance use research. Trainees also learn to develop skills in grant writing, manuscript preparation, and teaching. In short, our program relies on a combination of course work and research training aimed at integrating and translating bench and bedside approaches to produce scientists well prepared for productive and transformative careers in drug abuse research. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

Project Summary Excessive glutamate signaling through N-methyl-D-aspartate receptors (NMDARs) is implicated in altered forms of neuronal plasticity associated with opioid reward and dependence. The present Cutting Edge Basic Research Award proposes to functionally decouple signaling complexes downstream of NMDAR activation to eliminate aberrant NMDAR-dependent nitric oxide signaling and circumvent the development of opioid reward. In the United States, unintentional deaths due to prescription drug overdoses have more than tripled since 1990, more than deaths attributed to cocaine and heroin combined. The increase in unintentional drug overdose death rates has mainly been driven by increased use of opioid analgesics. Inadequate treatment for pain, exacerbated by incomplete analgesic efficacy and narcotic abuse liability, contributes to escalating drug use, resulting in socioeconomic costs estimated at $600 billion annually. Improving the safety, efficacy, side effect profile and abuse liability of opioid analgesics thus remains an urgent medical need. Excessive NMDAR stimulation triggers a signaling cascade involving activation of the enzyme neuronal nitric oxide synthase (nNOS), which catalyzes formation of the signaling molecule nitric oxide (NO), which promotes addiction- related behaviors. Inhibition of aberrant glutamatergic hyperexcitability and inhibition of nNOS reduces addiction-related behaviors in preclinical studies. However, the therapeutic potential of NMDA receptor antagonists and NOS inhibitors are limited by severe side effects. We propose to functionally decouple NMDARs from nNOS signaling to circumvent opioid reward without unwanted side effects of global NMDAR antagonists or nonselective NOS catalytic inhibitors. We propose to accomplish this objective by selectively disrupting the protein-protein interface between nNOS and postsynaptic density 95kDA, a scaffolding protein which tethers nNOS to NMDARs. Aim 1 will test the hypothesis that disruption of PSD95-nNOS protein-protein interactions will suppress morphine-induced reward using conditioned place preference and drug self- administration approaches. Aim 2 will test the hypothesis that disruption of PSD95-nNOS interactions will attenuate opioid-induced dopamine dynamics in the nucleus accumbens shell, a key component of the reward circuit. Disruption of protein-protein interactions was once considered an impossible target for drug development. Completion of these high risk, high impact studies will establish the feasibility of disrupting the PSD95-nNOS interface to eliminate opioid reward, while retaining therapeutic efficacy, bypassing unwanted side effects of both NMDAR antagonists and NOS catalytic inhibitors. Validation of our hypotheses will provide a strong rationale for undertaking lead optimization of PSD95-nNOS inhibitors for advancement toward clinical studies in opioid addiction, filling a major gap in an area of unmet therapeutic need.