The National Advisory Council on Alcohol Abuse and Alcoholism (NIAAA) convened for its 132nd meeting at 5:00 p.m. on February 6, 2013, at the Fishers Lane Conference Center in Rockville, Maryland, in closed session for a review of grant applications, a special Council review, and a Merit Award nomination/extension. The meeting recessed at 6:50 p.m. Dr. Abraham Bautista, Director, Office of Extramural Activities, presided over the closed session, which, in accordance with the provisions of Sections 552b(C)(6), Title 5, U.S.C. and 10(d) of Public Law 92-463, excluded the public for the review, discussion, and evaluation of individual applications for Federal grant-in-aid funds. The Council reconvened on February 7, 2013, in closed session, at which Dr. Tatiana Faroud presided over presentation of the Board of Scientific Counselors’ Report. Dr. Kenneth Warren, Acting Director, NIAAA, convened the Council in open session on February 7, 2013, at 8:45 a.m.
Council Members Present:
Andrea Barthwell, M.D.
Carol A. Casey, Ph.D.
Linda L. Chezem, J.D.
Fulton T. Crews, Ph.D.
Suzanne M. de la Monte, M.P.H., M.D.
Marianne L. Fleury
Joseph Thomas Flies-Away, J.D.
Andres G. Gil, Ph.D.
Kathleen Grant, Ph.D.
Paul J. Gruenewald, Ph.D.
Craig McClain, M.D.
Patricia E. Molina, M.D., Ph.D.
Gyongyi Szabo, M.D., Ph.D.
Chairperson: Kenneth R. Warren, Ph.D.
Executive Secretary: Abraham P. Bautista, Ph.D.
Vivian B. Faden, Ph.D.; Ralph Hingson, Sc.D., M.P.H., Keith Lamirande; Raye Litten, Ph.D.; Gary Murray, Ph.D.; Antonio Noronha, Ph.D.
Other Attendees at the Open Sessions:
Approximately 45 observers attended the open session, including representatives from constituency groups, liaison organizations, NIAAA staff, and members of the general public.
Call to Order of the Open Session, February 7, 2013
Dr. Kenneth Warren called the open session of the 132nd meeting of the Council to order at 8:45 a.m. on Thursday, February 7, 2013. He welcomed participants to the meeting, and Council members and NIAAA senior staff introduced themselves. Dr. Warren introduced new Council members Carol Casey, Ph.D.; Paul J. Gruenewald, Ph.D.; Sarah N. Matson Weller, Ph.D.; and Patricia E. Molina, M.D., Ph.D. Judge Joseph Thomas Flies-Away and Robert Messing, M.D., also have joined the Council.
Dr. Warren highlighted key recent Institute activities, referring to the written Director’s Report.
- In memoriam. Dr. Warren marked the recent death of esteemed scientist Daniel W. Hommer, M.D., who had served as Chief, Section of Brain Electrophysiology and Imaging, NIAAA.
- Functional integration with NIH Institutes. Dr. Warren reported that the National Institutes of Health (NIH) has decided to pursue functional integration of addiction research rather than merging NIAAA with the National Institute on Drug Abuse (NIDA).
- NIAAA Director search. NIH has initiated a search to recruit a permanent NIAAA Director. Council member Dr. Patricia Molina serves on the search committee.
- Legislation, budget, and policy. In FY 2012 NIAAA obligated $459.1 million in appropriated funds. In the absence of a budget for FY 2013, a continuing resolution has provided for the same level of funding for the Institute. Due to budget uncertainties NIH has adopted the mandated strategy to award noncompeting awards at 90% of the proposed award amount. Release of the FY 2014 President’s budget request for NIH and NIAAA has been delayed.
- NIAAA staff organization. Dr. Anita Bechtholt-Gompf has joined NIAAA’s Division of Neuroscience and Behavior as a Program Director. Mr. Fred Donodeo serves as Acting Chief, Communications and Public Liaison Branch. Dr. Yoshihiro Kashiwaya has left NIAAA’s Laboratory of Metabolic Control to return to his native Japan to practice neurology.
- Staff honors and editorial appointments. Dr. Rui Costa and Dr. Andrew Holmes received the Young Investigator Award and the 2012 Jacob P. Waletzky Award, respectively from the Society for Neuroscience. Dr. David Lovinger was appointed Editor in Chief of the journal Alcohol. Dr. John Matochik received the National Institute of Mental Health Director’s Merit Award. Dr. Pal Pacher was elected as an honorary member of the Hungarian Society for Experimental and Clinical Pharmacology, as well as elected to the Editorial Board of American Journal of Physiology-Cell Physiology. Dr. Christopher Ramsden will become an Associate Editor of the journal Prostaglandins.
