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-2, YEAR 7

6/01/07 to 5/31/08

The purpose of Center for the Translational Neuroscience of Alcoholism (CTNA) is to further basic and clinical approaches to understanding alcoholism in an integrated, translational program, and significant progress toward this goal was made during this second year of the Center’s renewal .  The original theme of GABA-GAD based mechanisms that tied all the projects together during CTNA-1 was advanced to characterize how disturbances in glutamate and dopamine neurotransmission within cortico-limbic circuitry contribute to the vulnerability to persistent heavy drinking and alcohol dependence during CTNA-2. Transdisciplinary research within projects and between/among projects and pilots has continued during this year. The Core elements have provided centralized support for all investigators, thereby facilitating relationships among teams.

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. These themes form the basis of the transdisciplinary work of the CTNA projects.

CTNA investigators have been very productive with publication of numerous papers from earlier studies, as well as presented material at many scientific conferences. As described in the Other Support sections, 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 2008 with workshops, presentations, and abstracts representing our progress to date.

  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 and perform analyses.

Second International Conference on the Applications of Neuroimaging to Alcoholism

The Second International Conference on the Applications of Neuroimaging to Alcoholism (ICANA-2) was the crowning achievement of the year. Sponsored by a conference grant from NIAAA, ICANA-2 was organized by CTNA Investigators to disseminate knowledge about the effects of alcohol on the brain from development to maturity.

John Krystal noted:

The field of alcoholism research is at a watershed, where advances in basic molecular research await the translation into important new clinical insights. Neuroimaging technologies are the most powerful new tools to characterize human brain structure, circuitry, function, and chemistry. They will play a critical role in the translation from basic insights into advances in treatment and prevention. Thus, it is timely to bring together the leading neuroimaging investigators from around the world to take stock of the advances in neuroimaging research and their applications to alcoholism.

ICANA-2 presented an outstanding array of neuroimaging research during two days of presentations. Equally important, it provided many opportunities for formal and informal scientific exchange.

We are confident that exciting new alcoholism research will grow from the camaraderie generated by this meeting.

Dr. TK Li, Director of NIAAA, welcomed attendees with these comments:

The NIAAA is committed to increasing the understanding of normal and abnormal biological functions and behavior related to alcohol use and abuse. To accomplish this, the NIAAA Strategic Plan emphasizes the need for research spanning the domains from genetic and molecular/cellular factors through to the study of environmental effects, with the understanding that these factors may impact drinking across the lifespan. Imaging can provide an important research tool to investigate questions related to the development of alcohol tolerance and dependence, organ damage, for translational research from animal experimental models to humans, and ultimately to clinical diagnosis and treatment. For example, positron emission tomography and magnetic resonance spectroscopy can offer insights into brain biochemistry underlying addiction, while structural magnetic resonance and diffusion tensor imaging can provide information on the effects of alcohol on brain structure. Imaging modalities vary in their spatial and temporal resolution and differ in their utility to measure various neural processes such as synaptic signaling and circuit formation. There is a need to increase spatial resolution to the sub-cellular level and to follow neural events over time. Recent developments in optical imaging may provide an avenue to increasing both spatial and temporal resolution and, combined with molecular/genetic reporter probes, may allows us to more closely observe neural events underlying development, degeneration, and plasticity in the central nervous system. It is important for researchers who are studying the effects of alcohol on brain and behavior to understand what is known about the metabolic pathways for alcohol in the brain. Does, for example, the metabolic conversion of alcohol to acetate represent another brain energy substrate that may influence alcohol use? Brain imaging can be a useful tool to help us further our understanding of the causes and consequences of alcohol abuse.   

CTNA-2: OVERALL PROGRESS  

Administrative Core

John Krystal, MD, Director

The data management support structure offered to individual projects is based in the Administrative Core.

Data Management & Biostatistical Center (DMBC) :

The Data Management and Biostatistics Center (DMBC) is based at the Yale Center for Medical Informatics (YCMI). Data are 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. 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.

The DMBC creates case report forms according to study specifications for the Clinical Core and individual projects. Data are entered directly onto the web-based system by study Coordinators. Data are cross-checked between source documents and electronic files before closing each subject’s file. Standard TrialDB reports and custom created for CTNA are used for data-checking, error-reporting, general and specific administrative reports to the administrator and the individual Study Principal Investigator.

All subjects enrolled in any CTNA-2 project or pilot study receive a CTNA ID number and complete the Clinical Core battery at their initial entry. Subjects participating in more than one tudy maintain their assigned CTNA ID number, receiving different study numbers for each study.

The Biostatistics component of the DMBC is led by Ralitza Gueorguieva, PhD, who is assisted by Brian Pittman, MS. The biostatisticians work closely with Dr. Brandt in coordinating all statistical, data management, and data systems. In particular, the biostatisticians are responsible for statistical considerations such as general study design, endpoints, sample size and accrual, analysis and interpretation of results of all data generated from the CTNA. They are involved in the preparation of DSMB and other progress reports and coauthor scientific manuscripts generated from the CTNA. Collected data are analyzed using the most appropriate and up-to-date statistical methods. All statistical analyses (except supplemental genomics/imaging analyses) are performed in the latest version of SAS software.

