Research Projects

The projects were selected on the basis of their scientific merit and their capacity to provide convergent and synergistic information in relation to the four over-arching hypotheses, around which the CTNA is organized.

OVERVIEW of CTNA YEAR 4
6/01/04 to 5/31/05

The Center for the Translational Neuroscience of Alcoholism (CTNA) has made enormous progress in all projects and pilots during the past year. Interim analyses provided sufficient information for us to conceptualize the next steps. The CTNA team met in a retreat at the beginning the Year 4 to start plans to a renewal application that was submitted in December 2004. The original theme of GABA-GAD based mechanisms that ties all the projects together in this grant cycle (called CTNA-1) was advanced to study mechanisms through which disturbances in glutamate and dopamine neurotransmission within cortico-limbic circuitry contribute to the vulnerability to persistent heavy drinking and alcohol dependence (CTNA-2). We will spend this final year of CTNA-1 completing the original projects and preparing for foll-on studies to be accomplished in CTNA-2. We will continue the transdisciplinary research within projects and between projects, and continue the highly productive Pilot Projects Core.
CTNA investigators have been very productive since our new start-up in June 2000. The group already has published numerous papers in leading journals, as well as presented material at many scientific conferences. As described in the Other Support sections of their Biographies, the investigators have generated extensive grant support from NIAAA, NIDA, VA and other agencies to sponsor a variety of alcohol research projects. Many of the collateral studies have benefited from the Core resources and structure of the CTNA. As in past years, CTNA Investigators will have a major presence at Research Society on Alcoholism meeting in June 2005 with workshops, presentations, and abstracts representing our progress to date.

Severl projects have made great strides toward understanding glutamate and dopamine neurotransmission. Dr. Eric Nestler and his colleagues identified novel molecular mechanisms underlying the rewarding effects of alcohol and other drugs of abuse and he linked these mechanisms to the propensity to self-administer these substances. This work highlighted a novel transcription factor, DFosB, that modulated glutamatergic neurotransmission. This work drew the CTNA toward consideration of glutamatergic neuroplasticity, particularly, links between addiction and learning.

CTNA expanded the Pilot Study led by Jane Taylor, Ph.D., by recruiting Patrick Allen Ph.D., another leading expert in the neurobiology and psychopharmacology of motivation/addiction, to probe the role of motivational circuitry in alcohol administration and related impulsivity. Building on pioneering research, CTNA focused on the role of glutamatergic (OFC, amygdala, hippocampus, thalamus) and dopaminergic (VTA) inputs to the nucleus accumbens (NAc) that converge on the dendritic spines of GABA neurons. These networks process shifts in environmental reward- and punishment-related contingencies and they generate motivational states. Thus, glutamate and dopamine systems are implicated in the acquisition and maintenance of motivational discriminations based on reward and punishment magnitude, delays between cues and reward, and the regulation of how "hard" animals will "work" for natural rewards and drugs of abuse. This literature suggests that addiction liability in animals is heightened by the convergent enhancement of NMDA and dopaminergic (D1 receptor>D2 receptor) receptor function. Further, this hypothesis implicates glutamatergic and dopaminergic signal transduction pathways and synaptic integrity in processes contributing to alcoholism development.

The CTNA Executive Committee meets regularly, usually combined with Investigator meetings. Investigators have been working with the Data Management and Biostatistical group to complete entry of current data nd perform analyses. The Data Safety Monitoring Board meets twice-yearly to review progress of the clinical projects within the Center. The Local and Regional Education Committees have sponsored or co-sponsored several scientific and educational symposia. A highlight was the 2004 annual Jellinek Lecturer, Nora Volkow, NIDA Director, who spoke about: "Why does the brain become addicted."

The Executive Committee has reviewed ongoing projects and considered proposals for new Pilot Studies. Dr. Godfrey Pearlson will be funded during year 5 to initiate pilot work that will be useful for the project he proposed for CTNA-2.