- New RFAs/PAs. The written Director’s Report included no new requests for applications and few program announcements.
- NIAAA communications and media coverage. NIAAA places great importance on research dissemination. Its fact sheet for the 2012 winter holiday seasonal outreach highlighting traffic safety generated more than 350 online postings by websites for local TV affiliates, blogs, and news outlets. NIAAA also initiated inexpensive billboard campaigns on well-traveled Northeast highways to “rethink holiday drinking.” The series was expected to reach1.5 million people weekly. A 15-second animated video shown on the David Letterman screen in New York’s Times Square reached an audience of 1.5 million people daily. NIAAA also disseminated two Halloween-themed photo messages for use across social media. Impact analysis for the summer billboard campaign revealed a 37% increase in NIAAA website visits over the previous quarter and 72% over the same quarter in 2011.
- Multimedia products. Dr. Warren highlighted the February issue of NIAAA’s journal Alcohol Research: Current Reviews and the Institute’s webzine, Spectrum. He noted that media conducted many interviews with NIAAA senior staff, and he acknowledged publication of Neural Immune Interventions in Brain Function and Alcohol-Related Disorders, a new book edited by Changhai Cui, Lindsey Grandison, and Antonio Noronha.
- Upcoming events. In May, Dr. Bankole Johnson will receive the Jack Mendelson Award and will deliver the Jack Mendelson, M.D., Honorary Lecture. NIAAA is partnering with the NIH Employee Assistance Program to offer free screenings for alcohol use disorder, and the Institute soon will release a publication on available treatment options for alcohol use disorders. The bi-annual meeting of Directors of Alcohol Research Centers will take place in April 2013.
Hughes Award Presentation
Dr. Warren presented Council member Mimi Fleury with the Senator Harold Hughes Award. The award recognizes a nonresearcher’s outstanding ability to create effective prevention and treatment programs grounded in scientific research and to connect the alcohol prevention, treatment, and policy making communities. Ms. Fleury founded the first Community of Concern in 1998 in her efforts with other parents to develop an effective way to explain to both parents and students the inappropriate use of alcohol and its risks. Together with professional researchers and counselors, Ms. Fleury’s organization developed a manual for parents that explained the dangers of substance use from scientific, legal, and social perspectives. The guide enjoyed success, and Ms. Fleury shared it with other schools in the Washington, D.C., area. Community of Concern consortia now operate in 36 states and several foreign countries. In thanking NIAAA for the honor, Ms. Fleury explained that she had rejected the conventional wisdom that “all kids drink in high school,” “keep them safe in the basement,” “just take their keys,” and “teach them to drink responsibly.” Students and parents needed to recognize the true consequences and dangers of alcohol use by young people, and to delay first use of alcohol. NIAAA and NIDA scientists contributed to the manual’s recent update to reflect the state of the science (see www.thecommunityofconcern.org).
Compliance with Inclusion Guidelines
Dr. Bridget Williams-Simmons, Health Scientist Administrator, Office of Science Policy and Communications, NIAAA, presented data for incorporation in NIAAA’s 2013 Biennial Advisory Council Report on Inclusion of Women and Minorities as Subjects in Clinical Research. The congressionally mandated report is based on the principle that all members of society should share in both the burdens and benefits of biomedical and behavioral research. The NIH Revitalization Act of 1993 requires that women and members of minority groups and their subpopulations be included in all NIH-funded clinical research; NIH must support outreach efforts to recruit and retain those populations in clinical studies; exclusions may be made solely by the NIH Director or Institute/Center (IC) Director based on a clear and compelling rationale; cost is not an acceptable reason to exclude these groups; and Phase III trials should be designed and implemented to analyze differences in intervention effects among gender groups and racial/ethnic groups.
Dr. Williams-Simmons described NIAAA’s procedures to ensure compliance: All funding opportunity announcements include a section on the inclusion policy; program officials answer questions from potential applicants; special attention is given to identification of Phase III clinical trials; scientific review officers (SRO) inform peer reviewers about the inclusion policy before and during reviews; peer review groups determine whether women and minorities have adequate representation; SROs summarize reviewers’ comments and code summary statements to reflect review groups’ comments and concerns; applications with unacceptable inclusion plans are barred from funding; for fundable studies, program officials discuss issues of noncompliance with applicants, who may modify the application to address reviewers’ concerns; upon award, program officials evaluate and approve required population target and cumulative enrollment reports of ongoing projects; and NIAAA provides staff training on the inclusion policy.