Data Management Activities-TrialDB

Work is continuing on organizing and optimizing the web-based data entry forms for the core and pilot projects working with the study coordinator. This will facilitate accurate data entry and less missing data. Form revision has also been required for the core study due to web-based computer memory limitations. Modification of web forms for active study requires data to be moved, checked and personnel retrained. A total of 81 unique web-based case report forms have been created and implemented for the core project and the 5 pilot projects with 25 users given access to TrialDB and appropriate training. Forms in a MS Word format are being put in web accessible help files to also facilitate accurate data entry and is ongoing.  Unique subject IDs were created and mapped to old study IDs in order to decrease the likelihood of enrolling duplicate subjects (as subjects participate in more than one study within the Center).

Data import and mapping from the previous data management computer system for 5 studies, 72 case report forms and about 6 thousand instances of forms was completed and validation of the import and mapping was performed by chart review.

Core Programming: Of the 25 web-based standard reports (several new and used by other studies), six reports were created for the CTNA project and pilot studies. These were tested by the coordinators and revised. Tables were added to the TrialDB database and several attributes to existing tables in order to accomplish needed features for CTNA.

For the new version of TrialDB, features have been added to provide the forms in an ordered (not alphabetical only) manner, allow data entry to be easier between forms, and to allow enrollment of subjects from the core into the pilot studies. Extracts and randomization have also been improved and modified. Work is continuing and beta testing by several user groups to make the new TrialDB user interface more functional for core and pilot related usage and data management. Biostatistics Activities

Across CTNA projects, the statisticians have routinely utilized the capabilities of the TrialDB user interface. Data entered into TrialDB are regularly extracted and downloaded by the statisticians for the generation of data validation reports, DSMB and other progress reports, and statistical analyses. The statisticians work closely with Project Investigators when interpreting results of analyses of data generated thus far from each project and have coauthored a number scientific manuscripts resulting from these data. The statisticians continually work with Dr. Brandt and her team to ensure sequential monitoring procedures are employed to facilitate the function of the Data Safety Monitoring Board, review procedures for randomization, data quality assurance, and data collection, and ensure communications between DMBC staff are in place.

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 met in October 2007 to review the CTNA-2 structure and assess plans for clinical projects and pilots.

CTNA Clinical Core

Stephanie O’Malley PhD, Director

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. The Clinical Core has provided input to the Data Safety Monitoring Board on the progress of the components. The Clinical Core has established common methodologies to facilitate cognitive and clinical research domains and allow us to compare and pool data across projects for secondary analyses. All subjects enrolled in a CTNA project or pilot study will complete the Clinical Core assessment battery. We recently decided to add measures relevant to Attention Deficit Disorder; the Assessment Committee will be finalizing these assessments over the upcoming weeks. Several enrollment criteria have been standardized across all studies:

The following table summarizes subject numbers and diagnostic groups expected for each of the major research projects with human subjects.

* Participants are drawn from Projects 3 and 5 and so are not unique participants. The specific breakdown by alcohol diagnosis is approximate.

1. Uniform Criteria Across Projects

Uniform criteria regarding definitions for family history positive and family history negative subjects are incorporated into all human projects in order to provide a unifying conception of family history status across studies. In addition, this step will allow pooling of subjects for comparisons of FHP and FHN subjects and for genetic analyses. Finally, additional age and alcohol related diagnostic criteria are specified, with these criteria and criteria for family history status established in conjunction with the Genetics Core, the Clinical Core and the Scientific Advisory Committee. These criteria for defining FH status are provided below:

1. For Family History Positive (FHP) Subjects: To be defined as FHP, subjects must have a biological father and another first or second-degree biological relative with a history of alcohol dependence as determined using the family history approach operationalized by the FHAM-Family History Assessment Module.

2. Family History Negative (FHN)Subjects: To be defined as FHN, subjects must not have any first or second degree biological relatives with alcohol dependence and must also be able to report on their biological parents. Moreover, they must have at least three biological relatives who are substantially through the age of risk (as defined as age 35 for the purposes of this study) to avoid the possibility that small family size could obscure the expression of alcoholism. No other psychiatric exclusion criteria will be applied to the family members of subjects in either the FHN or the FHP groups (Gelernter, Kranzler et al. 1999) .

3. Age and Alcohol Related Diagnoses in Subjects: All CTNA studies recruit participants in the age range of 21-25 in order to select individuals at risk for alcohol dependence by virtue of their family history, but who have not been shown to have persistent alcohol-related problems which may themselves influence responses on the measures being studied. For Project 2, the age range is expanded lower because the procedures involved have been safely applied to individuals in the 18-21 age range; so the pool of recruitment for this study may be broadened without substantially altering the target population. The one exception is Project 5 which tests the effects of a medication, memantine, on alcohol drinking. In this study, FHP and FHN participants will meet criteria for alcohol abuse and can be from the ages 21 - 50. In order to facilitate recruitment, we are evaluating the possibility of only requiring heavy drinking rather than diagnostic criteria for abuse in order to increase recruitment into the study.

