Joseph B. Justice - US grants

In order to understand the relationship of dopamine (DA) release to behavior, studies in which DA release, or an index of DA release, is monitored in intact animals are needed to complement studies using lesions of dopaminergic systems to produce behavioral deficits. One approach to the problem of monitoring DA release has been to use metabolite levels as indices of DA release. In order to use metabolite information effectively, the relationship of the extracellular levels of the metabolite to neurotransmitter release must be known. In the present proposal a model of the relationship of extracellular dihydroxyphenylacetic acid (DOPAC) to dopamine release is described and several experiments to test the model are proposed. In particular, in vivo voltammetry will be used to determine the kinetics of dopamine metabolism to extracellular DOPAC. This approach represents a new, nonpharmacological method for studying the regulation of neurotransmitter release. Rate constants for formation of DOPAC and its clearance from the extracellular space will be determined. This will be done by nonlinear regression on voltammetric data representing the increase in extracellular DOPAC following very brief electrical stimulation of the nigrostriatal dopaminergic pathway. The voltammetric data will be validated by high performance liquid chromatography of the extracellular fluid. The relative time course of dopamine release and change in extracellular DOPAC will be examined with regard to the use and limitations of using metabolite information as a temporal index of neurotransmitter release. Additionally, fundamental information on the dynamics of the intact nigrostriatal dopaminergic system will be obtained that is not possible to acquire by any other method.

One of the major issues in neuroscience is tying what we know about chemical messengers in the nervous system to actual behaviors. This project combines the expertise of investigators in the chemical and biological sciences to address the problem of the relationship of neurotransmitter release and behavior. The most studied neurotransmitter in the central nervous system is dopamine. Although the role that dopamine plays in behavior has been the subject of intensive investigation, its specific role in a number of behaviors is still not understood. Part of the reason for the slow progress has been the absence of methods which allow observation of dopamine release during behavior. Drs. Joseph Justice and Darryl Neill have developed new techniques which will be utilized to monitor neurotransmitter release in behaving animals. In particular the relationship of dopamine release and reward will be investigated, using intracranial self-stimulation, drug self-stimulation and food reward. Behavioral studies have indicated a role of dopamine in the reward of these three different stimuli. Direct observation of the neurochemical substrate thought to mediate these behaviors will significantly enhance our understanding of dopamine release and behavior.

Dopamine release in the nucleus accumbens (N ACC) has been shown to primarily mediate the reinforcing effects of cocaine and amphetamine. Using in vivo microdialysis procedures the proposed work is designed to quantify the extracellular concentration of dopamine (DA) in the N ACC during intravenous drug self- administration. The proposed project consists of two self- administration studies. The first study is designed to quantify DA concentrations maintained during self-administration of three doses of cocaine (0.25, 0.5 and 0.75 mg/inj). The aim of the study is to determine if the animals which self-administer different doses of cocaine will maintain DA concentrations at similar or different levels. The aim of the second study is to compare levels of DA maintained during cocaine, amphetamine and morphine self-administration. The second study will provide detailed information about the effects of a variety of abused upon the concentration of DA in the N ACC. As would be expected, administration of a drug which blocks the reuptake of DA (i.e., cocaine) should increases the extracellular concentration of DA. Furthermore, the administration of cocaine in short regular intervals should maintain the concentration of DA at some level above basal values. The emphasis of the proposed work is not to demonstrate that the above occurs. The major aim of the proposed projects is to quantify the concentration of DA in the N ACC that animals maintain over time during cocaine self-administration. Once the threshold level at which the DA concentration stabilizes during cocaine self-administration is quantified the information can be compared to DA levels maintained during amphetamine and morphine self-administration. These experiments will provide detailed information about the actions of a variety of abused drugs upon the concentration of DA in the nucleus accumbens. Future research can use this information to assess the effects of dependence, experience, withdrawal, tolerance, and also acquisition of drug self-administration. Moreover, the effectiveness of pharmacologial methods used to treat patients who abuse drugs can be evaluated. Quantification of DA thresholds in the nucleus accumbens during self-administration has the potential to provide immediate information and can be used to direct future studies.