Data for FY 2011 and 2012 reveal that women constituted 46% of the enrollment population in clinical studies. For FY 2011 the total minority enrollment was 43.8% and for FY 2012 it was 45%. In NIAAA’s Phase III clinical trials, the numbers of female and minority enrollees fluctuated greatly due to the small number of trials and the start of one and termination of another between fiscal years. The racial and ethnic populations with greatest representation in both 2011 and 2012 for both extramural clinical studies and Phase III trials were White, Black/African American, Hispanic or Latino, and Asian (in decreasing order). The numbers remained relatively consistent from year to year. Enrollment in Phase III clinical trials fluctuated greatly, while both male and female enrollment remained relatively consistent.
Discussion. Dr. Suzanne de la Monte urged NIAAA to ensure adequate enrollment of Latinos who do not speak English, and Drs. Williams-Simmons and Warren acknowledged NIAAA’s need to address the issue. Dr. Andres Gil urged NIAAA to manage the expectations of review committees on this issue; peer reviewers may have concerns about inadequate power and small samples. Dr. Patricia Molina observed that room for improvement exists for Hispanic enrollment. Dr. Warren noted that disparities may reflect the language issues and/or local study demographics. Dr. Williams-Simmons stated that NIAAA’s enrollment of Hispanics/Latinos was similar to NIH-wide levels.
Certification. Members of the NIAAA National Advisory Council voted unanimously to accept the report.
NIAAA Director Search
Dr. Stephen Katz, Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases, and co-chair of the search committee for the new NIAAA Director, informed Council members about the search and encouraged them to promote submission of applications by qualified applicants and to reach out to minority and underserved communities. Search committee members include co-chair Dr. Linda Birnbaum, Dr. Robert Croyle, Dr. Rueben Gonzales, Dr. John Krystal, Dr. Patricia Molina, Dr. Griff Rogers, and Dr. Constantine Stratakis. Dr. Katz stated that the ideal Director would be an outstanding scientist with a deep knowledge and understanding of research on the prevention and treatment of alcohol abuse and alcoholism, the ability to lead and manage a large and complex organization, the ability to work well collaboratively, and the ability to serve as the key spokesperson for NIAAA and NIH to the Congress.
Discussion. In response to Dr. Fulton Crews’s question, Dr. Warren stated that a new Director with an active research project could expect to transfer that program to NIH and be assured of adequate resources. Dr. Katz emphasized the search committee’s focus on candidates who can run the Institute at the highest level and make NIAAA’s mission a high priority across NIH. To questions from Dr. Andrea Barthwell and Dr. Gyongyi Szabo, Dr. Katz responded that, in his opinion, the Director must be able to be collaborative, adaptable, and appreciative of all dimensions of the work to advance NIAAA-supported science, and to communicate the mission’s importance, transmit scientific advances and the fruit of the Institute’s $500 million annual investment, focus on training the next generation of investigators, and understand the cross-cutting nature of NIAAA’s mission within NIH. Dr. Kathleen Grant commended the rapid pace at which NIH has begun the search for a new Director.
Consideration of the September and December 2012 Meeting Minutes and Future Meeting Dates
Council members unanimously approved the minutes of the NIAAA Council meetings held on September 19–20, 2012, and December 13, 2012. Future Council meetings will take place June 12–13 and September 18–19, 2013; February 5–6, June 4–5, and September 10–11, 2014; and February 4–5, June 10–11, and September 16–17, 2015. Joint NIAAA and NIDA Council meetings may be held in conjunction with the Institutes’ February meetings.
Chronic Alcohol Reshapes the Neural Circuits Mediating Executive Functions
Dr. Andrew Holmes, Chief, Laboratory of Behavioral and Genomic Neuroscience, NIAAA, described his work on a framework and approach to investigate how alcohol shifts key brain circuits in animals to affect high-order behaviors. Inspiration for this work stems from the case of Phineas Gage who, in the1800s suffered brain injury when an explosion propelled an iron bar through his brain, resulting in personality and behavioral changes including impulsivity, drinking, and gambling. Since then it has been learned that the prefrontal cortex (PFC) plays an important role in executive functions such as working memory, impulse control, and emotional regulation. In studies alcoholics show impaired PFC-mediated impulse control and flexibility, and abnormal PFC activation during presentation of alcohol cues. The emerging literature on animals shows similar behaviors; for example, rats given alcohol chronically show deficits in prefrontal-mediated behaviors and some associated changes in the structure of the PFC molecular profile. Dr. Holmes pointed out that many diagnostic criteria for alcoholism can be viewed as problems of prefrontal function. His laboratory works from the premise that with addictions such as alcoholism progressive degradation occurs in the functional integrity of PFC, with a shift in control to more primitive structures such as the amygdala and the limbic system in the dorsal striatum.