Clinical Core Assessments

All subjects enrolled in CTNA studies will be asked to complete the Clinical Core Assessments. This battery will provide uniform data across the whole pool of subjects that will help define issues of vulnerability.

Genetics Core

Joel Gelernter MD, Director

The goal of the Genetics Core is to support the genetic components of each of the projects participating in this Center to help understand the nature of genetic influences on the phenotypes measured, and to allow for the ascertainment of genetic covariates that might affect those outcomes. All subjects will be asked to provide a DNA sample. Information on family history and other characteristics will be collected in the Clinical Core to provide the basis for analyses with DNA information.

Specific candidate genes have been identified as potentially important for each of the component projects in the center. We have the goal of sampling DNA from 100% of study subjects. The major function of the Genetics Core will be to support genotyping for each of the projects (including pilot projects), for the purpose of identifying genotype/phenotype correlations.

The Genetics Core will support genotyping at each of these loci including selection of specific single nucleotide polymorphisms (SNPs) to be genotyped. Genes to be studied in all subjects will include the following: spinophilin (PPP1R9B); GABA receptor, A2 subunit (GABRA2); alcohol dehydrogenase 4 (ADH4); catechol O-methyltransferase (COMT); D2 dopamine receptor (DRD2); and serotonin transporter protein (SLC6A4). Our initial focus on the pinophilin locus exemplifies the translational nature of the Center.

It is often the case that either the functional SNPs at a locus of interest are not known, or there are more than one such relevant SNP. Therefore, it is important to ascertain and understand haplotypes at the loci studied. The Genetics Core will provide statistical support for reconstruction and understanding of haplotypes at all of the loci studied.

The goals of the clinical components of the Center require study of ethnically heterogeneous populations, but study of stratified samples that differ in allele frequency and phenotype for candidate loci of interest can create artifactual association. We will therefore apply structured association methodology to measure and if necessary statistically correct for the effects of population stratification. A previously described (Stein et al, 2004; Kaufman et al, 2004; Yanget al, submitted ms) set of ancestry-informative markers (AIMs) will be genotyped for each subject.

OVERVIEW OF PROJECTS

We finish Year 7 with 4 clinical projects and 6 pilot projects, each of which is summarized below.

Project 2: A. Abi-Dargham. PET Ventral Striatum Dopamine Release Deficits With Family History of Alcoholism.

During Year 6, Dr. Abi-Dargham’s group changed the design of their study to assess effects of alcohol challenge and MIDT on dopamine release as measured with [11C]raclopride displacement. This year, progress was made in adapting the MIDT for use with PET scanning and concomitant alcohol challenge. Thus far, five participants have completed both the placebo and alcohol challenge PET scans, and two additional participants have completed one of the PET scan days. No differences in dopamine binding between MIDT and the control task have been observed yet in this small sample. Ten individuals have participated in fMRI scanning; data are still being analyzed.

Project 3: I. Petrakis. Family History and Spinophilin Genotype Influence on NMDA Receptor Function and Ethanol Response.

This study will test the hypothesis that both ketamine and alcohol response (using the alcohol clamp procedure to minimize pharmacokinetic issues) will be similarly influenced by family history of alcoholism and by the spinophilin SNP, strengthening the hypothesis that the NMDA receptor antagonist component of ethanol action contributes to the abuse liability of ethanol.

Data collection is ongoing. Thus far, 82 individuals have completed all three test days for the alcohol challenge study, and 33 individuals have completed all three test days for the ketamine challenge study.

This project has resulted in several publications this year. One manuscript, which is currently under review, characterizes behavioral effects of the GABA-A agonist thiopental and ketamine. Both medications produced cognitive and subjective effects similar to ethanol, highlighting the importance of GABA and NMDA receptor systems to the effects of alcohol intoxication. In a related study, the investigators examined the influence of ketamine and thiopental on ERP response during a visual oddball task. Both drugs attenuated neural response, demonstrating the role of GABA and NMDA mechanisms in processing of novel stimuli. Both of these projects provide a framework for understanding the mechanisms by which altered glutamate and GABA receptor systems may affect cognitive functioning and behavioral response to alcohol among FHP individuals.

Another manuscript, which is in press, examines the influence of ethanol administration on electric shock-induced pain sensitivity among FHP and FHN individuals. The authors found a concentration-dependent analgesic effect for alcohol; this effect did not differ between FHP and FHN groups. Noxious electrical stimulation may only probe one facet of alcohol’s analgesic properties. Therefore, future efforts will produce pain via capsaicin administration to more fully examine the ability of alcohol to reduce different aspects of pain. In particular, the capsaicin paradigm may more adequately probe alcohol’s effects on central sensitization, which may be relevant to the neurobiology of alcoholism risk, and better differentiate between FHP and FHN individuals.

Project 4: G. Pearlson. Ventral Striatal Functional Deficits and Familial Alcoholism Risk: Impact of Genotype.