CTNA Administrative Core:
The support structure offered to individual projects is based in the Administrative Core.
Data Management & Biostatistical Center (DMBC):
     The Data Management and Biostatistics Center (DMBC) had been based at the Department of Veterans Affairs Cooperative Studies Program (West Haven). During the past year, CTNA has transitioned to a new system based at the Yale Center for Medical Informatics (YCMI) (http://ycmi.med.yale.edu/). Data now will be entered using a web-based system (Trial/DB) that was developed by YCMI. CTNA is fortunate to collaborate with YCMI as a service of the General Clinical Research Center (GCRC) led by YCMI Assistant Director Cynthia Brandt MD, MPH. Dr. Krystal and other investigators have used GCRC facilities and services for many years. YCMI uses highly productive methodology for data management that serves all types of data management functions associated with clinical research. They are experienced in study design, preparation of study forms, management of data, preparation of reports related to the progress of each study on a twice yearly basis for the Data Safety Monitoring Board and preparation of annual progress reports to NIAAA and Scientific Advisory Board. Trial/DB (formerly called ACT/DB) is a powerful, flexible, Web-accessible database designed to support clinical trials and clinical research and has been used extensively at both a local and a national level. Trial/DB is currently being used at Yale to support clinical trials and clinical research in several clinical domains, including cancer, cardiology, endocrinology, and psychiatry. Several Yale-based trials involve multi-institutional collaborations where data is entered from multiple sites nationwide, using Trial/DB's Web interface with appropriate security and confidentiality safeguards. Web data-entry interface to Trial/DB allows data entry to be performed from anywhere on the Internet and uses 128-bit secure sockets layer (SSL) security to protect the confidentiality of the data, under HIPPA guidelines. CTNA will be able to share this system with other NIAAA Centers within the next year.
     This transition to TRIAL/DB also led to a change in statistical support. We are fortunate to have recruited Ralitza Gueorguieva Ph.D. to CTNA. She has worked in the Department of Psychiatry with CTNA investigators for several years on a variety of projects. Addition of CTNA projects represents familiar concepts for her while providing investigators with opportunities to use her skills at longitudinal analyses.

Data Safety Monitoring Board (DSMB):
The DSMB continues to review all clinical programs to assure patient safety. Under the thoughtful leadership of Robert Swift MD, PhD, Chairman, the DSMB also considers whether projects have answered their proposed scientific aims or are unlikely to be concluded successfully, thus placing subjects at avoidable potential risk. The Data Management & Biostatistical Center provides detailed reports to the DSMB on safety and efficacy for each clinical study. Members are: Robert Swift MD, PhD, Chairman (clinical researcher, Brown Univ.), Robert Hitzemann, Ph.D. (basic science researcher, Univ. Oregon), Lisa Newton, Ph.D. (ethicist, Fairfield Univ.), Robert Stout PhD (statistician), Howard Zonana MD (forensic psychiatry and bioethics, link to Yale IRB). The DSMB fully supported the renewal application for CTNA-2.

Scientific Advisory Board (SAB):
The SAB met in July 2004 to provide critical and helpful comments on all projects and pilot studies to assure that the transdisciplinary goals of the CTNA were being achieved. SAB members (Charles O'Brien MD PhD, John Crabbe, PhD, Ivan Diamond, MD, Ph.D., Kirk Frey MD PhD, David Goldman, MD, Kathleen Grant, PhD, Victor Hesselbrock, .D, Perry Renshaw, M.D., PhD) also provided valuable advice on how to restructure for the CTNA-2 renewal application.

Local/Regional Education Committee (LEC/REC):
The highlight of CTNA educational activities since its inception was the successful International Conference on Applications of Neuroimaging to Alcoholism (ICANA) supported by NIAAA in January 2004. CTNA Investigators continue to reap the benefits from the associations they made with international colleagues during that event. The CTNA web site (http://info.med.yale.edu/CTNA) includes information describing the purpose and background of CTNA projects, information for research professionals, clinical professionals, and the lay public. The site also is used to recruit subjects for studies. The website (http://info.med.yale.edu/CTNA/ICANA) was the focal point for information about ICANA. We have uploaded slide and videotapes of selected ICANA presentations to the site to disseminate these educational materials beyond those who were able to attend the sessions.
Other educational activities included the Annual Jellinek Symposium: Nora Volkow, PhD, Director NIDA who spoke on: "Why does the brain become addicted." A videotape of this lecture also is posted on the website: http://info.med.yale.edu/ctna/training.html.