This award will support collaborative research between Dr. Joseph Justice, Emory University, and Dr. Urban Ungerstadt, Department of Pharmacology, Karolinska Institute, Stockholm, Sweden. The objective of the research is to investigate dopamine neurochemistry and to better characterize microdialysis, a new method of monitoring the chemistry of dopamine and other neuro-transmitters, metabolites and related compounds. The two laboratories cooperating in this project are the leading ones involved in the rapidly emerging technique of in vivo dialysis. This technique has already been shown to be useful to examine the chemical composition of the extracellular fluid of the brain. The proposed research will continue the investigation of the effect of dialysis on the surrounding tissue, investigate ways to relate the chemical information obtained to the synaptic region, and specifically examine the role of uptake of catechola- mines in dopaminergic neurons. The project will involve mathema- tical modeling of the dopaminergic nerve terminal. New data from the two laboratories will be incorporated into the model. Uptake and inhibition of uptake will be a particular focus of effort. In addition, the microdialysis sampling methodology in use in both laboratories to study dopamine will be characterized. The charac- terization will consist of a mathematical analysis of the diffusion profiles created during sampling of the extracellular fluid. This will provide important new information about a technique that is being increasingly used in many laboratories in the United States and Europe.

There are noticeable differences in the amount of drug exposure necessary for individual animals and humans to become addicted. A subject's locomotor response to a novel environment appears to predict vulnerability to psychomotor stimulants. The proposed experiments are designed to determine the specific neuroanatomical areas of the mesolimbic system responsible for individual differences in vulnerability to amphetamine. The areas of the mesolimbic system to be examined are the ventral tegmental area (VTA), nucleus accumbens (NACC), and medial frontal cortex (MFC) since they primarily mediate the behavioral responses to drugs such as amphetamine and to novelty. The role of dopamine in these regions in determining individual vulnerability to amphetamine will also be examined. The proposed project consists of one behavioral and two neurochemical experiments to determine individual vulnerability to drugs of abuse. The first experiment uses behavioral measures following repeated intracranial administration of amphetamine and a subsequent systemic challenge to determine which neuroanatomical structures are most responsible for differences in sensitization to the drug. The two neurochemical experiments are designed to use in vivo microdialysis to determine differences in dopaminergic basal state and responsiveness in the VTA, NACC, and MFC. These experiments will provide detailed information about the role of neuroanatomical and neurochemical basis of vulnerability to drugs of abuse.

DESCRIPTION (provided by applicant): Knowledge of the structure and mechanism of the dopamine transporter is an important step in understanding this key membrane bound protein in dopaminergic neurotransmission. While dopamine and the related neurotransmitters norepinephrine and serotonin are implicated in several mental disorders with severe social impact, the structure and mechanism of the key proteins involved in their signaling remain elusive. One example for the application of such knowledge is the development of medications to treat cocaine abuse, an important goal in addressing the national problem of drug abuse. In order to develop new drugs which prevent the binding of cocaine to its target, the dopamine transporter, it would be very useful to know where both cocaine and dopamine bind on the transporter protein. One approach to localization of the binding site for cocaine has been to explore the structural features of cocaine analogs that affect binding to the transporter. An extension of this approach has been to develop inhibitors which bind irreversibly to the transporter. This class of compounds reacts with the protein to form a covalent bond between the inhibitor and the protein. Knowledge of where this bond is formed, that is, which amino acid in the sequence is involved, allows the binding site to begin to be localized. Mutagenesis studies can then further probe the region. With a range of irreversible inhibitors, including irreversible cocaine analogs, a map of the cocaine binding site can be constructed. To identify the substrate binding region, analogs of the substrate dopamine, will be used that irreversibly react with the transporter. Thus regions important in both inhibition and transport will be identified. The combined results will help to construct a picture of the ligand binding regions critical for human dopamine transporter function and inhibition. Without adequate structural information, it is not possible to understand the mechanism of this important membrane bound protein when functioning normally or in response to acute or chronic drugs of abuse. Identification of domains, and residues within domains, involved in substrate and inhibitor binding is an important first step in understanding the mechanism of this and related transporters.