Participant recruitment is ahead of schedule. Thus far, 27 FHP and 21 FHP individuals have performed the monetary incentive delay task during functional MRI acquisition. Results show that FHP individuals demonstrate significantly less ventral striatal activation during reward anticipation compared to FHN, suggesting reduced reward sensitivity among individuals with a family history of alcoholism. Moreover, greater impulsivity was correlated to less activation in the nucleus accumbens. These results were presented in several posters and a talk at ICANA. Subject recruitment is ongoing in order to further investigate the influence of alcohol abuse diagnosis.

Project 5: S. Krishnan-Sarin. NMDA Antagonist Efficacy in Reducing Human Alcohol Consumption: Impact of Family History.

Recruitment for this project increased this year after changes to exclusion criteria were made. Thus far, 25 participants have been randomized and 22 have completed all phases of the study. One participant withdrew due to family constraints, and two withdrew due to anticipated medication side effects. An additional site has been added to facilitate running more subjects.

PILOT CORE:

The Executive Committee has reviewed ongoing projects and considered proposals for new Pilot Studies. We received 15 concepts for pilot projects, and the Executive Committee reviewed and funded applications that were most closely related to the goals of the CTNA. Requests for new pilot concepts will be announced in April 2008. Following Executive Committee review, awards will be made in May, for funding during Year 8, starting June 1, 2008. The total amount of funds for the Pilot Core ($44,500) is less than 10% of the overall annual budget. Following are descriptions of current Pilot Studies:

Pilot 1: R. Duman. Gene Expression Profile of Postmortem, Alcohol-dependent Brains .

Continuing project. This pilot project is identifying dorsolateral prefrontal cortex gene expression and the relationship to cellular changes in alcohol dependence. Methodological progress has been made, optimizing collection and analysis of RNA, resulting in a publication on prefrontal cortex gene expression in major depression. Data from alcohol dependent patients is ready to be analyzed.

Pilot 2: G. Mason. Evaluation of Alcohol Effects on Glutamate and GABA neurotransmission in FHP and FHN Subjects using [1H] and [13C] MRS.

Continuing project.

This pilot project evaluates changes in brain GABA and glutamate levels and turnover in response to an acute ethanol challenge in family history positive and family history negative healthy adults. This project has made significant methodological progress this year, refining the ethanol clamp procedure for use in the magnet. Using 1H MRS during ethanol infusion, the investigators have observed a rise in GABA followed by a return to baseline, as well as a decrease in myo-inositol levels. Plans are to test the ethanol and glucose co-infusion procedure bedside and make any necessary modifications before 13C MRS scanning. 

Pilot 3: W. Corbin. Effects of Alcohol and Prior Gambling Outcomes on Gambling Behavior.

Continuing project.

This project investigates the influence of alcohol consumption and impulsivity on a computerized gambling task. Thus far, data are available for 126 participants, all of whom are moderate to heavy drinkers and regular gamblers. Preliminary results demonstrate that participants bet more during the alcohol condition relative to the placebo condition. Further analyses will elucidate the possible moderating and mediating fators, such as impulsivity and gambling related cognitions. This research contributes to the understanding gambling behaviors related to pharmacological and cognitive effects of alcohol, and may help identify risk factors and intervention strategies for individuals with problem drinking and gambling behaviors. This project provided pilot data for a now funded R21 application.  

Pilot 4: A. Simen. Epigenetic factors in stress mediated vulnerability to alcohol abuse and dependence. New project.

This pilot project examines the effects of early life maternal separation or restraint stress on prefrontal cortex gene expression and alcohol preference in mice. This year, the investigators made substantial methodological progress by developing a new paradigm involving maternal separation and early weaning to reliably produce behavioral changes indicative of stress in mice. Pilot analyses of gene expression changes in prefrontal cortex have also been conducted. Plans include piloting and implementing an alcohol preference paradigm to test the effects of early life stress on alcohol consumption.  

Pilot 5: J. Taylor. Analysis of protein expression in a genetic animal model of alcoholism.

Continuing project.

This pilot project is examining differences in protein expression in alcohol preferring rats in order to determine the protein expression changes that correspond to genetic vulnerability. Thus far, data have been analyzed for one rat bred to consume large amounts of alcohol and three non-alcohol rats. With this small sample, approximately 20 proteins have been identified as being differentially regulated in the alcohol preferring rat. Data collection and analyses are continuing in order to identify genetic factors related to alcoholism risk. 

Pilot 6: S. O’Malley. Targeted Naltrexone for Heavy Alcohol Use in Young Adults.

Continuing project.

This pilot project examined the utility of naltrexone administration in reducing heavy drinking among young adults, which is a population in need of more effective treatment strategies. Participants received naltrexone and behavioral counseling for 8 weeks. Following treatment, participants significantly reduced drinks per drinking day, percentage of heavy drinking days, and alcohol-related consequences. These important findings support the use of naltrexone to reduce heavy drinking in young adults. This pilot project has finished collecting data; it provided pilot data for a now funded R01 application.