CTNA Clinical Core (Stephanie O'Malley PhD):
The Clinical Core continues to support clinical assessment tools be used throughout CTNA studies, maintenance of reliability of clinical assessments, and support for recruitment of subjects into clinical studies. A decision was made this year to discontinue use of the SSADDA interview throughout CTNA because it is too cumbersome for diagnostic purposes. Investigators need a DSM diagnosis quickly whereas the SSADDA is not easily scored. The designation of family history of alcoholism is made with the FHAM instrument that also is easily administered and scored. Thus, the position of SSADDA interviewer will be discontinued during year 5. Those funds will be reallocated to support pilot studies.

This Core has four components:
1. Ligand Development (directed by Marc Laruelle MD) is complete with the selection of raclopride for future PET imaging studies. Dr. Laruelle will conclude this work at the end of year 4. The effort to develop imaging agents for the glutamate system has proceeded with a focus on the NMDA (glycine sites and NR2B) and AMPA receptors.

A. NMDA receptors.
Efforts are ongoing to develop radiotracers for the glycine site of the NMDA receptor and the NR2 subunit. We have proposed to synthesize compound C, which has an in vitro binding affinity of 1.6 nM (Ki) for the NMDA glycine site, as a potential PET imaging agent for the glycine site. Preparation of this compound is progressing. We have synthesized the cold standard A and the radiolabeling precursor B according to the synthetic pathway. Radiosynthesis of compound C will commence soon.

B. NR2B subunit.
We have proposed to synthesize compound D, a selective NR2B antagonist with a Ki of 0.7 nM, and test its binding in vivo. The standard (E) and precursor (F) were prepared as described in Scheme 2. The radiolabled compound D was prepared from its precursor by reaction with C-11 methyl triflate in high radiochemical yield. Compound D was tested in a baboon. Unfortunately, very little uptake was detected in the brain. We are continuing to search for appropriate ligands to label the NR2B subunit of the NMDA receptor in vivo.

C. AMPA receptors
We proposed to prepare a series of positron-labeled analogs of CP-465022 and evaluate their potential for imaging AMPA receptors in vivo using PET. A. To date, we have successfully prepared the [C-11]-methoxy derivative of CP-465022 (compound 1). Preliminary PET study in baboon showed that [C-11]-1 displayed homogenous brain uptake. Blocking studies with CP-465022 and the unlabelled compound 1 are in progress. B. The preparations of dimethyl-derivative of CP-465022, compound 2, has been achieved in our laboratory and the preparation of corresponding desmethyl precursor for C-11 labeling of 2 is in progress. Various attempts to prepare the F-18 labeled derivative, compound 3, were not successful. C. In addition, efforts are being made to prepare [C-11]-labeled CP471236, which has recently been demonstrated as a potent ligand to study AMPA receptors. We have prepared the key intermediate 5, which is needed for preparing the trimethyltin substituted CP471236 derivative. Once compound 6 is available, it will be used for the preparation of [C-11]-CP471236. We also propose to prepare [F-18]-labeled compound 4, according to the reactions. Once the tracers are successfully prepared, they will be evaluated in vitro and in vivo.

2. Genetic studies (directed by Jaakko Lappalainnen MD, PhD). DNA samples are being collected from subjects enrolled in all clinical CTNA projects. Subjects also are recruited for assessment of Axis 1 diagnoses and family history of alcoholism. Family members who are heavy drinkers are included in testing for trios.

3. Clinical Electrophysiology (directed by Daniel Mathalon PhD, MD)
The section supports evoked potential recordings for clinical studies. This continues to be an important core resource.

4. Recruitment Support (directed by Stephanie O'Malley PhD) is an ongoing process of updating referral lists among Research Assistants so they can determine which of the many projects would be most suitable for any person who responds to an advertisement. The system has utilized a trained Recruiter who used a mobile telephone to answer calls during evenings and weekends to supplement calls taken during the work day. Advertising as a group has avoided duplication of efforts and competing advertisements. Posters have been placed at local colleges to attract healthy, young volunteers for laboratory studies. Newspaper advertisements remain the best source for heavy drinkers.

CTNA Molecular Core (Joel Gelernter MD):
The Molecular Core supports basic marker testing, marker panel preparation and testing, and creation of new markers, in support of greater genotyping effort in patient populations. The Director together with the Scientific Directors decided to rebudget approximately 40% of the funds that were to be devoted to this purpose, and some functions have been moved into Dr. Gelernter's project (Project 2). Accordingly, the scope of the work in the Molecular Core has been reduced, and now excludes genotype creation in large samples of alcohol dependent subjects.