9412703 Justice Much of our understanding of how the brain controls behavior depends upon our ability to accurately describe how cells within the brain communicate with one another. Some general information on this process has been obtained by removing brain tissue and analyzing it chemically, but this provides no information on the dynamics of chemical signalling that actually takes place during episodes of behavior. With this grant, Dr. Justice will refine techniques for measuring the flux of chemical neurotransmitters within the brain during bouts of natural behavior. Dr. Justice's group will concentrate upon the dynamics of the neurotransmitter dopamine. This work will make it possible to describe the chemical dynamics of the dopamine system (and also, to some extent, serotonin and acetylcholine) in a precise way that is directly correlated with organismic behavior. It should greatly stimulate our understanding of normal brain function, the action of several classes of drugs on the brain, and the processes associated with abnormal function of neurotransmitter systems. ***

DESCRIPTION: (Applicant's Abstract) The transporters for dopamine, norepinephrine, and serotonin are targets for drugs of abuse such as cocaine and amphetamine, and for therapeutic drugs such as the antidepressants. The structure and mechanism of these membrane spanning protein remains unknown. However, the recent cloning and expression of these transporters has allowed progress to be made in better understanding how these proteins work. The proposed experiments build on this recent progress by combining the developments in molecular biology with recent advances in electrochemical measurement technology. The kinetics and mechanism of the human norepinephrine transporter will be examined with rapid voltammetric methods. Hypotheses concerning complimentary alterations in substrate structure and mutation in the transporter will be tested. Interactions of the amino group of the substrate with an amino acid residue in transmembrane region one constitute one set of experiments. Interactions of the ring hydroxyls with residues in transmembrane region seven compromise a second set of experiments. An additional experiment is designed to characterize the transport characteristics of a substrate designed to act as photoaffinity label for probing the active site of transport.

DESCRIPTION(Adapted from applicant's abstract): Development of medications to treat cocaine abuse is an important goal in addressing the national problem of drug abuse. In order to develop new drugs which prevent the binding of cocaine to its target, the dopamine transporter, it would be very useful to know where both cocaine and dopamine bind on the transporter protein. One approach to localization of the binding site for cocaine has been to explore the structural features of cocaine analogs which affect binding to the transporter. An extension of this approach has been to develop inhibitors which bind irreversibly to the transporter. This class of compounds reacts with the protein to form a covalent bond between the inhibitor and the protein. Knowledge of where this bond is formed, that is, which amino acid in the sequence is involved, allows the binding site to begin to be localized. The proposed work uses mass spectrometry to identify the sites of attachment of the irreversible ligands at the human dopamine transporter. With a range of irreversible inhibitors, including irreversible cocaine analogs, a map of the binding site can be constructed. In addition, analogs of dopamine, the substrate for the transporter, that irreversibly react with the transporter will be used to identify the substrate binding region. Thus, regions important in both inhibition and transport will be identified. The combined results will help to construct a picture of the ligand binding regions critical for human dopamine transporter function and inhibition. Without adequate structural information, it is not possible to understand the mechanism of this membrane bound protein when functioning normally or in response to acute or chronic drugs of abuse. Identification of domains involved in substrate and inhibitor binding is an important first step in understanding the mechanism of this and related transporters.

With support from the Chemistry Research Instrumentation and Facilities (CRIF) Program, the Department of Chemistry at Emory University will acquire a Circular Dichroism Spectrometer. This equipment will enhance research in a number of areas including a) the design of polypeptide materials of precise and controllable molecular structure (Conticello); b) studies on flavoenzyme systems (Edmondson); c) peptide structure and self-assembly (Lynn); d) transition metal-DNA interactions (Marzilli); and e) bio-organic chemistry of protein-DNA complexes (Mohler).