OVERVIEW of CTNA-2, YEAR 6

6/01/06 to 5/31/07

The Center for the Translational Neuroscience of Alcoholism (CTNA-2) started its first year of the competitive renewal after a delay in funding. Yale continued to pay salaries but would not consider other payments until the notice of grant Award was received. The uncertainty of the award made it difficult for Investigators to transition smoothly into the sixth year. Nonetheless, progress was made in analyzing data collected during the earlier grant period, as well as planning for project to be carried out during this grant cycle. Analyses provided sufficient information for us to conceptualize the next steps. The original theme of GABA-GAD based mechanisms that tied all the projects together during 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 during CTNA-2.

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. Transdisciplinary research within projects and between/among projects and pilots has continued during this year.

CTNA investigators have been very productive with publication of numerous papers from earlier studies, 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 July 2007 with workshops, presentations, and abstracts representing our progress to date. Although the basic science project led by Jane Taylor PhD and Patrick Allen PhD was deleted from the renewal grant, they have resubmitted a request for supplemental funding to rejoin CTNA-2. Their work probes the role of motivational circuitry in alcohol administration and related impulsivity. We expect to have them rejoin CTNA to extend our translational activities.

  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 and perform analyses. Projects 2 and 5 were delayed starting because they needed to make changes in protocols, requiring full IRB re-approval.

The Executive Committee has reviewed ongoing projects and considered proposals for new Pilot Studies. Dr. Godfrey Pearlson (Project 4) is a new addition whose earlier Pilot studies is now a full Project.

CTNA-2: OVERALL PROGRESS

 Administrative Core :

The data management support structure offered to individual projects is based in the Administrative Core.

Data Management & Biostatistical Center (DMBC) :

The Data Management and Biostatistics Center (DMBC) is based at the Yale Center for Medical Informatics (YCMI). Data are 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. 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.

The DMBC creates case report forms according to study specifications for the Clinical Core and individual projects. Data are entered directly onto the web-based system by study Coordinators. Data are cross-checked between source documents and electronic files before closing each subject’s file. Standard TrialDB reports and custom created for CTNA are used for data-checking, error-reporting, general and specific administrative reports to the administrator and the individual Study Principal Investigator. Descriptive data on the projects that have transferred from CTNA-1 to CTNA-2 are listed in the Appendix.

All subjects enrolled in any CTNA-2 project or pilot study will receive a CTNA ID number and complete the Clinical Core battery at their initial entry. Subjects participating in more than one study will maintain their assigned CTNA ID number, receiving different study numbers for each study.

Data Management Activities-TrialDB

Work is continuing on organizing and optimizing the web-based data entry forms for the core and pilot projects working with the study coordinator. This will facilitate accurate data entry and less missing data. Form revision has also been required for the core study due to web-based computer memory limitations. Modification of web forms for active study requires data to be moved, checked and personnel retrained. A total of 81 unique web-based case report forms have been created and implemented for the core project and the 5 pilot projects with 25 users given access to TrialDB and appropriate training. Forms in a MS Word format are being put in web accessible help files to also facilitate accurate data entry and is ongoing.  Unique subject IDs were created and mapped to old study IDs in order to decrease the likelihood of enrolling duplicate subjects (as subjects participate in more than one study within the Center).

Data import and mapping from the previous data management computer system for 5 studies, 72 case report forms and about 6 thousand instances of forms was completed and validation of the import and mapping was performed by chart review. Core Programming: Of the 25 web-based standard reports (several new and used by other studies), six reports were created for the CTNA project and pilot studies. These were tested by the coordinators and revised. Tables were added to the TrialDB database and several attributes to existing tables in order to accomplish needed features for CTNA.

For the new version of TrialDB, features have been added to provide the forms in an ordered (not alphabetical only) manner, allow data entry to be easier between forms, and to allow enrollment of subjects from the core into the pilot studies. Extracts and randomization have also been improved and modified. Work is continuing and beta testing by several user groups to make the new TrialDB user interface more functional for core and pilot related usage and data management.

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 met in October 2007 to review the CTNA-2 structure and assess plans for clinical projects and pilots.

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.

Clinical Core Assessments: All subjects enrolled in CTNA studies will be asked to complete the Clinical Core Assessments. This battery will provide uniform data across the whole pool of subjects that will help define issues of vulnerability.

Genetics Core, PI: Joel Gelernter MD

The goal of the Genetics Core is to support the genetic components of each of the projects participating in this Center to help understand the nature of genetic influences on the phenotypes measured, and to allow for the ascertainment of genetic covariates that might affect those outcomes. All subjects will be asked to provide a DNA sample. Information on family history and other characteristics will be collected in the Clinical Core to provide the basis for analyses with DNA information. The Genetics Core supports genotyping of all subjects enrolled in CTNA studies. Dr. Gelernter will advise CTNA Investigators on issues related to genetics studies. Specifically, this will include (a) consultation on genetics-related human subjects issues; (b) consultation regarding study design (which may include preselection of subjects by genotype in some cases, as well as selection of genes and specific markers for study); (c) statistical analyses services (including implementation of analyses results involving AIMs); and (d) consultation to aid in interpretation of results.

He expects to be able to initiate genotyping and pilot analyses of CTNA-based DNA samples during collection of samples across studies.