Dr. Holmes described the mouse model of chronic alcohol exposure, which introduces fear extinction as a behavioral assay of chronic alcohol’s effects on heavy PFC-dependent behavior. Pavlovian fear extension measures animals’ capacity to use new information about a trauma-associated cue to learn to suppress anxiety. The animal undergoes classical conditioning with a tone associated with foot shock and then undergoes extinction training using the conditioned stimulus (tone) with no foot shock, so the animal learns that the tone is associated with no foot shock. Later the stressful tone is administered alone to measure how much fear it elicits in the animal, testing the animal’s ability to acquire and express this extinction memory in time. This work has relevance to human anxiety disorders in that people with PTSD have deficits in fear extinction, specifically the retention of fear extinction memories. Alcoholics are at elevated risk of experiencing major stressors, and anxiety disorders commonly co-occur with alcoholism. If alcohol impedes the fear extinction process, individuals may be at greater risk for lasting effects of stress and trauma exposure.
Fear extinction relies on ventromedial PFC (vmPFC), providing a highly sensitive assay for loss of PFC function. Chronic exposure of mice to alcohol vapor to induce dependence was found not to affect acquisition of the initial fear response, suggesting that the animals were not in a high-anxiety state and that their general learning processes were unaltered. Only modest impairment was found in the rate of extinction acquisition in the animals exposed to alcohol. Importantly, the alcohol-exposed animals failed to retain fear extinction training, unlike the control animals that had received air instead of alcohol vapor. To tie the findings to vmPFC, Dr. Homes subjected animals to four cycles of exposure to alcohol vapor, reconstructed neurons from the vmPFC, and found profound alteration to dendritic arborization—but no change in other brain regions of the same animals that showed hypertrophy in the vmPFC. Morphological changes produced by alcohol, then, are highly localized to this brain region important for fear extinction.
This research provides a static snapshot of potential functional PFC changes, so the next step involved a more dynamic measure of prefrontal function, single-unit recording of the neurons in vivo. Early rat studies show a sharp increase in prefrontal activity as rats retrieve extinction memory, and the magnitude of the activity predicts well how they will retrieve the extinction memory. Earlier work derived similar increases in activity in mouse vmPFC during extinction retrieval. The investigators repeated the chronic alcohol procedure and, using 16-channel fixed arrays directed at the brain region, recorded neural activity as the animals retrieved the extinction memory. They found a strong correlate of the extinction impairment in the in vivo measure. This finding might be interpreted as a loss of neuronal encoding of extinction as a result of chronic alcohol exposure.
The investigators next learned whether potential molecular changes underlie the physiological, structural, and behavioral changes with alcohol by sorting neuronal activity into bursts, or clusters of spiking activity. On the retrieval component of the behavioral task, the amount of activity was significantly less in the alcohol-exposed animals. Earlier work on rats had shown that bursting in the same region was dependent on NMDA receptors. The study showed that administering the same dose of an NMDA receptor antagonist was sufficient to impair fear extinction, suggesting a link between the amount of NMDA-driven bursting and the efficacy of extinction memory and retrieval.
In a study using Western blotting to look at the expression of NMDA receptors, the investigator found a marked decrease in the expression of the NR1 subunit in the PFC. Most past literature had shown that chronic alcohol exposure in other brain regions, and at least one case in the PFC, produces an upregulation of NMDA receptors. An experiment to investigate the presence of a link between loss of NMDA receptor function—the fear extinction phenotype—by infusing an NMDA receptor antagonist into the vmPFC of alcohol-naïve mice reduced the function of NMDA receptors in the PFC akin to the effect of chronic alcohol. Dr. Holmes identified the need for further investigation to learn whether reductions in NMDA receptor function drive these abnormalities. The current heuristic model suggests that chronic alcohol reduces molecular disruptions in this brain region and disrupts the ability of the pyramidal neurons in this area to encode important behaviors and, as a result, produces behavioral deficits such as impairment in fear extinction.