Circular dichroism spectroscopy is an extremely useful tool in modern analytical chemistry. It provides a very reliable and sensitive method for assigning absolute molecular configurations. The results from these studies will have an impact in a number of areas, in particular, biochemistry.

Basing their work on their expertise in science and mathematics, teams composed of teachers, and graduate and undergraduate Fellows are developing new materials and adopting and adapting existing materials for problem- based learning (PBL) and interactive case based learning (ICBL) cases that integrate grade appropriate science and math content into locally relevant classroom modules. The Fellows are assisting teachers in introducing and testing these cases and problems in a variety of middle and high schools in the Atlanta area. In preparation for this work, the teams are participating in workshops on PBL and ICBL pedagogy, as well as teacher- designed introductions to urban education. Each team is collaborating on developing plans that include a needs assessment, an action plan, an implementation plan and a communication plan. The teams are also providing professional development for other teachers in the school. The broader impacts of these activities include benefits to the Fellows, the teachers and the K-12 students involved. The Fellow are gaining the skills and knowledge necessary to: engage in active learning teaching pedagogies and reflective practice; practice the teamwork required for interdisciplinary science collaboration that leads to success in modern scientific research; communicate science effectively to a broader audience; and to include outreach activities in their career goals when they are practicing scientists. The teachers are enhancing and updating their Math /Science content knowledge and are sharing this knowledge with other teachers. They are benefiting as well from enhanced articulation and communication between middle school and high school teachers and between teachers and college faculty and students. The high school and middle school students involved are benefiting not only from the classroom modules being developed but are also interacting with role models to whom they can relate, leading to improved student interest in science and mathematics careers. Part of the support for this project comes from the Directorate for Mathematics and Physical Sciences

Title: Problems and Research to Integrate Science and Mathematics (PRISM)
Institution: Emory University
PI/Co-PI: Joseph B. Justice; Patricia Marsteller, Preetha Ram
Partner School Districts: DeKalb County, City Schools of Decatur, Fulton County, Atlanta Public
Funding: $1,493,055
# of Fellows/yr: 10 Graduate, 10 Undergraduate
Setting: Urban
Grade Band: middle-high school
Disciplines Affected: Science and Mathematics

With support from the Chemistry Research Instrumentation and Facilities (CRIF) Program, the Department of Chemistry at Emory University will acquire an isothermal titration calorimeter and a differential scanning calorimeter. This equipment will enhance research in a number of areas including a) engineering and structure/function studies of proteins; b) binding of reactant, inhibitors and coenzyme to B12-dependent ethanolamine deaminase; c) design of self-assembling peptide materials; d) template-directed polymer synthesis; and e) exploration of colloidal systems.

Calorimetry is an extremely useful tool in modern analytical chemistry. Calorimetric analysis is used to measure the thermal energy (heat) exchange that occurs during molecular interactions and reactions. Thus it can provide a very reliable and sensitive method for determining the thermodynamic properties of materials such as changes in heat capacity of liquid and solid samples.

This proposal describes a Track 2 project that builds on 15 years of experience at establishing a sustainable community partnership that enhances Emory's research, teaching and service missions. The primary goal of PRISM II is to fully institutionalize the partnership established by PRISM I. Specifically, the objectives are to engage teachers and graduate fellows (GF) in learning pedagogies and reflective practice, enhance skills in teamwork and research practices, improve skills in communication with the broader public, improve content in science and mathematics, and increase the number of role models available for students and teachers. To accomplish these goals and objectives, Emory will partner with Clark-Atlanta University, Atlanta Public Schools, DeKalb County School System, City Schools of Decatur, and Fulton County Schools. The project will target ten teacher-GF teams per year with the potential to impact hundreds of students.