OVERVIEW OF PROJECTS

We start the first year with 4 clinical projects, three of which are continuations of protocols with new hypotheses, and one of which is altogether new. CTNA also includes 3 clinical pilots, 1 new and 2 continuing.

Project 2: A. Abi-Dargham. PET Ventral Striatum Dopamine Release Deficits With Family History of Alcoholism. Continuation of a CTNA project

This study was planned to determine whether healthy FHP individuals show reductions in ventral striatal dopamine release compared to healthy FHN individuals. However, another group recently reported data on exactly this study design. Therefore, Dr. Abi Dargham has revised the hypotheses and initiated a similar that meshes with the overall CTNA program. She will first determine the best paradigm to probe dopamine response by comparing alcohol challenge versus the Monetary Incentive Delay Task.

Project 3: I. Petrakis. Family History and Spinophilin Genotype Influence on NMDA Receptor Function and Ethanol Response. Continuation of a CTNA project

This study will test the hypothesis that both ketamine and alcohol response (using the alcohol clamp procedure to minimize pharmacokinetic issues) will be similarly influenced by family history of alcoholism and genetics, strengthening the hypothesis that the NMDA receptor antagonist component of ethanol action contributes to the abuse liability of ethanol.

The study is based on the hypothesis that disturbances in the interplay of glutamate and dopamine systems contribute to the vulnerability to alcohol dependence by: 1) impairing the normal engagement of reward/motivation circuitry and thereby increasing the relative motivational impact of alcohol-related rewards; and 2) by shifting the reward valence of the N-methyl-D aspartate (NMDA) glutamate receptor antagonist component of ethanol action toward reward, making ethanol a more addictive substance for those at risk. Dr. Petrakis established the IV-Ethanol infusion methodology during CTNA-1, with assistance from Dr. S. O’Connor, enabling an early start of the new study.

Project 4: G. Pearlson. Ventral Striatal Functional Deficits and Familial Alcoholism Risk: Impact of Genotype. New project. This study will determine whether FHP healthy subjects and FHP alcohol dependent patients show deficits in ventral striatal activation associated with the anticipation of reward. The first component of the project will explore whether deficits in ventral striatal activation in family history positive healthy subjects related to increased NMDA receptor function genotype. This study is off to a good start with more than the expected number of subjects completed during the first year. Dr. Pearlson reports that early results show that ventral striatum and ventral mesial PFC are active during the Reward Anticipation period.

Project 5: S. Krishnan-Sarin. NMDA Antagonist Efficacy in Reducing Human Alcohol Consumption: Impact of Family History. Continuation of a CTNA project

This project evaluates the dose-related efficacy of memantine in reducing alcohol consumption in FHP/FHN alcohol dependent patients. It also tests whether memantine, perhaps through “normalizing” NMDA receptor function, reduces alcohol cue-induced craving, while modified by familial alcoholism history. The protocol was altered after approval, resulting in a delay before starting recruitment. Dr. Krishnan-Sarin expects to catch-up quickly now that recruitment is underway.  

PILOT CORE

The Executive Committee has issues a Request for Proposals for new and renewing Pilot Studies related to the CTNA-2 theme. Awards will be made in May, for funding during year 7, starting June 1, 2007. The total amount of funds for the Pilot Core will be less than 10% of the overall annual budget. Following are descriptions of current Pilot Studies.

Pilot 1: R. Duman. Gene Expression Profile of Postmortem, Alcohol-Dependent, Prefrontal Cortex

This Pilot Project will identify gene expression changes in alcohol dependence. Microarray analysis of gene expression using RNA from frozen brain sections will be performed to identify genes that are either up-regulated or down-regulated in alcohol dependence. Dr. Duman has developed new techniques that allow for analyses of these unique samples of brain tissue. This Pilot study will be completed in year 2.

Pilot 2: G. Mason. Evaluation of Alcohol Effects on Glutamate and GABA neurotransmission in FHP and FHN Subjects using [1H] and [13C] MRS

This Pilot Project will evaluate changes in brain GABA and glutamate levels and turnover in response to an acute challenge of ethanol in healthy adults, according to status of family history positive or negative.

The primary hypothesis is that subjects who are family history negative will show an increase in brain GABA when drinking. Dr. Mason continued to develop the 13C and 1H MRS GABA acquisition measurements over the past year, including running IV-ethanol infusions. They are able to measure the rate of GABA synthesis in individual subjects. This Pilot study will be completed in year 2.

Pilot 3: M. Potenza. Influence of Temporal Delays on Neural Correlates of Reward Processing in Adults Family History Positive for Alcoholism .

This pilot project investigated the neural correlates of reward processing in adults family positive and adults family history negative for alcoholism using the Experiental Discounting Task. The hypothesis is that the anticipation phases of reward processing (anticipation of working for reward and anticipation of receiving reward) will be associated with activation of ventral striatum, and that individuals with a family history of alcoholism will show relatively less activation of ventral striatum during the anticipation phases. Dr. Potenza expects to complete data collection by the end of year 1 with data analysis continuing into year 2 to provide the basis for future studies.