This work with mice suggests that a history of chronic alcohol abuse and alcoholism may impair one’s ability to recover from a traumatic stressor. Open questions include: Could impaired recovery contribute to high rates of anxiety disorders in alcoholics? Could impaired extinction provide an early diagnostic marker for alcohol-induced prefrontal damage? “Big-picture” questions include whether investigators can show a similar role of the vmPFC in such behaviors as drinking control over reward seeking. The investigators also have interest in pursuing the notion of individual differences in genetic risk. Individuals differ greatly in their risk of alcoholism, and the mouse offers a way to leverage and test that question. The goal is to find ways to translate this knowledge into pharmacological treatments that restore vmPFC function.
Discussion. Dr. Holmes responded to Dr. Gary Murray that whether prefrontal changes are reversible remains unknown to date. With an eye to specifically targeting and manipulating those circuits to reverse alcohol’s effects, the investigators are trying to reengage circuits with optogenetic tools by expressing light-sensitive viruses in vmPFC and downstream predictions in the PFC to modulate the amygdala.
Integrated Neuroscience Initiative on Alcoholism—West (INIA-West) Consortium: Progress Report
Dr. George Koob, Director, NIAAA-supported Alcohol Research Center on Central Nervous System Effects of Cellular Neurobiology, and Director, INIA-West Consortium, stated that the 20-project NIAAA-supported consortium aims to confirm gene targets nominated by expression assays or other methods by use of transgenic, knockout, inducible knockouts, site-specific knockouts, RNAi, in situ hybridization, in vivo electrophysiology and imaging, and next generation sequencing. It also seeks to identify drug-able targets that are most promising for medications development for alcoholism treatment by use of novel molecules in concert with existing FDA-approved molecules in those animal models with the most predictive ability. In addition INIA-West works to attract new and innovative investigators to alcohol research by recruiting individuals for U01 grants and pilot projects, and by making informatics integrated data sets accessible, searchable, and interactive with other data sets for all scientists interested in alcoholism research. The administrative core provides leadership for the consortium of basic researchers who investigate the neurobiological bases of excessive drinking, provides scientific project management and oversight, and facilitates integration, dissemination, and translation of data. The research core and research projects, which interact directly, are integrated by the administrative core. The structure incorporates pilot projects, steering committee, and scientific advisory board. Since 2006, 3 of INIA-West’s 11 pilot projects have become research U01 proposals; 6 others have been or will be submitted as R01s. Dr. Koob identified a number of new investigators who have remained with INIA-West. He stated that INIA-West highly values dissemination of findings on its multiple websites, which present data and sophisticated research tools.
Dr. Koob described several of the consortium's unique findings on alcoholism. INIA-West has refined animal models of excessive drinking, in part in collaboration with INIAstress; identified new gene targets for excessive drinking; and developed novel approaches to functional validation. Through selective breeding for blood alcohol level, INIA-West has developed excessive drinking models such as Drinking-in-the-Dark, which features mice that drink lots of alcohol in 2-hour sessions at the beginning of the dark cycle, and has established, and also exploited the use of other, excessive drinking animal lines. INIA-West also has used a hybrid mouse line to identify genes that are over expressed in high-drinking mice. A study of chronic intermittent ethanol exposure revealed changes in the brains of mice that lever pressed for alcohol over a 1-hour session; these animals persist in high alcohol intake well into abstinence. INIA-West has developed a conceptual framework for the neurobiological bases of the transition to excessive drinking to identify how molecules generate the changes in synaptic plasticity that generate the change in weighting of the various specific neural circuits associated with addiction and alcoholism.
Based on its extensive gene expression studies using gene arrays, INIA-West has identified priority gene targets in four pathways: neuroinflammation, ion channels, second messenger systems, and epigenetic mechanisms. In addition to mice, INIA-West studies drosophila and rats, and, in conjunction with others, macaques and human brain material. In describing this work, Dr. Koob referenced Dr. Crews’s construct of the neuroinflammatory cascade in alcoholism. The processes and targets on which INIA-West focuses include the toll-like receptor (TLR4), proteins in the regulation of NF-kappaB, peroxysome proliferation activating receptor agonists (which modulate NF-kappaB, the transcription factor that drives the production of pro-inflammatory cytokines), possibly modulating the pro-inflammatory cytokines with the use of phosphodiesterase-4 inhibitors. INIA-West is applying small molecules to this work in hopes of moving into human laboratory studies in the near future, upon validation of the molecules. Dr. Koob stated that the work with TLR4 agonists shows that this treatment blocks excessive alcohol consumption with no nonspecific effects. Other important findings: CD14 knockout mice that do not drink a lot of alcohol in excessive drinking paradigms have a dramatically blunted electrophysiological response in the amygdala; GABA released from interneurons in the central nucleus amygdala, which is known to interface with CRF to drive anxiety-like behavior, is abolished in CD14 knockout mice. Dr. Koob stated that INIAstress is studying the possibility of a link between the neuroinflammatory system and the brain’s emotional systems through some of the brain’s stress transmitters, such as CRF.