Collaborative work with other CTNA Investigators:
     The Molecular Core has taken on a role in leading collaboration with other CTNA investigators, in identifying candidate variants for study and implementing DNA preparation and genotyping. CTNA investigators routinely collect blood for DNA extraction for investigating genotype-phenotype correlations. We have started to develop preliminary results from these collaborations; one example is given below.

Family History and PPP1R9B (Spinophilin) Genotype Influence on NMDA Receptor Function
Dr. Petrakis has collected DNA from all subjects participating in CTNA studies. This challenge employs a 60 minute challenge; the outcomes include measures of ethanol effects, ERP measurements and genotyping. To date 16 individuals have completed this ongoing study, demonstrating the feasibility of the proposed research. Ketamine responses were compared in healthy subjects, between the ages of 21-30, with a family history of alcoholism (FHP) (n=5) and healthy control subjects without a family history of alcoholism (FHN) (n=19). Subjects completed two test days involving a 60-minute intravenous infusion of each of 2 conditions: saline and ketamine 0.812 mg/kg, in a randomized order under double-blind conditions. Subjects had blood drawn for DNA extraction and genotyping, and outcomes included the Biphasic Alcohol Effects Scale (BAES), visual analog scales (VAS) for high, similarity to ethanol and mood states, the Sensation Scale (ethanol-like sensations) and the on prefrontal cortical (PFC) function as measured by P300 amplitude. We genotyped marker PPP1R9B rs847680. This study suggests there is a significant difference in ketamine response by spinophilin genotype.

PROGRESS REPORTS FOR PROJECTS AND PILOTS

Project 1: Transcription Factors, Glutamatergic Function, Ethanol Reward, and Adaptation to Chronic Ethanol (Dr. Nestler). May 2005 progress report.
Dr. Nestler has focused his work on a systematic mapping of dFosB induction in brain by chronic alcohol administration, using several methods of alcohol exposure. He has characterized the specific cell types within these regions where this alcohol induction of dFosB occurs, in the search for target genes for dFosB, and hence for alcohol. The data indicate that alcohol-induced accumulation of dFosB in nucleus accumbens, dorsal striatum, amygdale, and prefrontal cortex. Working in collaboration with George Koob (Scripps Research Institute) and Adron Harris (University of Texas at Austin), they found that dFosB may reduce the effects of alcohol on balance, while enhancing the anxiolytic effects of alcohol.
He has asked for a reduced budget while work is completed during year 5. This completed project will not be continued in CTNA-2.

Project 2: Guided Family-Controlled Linkage Disequilibrium Scan for Alcohol Dependence and PFC-Related Endophenotypes (Drs. Gelernter, Lappallainen) May 2005 progress report.
Dr. Gelernter will continue SNP-based fine-mapping of regions of chromosome 1 and 11 that show LD with alcohol dependence. He will continue collaboration with other CTNA investigators to identify genetic association with EtOH-related endophenotypes. He also will add new SNFs to the genotyping panel as they are recruited through the CTNA clinical core.

Project 3: A Simultaneous Assessment of PFC Gray Matter Glutamate Turnover and Glucose Metabolism in Alcoholism with [13C]MRS (Dr. Mason). May 2005 progress report.
Dr. Mason continued to develop the 13C and 1H MRS GABA acquisition measurements over the past year. They are able to measure the rate of GABA synthesis in individual subjects.

Project 4: Presynaptic and Postsynaptic 5-HT Receptor Abnormalities in Alcoholism: A PET Study (Dr. Abi-Dargham). May 2005 progress report.
Dr. Abi Dargham's protocol focuses on a comparison of 5HT transporter binding potential (BP) between alcohol dependent subjects and healthy controls. It combines [11C]WAY100635 and [11C]DASB, as a superior tracer for the 5HT transporter Alcohol dependent subjects were found to have a decrease in D2 receptor availability in the ventral and dorsal regions of the striatum compared to healthy controls and the reduction in striatal D2 receptors correlated with the daily intake of alcohol. Although there was a temporary closure of the PET Center, they had already completed data acquisition. The down-time has been used for data analysis. This project will be expanded in CTNA-2

Project 5: Altered NMDA Receptor Function with Familial Alcoholism Risk (Dr. Petrakis). May 2005 progress report.
Dr. Petrakis found that people with a family history of alcoholism (FHP) have altered sensitivity to the behavioral effects of ethanol, and are at higher risk of developing alcoholism, relative to healthy individuals without a family history (FHN). She found that FHP individuals have an altered response to the NMDA antagonist, ketamine, compared to FHN individuals. A follow-up study evaluating an IV ethanol challenge versus placebo using an IV alcohol clamping method is in progress. This project will be expanded for CTNA2.