Pilot 4 (PI: W. Corbin): Current Project: Alcohol Effects on Gambling Persistence and Betting Strategy Using a Simulated Slot Machine.

This study was designed to examine the effects of alcohol on gambling persistence and betting strategy under conditions of initial loss, breaking even, or initial gains. The primary study hypothesis is that alcohol will lead to increased gambling persistence and heavier betting under conditions of loss. In other words, alcohol consumption is expected to facilitate “chasing” behavior that is well documented among pathological gamblers. This study had been supported by other funds, but will given a small amount of Pilot support in year 2 while Dr. Corbin awaits RO1 funds.

Pilot 5: S. O’Malley: Targeted Naltrexone for Heavy Alcohol Use in Young Adults

This pilot was initiated to develop data for an RO1 submission on the feasibility of the protocol and to provide preliminary data that will inform the conduct of a subsequent double-blind placebo-controlled trial testing the efficacy of daily and targeted naltrexone. The primary hypothesis is that compared with baseline rates, naltrexone use and counseling will be associated with significant decreases in heavy drinking days and overall frequency of consumption. Dr. O’Malley will complete this Pilot in year 2 while preparing an RO1 proposal.

Request for Proposals for Pilot Studies

An RFP was issued in February 2007 to encourage submission of ideas from a broad range of Investigators in the Yale neuroscience community. Although some funds have been committed to complete the Pilots described above, some funds are reserved for new proposals. The Executive Committee will use the Pilot Core to expand research on the CTNA theme with hopes of attracting new investigators to study alcoholism.

Summary

This first year of CTNA-2 has been highly productive. Always mindful that we are a Center, each Investigator demonstrated the associations among and between projects and use of Core facilities, with discussions of inter-relationships among projects and pilots. We expect greater recruitment of subjects in all studies, as well as ongoing data collection in the next year to maintain the original goals for CTNA-2.

OVERVIEW of CTNA YEAR 5

6/01/05 to 5/31/06

The Center for the Translational Neuroscience of Alcoholism (CTNA) has accomplished an enormous amount of work in all projects and pilots since its inception in June 2000. These efforts were recognized with the award of a renewal grant that will allow us to extend and expand upon the initial projects and pilots during the next 5-year grant cycle.

The theme of this first grant (called CTNA-1) focused on GABA-GAD based mechanisms to study disturbances in glutamate and dopamine neurotransmission within cortico-limbic circuitry. CTNA investigators have been highly productive during the past 5 years, with numerous papers in leading journals, presentation at many scientific conferences, and spin-off grant support from NIAAA, NIDA, VA and other agencies to sponsor a variety of alcohol research projects. CTNA Investigators have a major presence at Research Society on Alcoholism meetings with workshops, presentations, and abstracts representing our current findings. These activities are fully described in the individual reports. Both the projects and collateral studies have benefited from the Core resources and structure of the CTNA.

The focus of CTNA-1 on glutamate and dopamine neurotransmission has been advanced by work accomplished from Projects and Pilots. Dr. Eric Nestler and his colleagues identified novel molecular mechanisms underlying the rewarding effects of alcohol and other drugs of abuse. His studies have linked these mechanisms to the propensity to self-administer these substances. The novel transcription factor, D FosB, was shown to modulate glutamatergic neurotransmission, suggesting glutamatergic neuroplasticity, with links between addiction and learning.

This work has been extended in a Pilot Study by Jane Taylor, Ph.D. and Patrick Allen Ph.D., experts in the neurobiology and psychopharmacology of motivation/addiction, who were recruited to the field of alcohol research within CTNA-1. They have probed the role of motivational circuitry in alcohol administration and related impulsivity. Their work exploring the role of glutamatergic and dopaminergic inputs to the nucleus accumbens that converge on the dendritic spines of GABA neurons will be extended into CTNA-2. To date, they have studied networks that process shifts in environmental reward- and punishment-related contingencies and they generate motivational states. Their pilot work showed that 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 concept has provided evidence implicating glutamatergic and dopaminergic signal transduction pathways and synaptic integrity in processes contributing to alcoholism development .

The CTNA Executive Committee managed the process of the competing renewal grant cycle, with extensive support from all Investigators and collaborators. The Data Management and Biostatistical group worked smoothly to provide data for renewal proposals as well as reports on the conclusions from studies. The Data Safety Monitoring Board met twice-yearly to review progress of the clinical projects, end endorsement of continuation for the renewal application. The Local and Regional Education Committees have sponsored or co-sponsored several scientific and educational symposia, the highlight of which was the International Conference on the Applications of Neuroimaging to Alcoholism (ICANA).

CTNA Administrative Core :

The support structure offered to individual projects is based in the Administrative Core.

Data Management & Biostatistical Center (DMBC) :

The DMBC has worked closely with Investigators to help with study design, preparation of study forms, management of data, as well as 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.

Data Safety Monitoring Board (DSMB ):

The DSMB has met twice-yearly to review clinical programs to assure patient safety. They also determine whether projects have answered their proposed scientific aims or are unlikely to be concluded successfully, thus placing subjects at avoidable potential risk. The DSMB fully supported the renewal application for CTNA-2.

Scientific Advisory Board (SAB):

The SAB has provided helpful comments on all projects and pilot studies to assure that the transdisciplinary goals of the CTNA were being achieved. They also provided valuable advice on how to restructure for the CTNA-2 renewal application.

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.

Thi s Core has several components:

Genetic studies (directed by Joel Gelernter MD): DNA samples were collected from subjects enrolled in all clinical CTNA projects. These data were used as covariates for clinical projects.

Clinical Electrophysiology (directed by Daniel Mathalon PhD, MD): The section supports evoked potential recordings for clinical studies. These data were used as covariates for projects, as well as for independent analyses that will be published separately.

Recruitment Support ( directed by Stephanie O’Malley PhD): The centralized subject recruitment planning was successful in enrolling subjects into all projects and pilots.

 CTNA Molecular Core (Joel Gelernter MD):

The Molecular Core supported basic marker testing, marker panel preparation and testing, and creation of new markers, in support of greater genotyping effort in patient populations. They are working with Investigators to identify candidate variants for study and implementing DNA preparation and genotyping.

Project 1

PI: Eric Nestler MD, PhD

TRANSCRIPTION FACTORS, GLUTAMATERGIC FUNCTION, ETHANOL REWARD, AND ADAPTATION TO CHRONIC ETHANOL

Dr. Nestler has focused his work on a systematic mapping of ∆FosB 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 ∆FosB occurs, in the search for target genes for ∆FosB, and hence for alcohol. The data indicate that alcohol-induced accumulation of ∆FosB 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 ∆FosB 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

PI: Joel Gelernter MD

GUIDED FAMILY-CONTROLLED LINKAGE DISEQUILIBRIUM SCAN FOR ALCOHOL DEPENDENCE AND PFC-RELATED ENDOPHENOTYPES

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

PI: Graeme Mason PhD

13 C MRS MEASUREMENT OF GLUTAMATE NEUROTRANSMITTER CYCLING IN INDIVIDUALS WITH FAMILIAL VULNERABILIUTY TO ALCOHOLISM, RECENTLY DETOXIFIED ALCOHOLICS, LONG-TERM SOBER ALOCHOLICS, AND HEALTHY INDIVIDUALS

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

PI: Anissa Abi-Dargham MD

PRESYNAPTIC AND POSTSYNAPTIC 5-HT RECEPTOR ABNORMALITIES IN ALCOHOLISM: A PET STUDY

Dr. Abi Dargham’s protocol focuses on a comparison of 5HT transporter binding potential (BP) between alcohol dependent subjects and healthy controls. It combines [ 11 C]WAY100635 and [ 11 C]DASB, as a superior tracer for the 5HT transporter Alcohol dependent subjects were found to have a decrease in D 2 receptor availability in the ventral and dorsal regions of the striatum compared to healthy controls and the reduction in striatal D 2 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

PI: Ismene Petrakis MD

ALTERED NMDA RECEPTOR FUNCTION WITH FAMILIAL ALCOHOLISM RISK

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

PI: Suchitra Krishnan-Sarin PhD

DIFFERENTIAL INTERFERENCE WITH ETHANOL SELF-ADMINISTRATION BY NALTREXONE IN ALCOHOLIC INDIVIDUALS WITH OR WITHOUT FAMILIAL ALCOHOLISM

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:

Pilot 2

PI: Daniel Mathalon PhD, MD

NEURAL CIRCUITRY OF P300 ABNORMALITIES IN ALCOHOLICS: RELATIONSHIP TO ALCOHOL CUE REACTIVITY

This pilot study resulted in an ABMRF grant to extend the preliminary findings funded by CTNA.

Pilot 3:

PI: William Corbin PhD

ACUTE EFFECTS OF ALCOHOL ON INFORMATION PROCESSING AND BEHAVIORAL CONTROL

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 5:

PI: Jane Taylor PhD

BEHAVIORAL AND MOLECULAR CONSEQUENCES OF CHRONIC ETHANOL ADMINISTRATION IN THE RAT.

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 6:

PI: Julie Staley PhD

[ 123I]5-IA-85830 SPECT IMAGING OF β2-NICOTINIC ACETYLCHOLINE RECEPTORS IN ALCOHOLISM

Dr. Staley has studied whether thalamic and cortico-limbic β2 nicotine acetylcholine receptors (β2~nAChR) are increased during sobriety in alcohol-dependent nonsmokers with a family history of alcoholism. She is using SPECT imaging of β β2~nAChR to assess adaptations during the early development of sobriety. This pilot will be continued during year 5.

Pilot 7

PI: Godfrey Pearlson MD

VENTRAL STRIATAL FUNCTIONAL DEFICITS AND FAMILIAL ALCOHOLISM RISK

Dr. Pearlson will perform pilot studies this year to refine methods for a project that is included in CTNA 2. He uses fMRI to assess impulsivity in healthy subjects with and without a family history of alcoholism.

Summary :

This five-year program of CTNA-1 has been highly productive. We are preparing for the start of CTNA-2 with some changes in Investigators and Projects to focus on glutamate.

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.