Project 6: Differential Interference with Ethanol Self-Administration by Naltrexone in Alcoholic Individuals With or Without Familial Alcoholism (Dr. Krishnan-Sarin). May 2005 progress report.
Dr. Krishnan-Sarin is completing this project with the enrollment of additional family-history positive drinkers. This additional effort will balance the sample sizes between groups to validate the initial findings using the laboratory paradigm for detecting the effects of opiate antagonists on alcohol drinking. She has found that the effect of naltrexone on drinking occurs primarily in FHP subjects. This project will be expanded to include memantine as a treatment group for CTNA2.

PILOT CORE
CTNA Pilot Projects: Request for Proposals Instructions.

Pilot Project 2: Neural circuitry of P300 abnormalities in alcoholics: relationship to alcohol cue reactivity (Dr. Mathalon). May 2005 progress report
Dr. Mathalon has concluded this pilot project with the successful award of an ABMRF grant. Pilot results showed that automatic processing of brief, task-irrelevant, alcohol cues activates craving-related circuitry in alcohol dependent subjects more than in healthy controls. An attentional bias may be mediated by cue-craving circuitry invoked subsequent to initial orienting of attention. This study will conclude a the end of year 4, to be followed by a full study under the ABMRF grant.

Pilot Project 3: Alcohol effects on information processing and behavioral control (Dr. Corbin). May 2005 progress report.
Dr. Corbin is studying the effects of alcohol on inhibition of behavioral response, and the relation between behavioral inhibition and risk factors for alcohol-related problems. He found a trend toward alcohol impairment on sensitivity to the stimuli presented in the task (go and stop signals). This effect is apparent despite attempts among participants in the alcohol condition to slow themselves down to compensate for impairment. He is preparing a follow-up study using a different easure of behavioral inhibition with a slot machine task. This Pilot Study will have continued support to collect data toward submission of a full proposal. He is collaborating with Dr. Pearlson and Dr. Potenza in the development of a new project for CTNA2.

Pilot Project 5: Behavioral and molecular consequences of chronic ethanol administration in rat (Drs. Taylor & Allen). May 2005 progress report.
Dr. Taylor is examining how molecular adaptations in the mesocorticolimbic structures contribute to neurocognitive effects of ethanol. Current studies focus on spinophillin, the controller of protein phosphatase 1 (PP1) that is an antagonist of NMDA activity through inhibition of channel conductance. Working with a new collaborator, Patrick Allen PhD, they will investigate how regulatory mechanisms affect incentive learning and cognitive/ibhibitory functions in alcoholism. This concept of "impulsivity" will be a main theme in CTNA2, carried through from this animal model to humans.

Pilot Project 6:[123I]5-IA-85830 SPECT IMAGING OF ß2-NICOTINIC ACETYLCHOLINE RECEPTORS IN ALCOHOLISM (Dr. Staley). May 2005 progress report.
Dr. Staley has studied whether thalamic and cortico-limbic ß2 nicotine acetylcholine receptors (ß2~nAChR) are increased during sobriety in alcohol-dependent nonsmokers with a family history of alcoholism. She is using SPECT imaging of ß ß2~nAChR to assess adaptations during the early development of sobriety. This pilot will be continued during year 5.

Summary

This fourth year of CTNA has been highly productive in all Projects. We started to prepare for the renewal application six-month in advance. The first activity was a Scientific Advisory Board where Investigators proposed projects and heard critiques. Always mindful that we are a Center, each Investigator demonstrated the associations among and between projects and use of Core facilities. The submission of the CTNA-2 proposal at the end of 2004 brought us back to regular Investigator meetings for discussion of how to bring all current projects to successful conclusions while preparing for the next stages.