In its work on other targets, INIA-West has found that one gene reappears in most of the consortium's array studies. Two ion channels may be involved in binge intoxication, and two sodium channels and two potassium channels may indicate where alcohol works to produce initial intoxication. Components of signal transduction pathways repeatedly appear in INIA-West’s work—a novel G-protein–linked receptor and a diacylglycerol zeta kinase. INIA-West also has developed novel approaches to functional validation. In collaboration with INIAstress, three projects at INIA-West use next-generation transcriptome sequencing to provide big-picture information on differential expression of genes, nucleotide by nucleotide; this new approach is poised to supplant microarray-based approaches for transcriptome analysis and provides a mechanism to understand underlying regulatory code. This approach enabled seeing a translation from mouse to rat to monkey.
Representing a major change in the consortium's direction, investigators have identified small molecules to target proteins identified by INIA-West genes, use animal models of excessive drinking to validate the effects of small molecules, and then will move these into human laboratory testing upon FDA approval. INIA-West currently focuses on the TLR4 antagonist, IKK-beta antagonist, and PD4 inhibitors.
Discussion. Dr. Adron Harris, Director, Waggoner Center for Alcohol and Addiction Research, and Scientific Director of the INIA-West Consortium, moderated the discussion. Dr. Koob concurred with Dr. Szabo’s suggestion that INIA-West share animal tissues with other investigators of inflammation. Dr. Crews underscored the fact that genomics is body wide, and that the gut and liver both contribute to the drinking transcriptome. He stated his anticipation that new therapies will emerge from these studies of the circuitry of addiction. Dr. Brooks inquired about the relationship of these processes to alcohol-induced leaky gut. Dr. Harris explained that injecting lipopolysaccharide (LPS), which is implicated in inflammatory liver disease in humans, into animals produces long-lasting increases in alcohol consumption. Dr. Koob noted that a leaky blood-brain barrier may exist as well. Dr. McClain added that the brain’s tight junctions act similarly to those in the intestine and lung, and that anti-inflammatory therapeutics good for the liver also should be good for addiction, intestine, lung, brain, and whole system.
INIAstress Consortium Progress Report
Dr. Kathleen Grant, Professor, Department of Behavioral Neuroscience, Oregon Health and Science University, explained that the INIAstress Consortium focuses on interactions between stress and heavy drinking. With a structure similar to that of INIA-West, INIAstress has organized itself to be translational and integrative in the programmatic areas of behavior/neuroendocrine, brain circuitry, and genetics. In the neuroendocrine area, INIAstress begins with the phenotype of excessive drinking examined through standard procedures such as alcohol vapor inhalation and a monkey model developed by Dr. Grant, which permits integrative research using the same phenotype. Inhalation chambers now exist in seven labs in five states and one European country. INIAstress focuses on the transition to dependence and altered stress response using neuroendocrine measures. The addiction circuitry is similar across all the labs that study addiction. In the monkey, the consortium focuses on the striatum and habitual vs. goal-directed drinking behaviors and stress reactivity. The mouse group design allows for additional research. The neurochemistry group presents opiate challenges and stress challenges to humans. Investigators seek to translate the research by using the same agent and post to the same dose range for monkeys in the opiate challenges, and then look at the same circuitry in mice. Genetic analysis involves SNP and GWAS blood epigenetics and genomics, which is translational with monkeys and which add opportunities to study longitudinal blood and brain samples.
A main INIAstress hypothesis is that excessive, prolonged, and intermittent exposure to alcohol affects the stress system, a combination of neural and physiological adaptations unique to ethanol. Under this hypothesis chronic ethanol results in unique changes to the central nervous system that promote ethanol self-intoxication. In monkeys the induction of drinking leads to some changes in the stress response, which become associated with excessive drinking and in turn result in greater deviations from homeostasis and thereby form a forward loop. According to the second main hypothesis of the consortium, once these changes have occurred in the animal, stressors other than alcohol intoxication are then channeled through these adaptations. The other stressors then promote excessive drinking leading to a further, perhaps irreversible, disruption in homeostasis.
In describing excessive drinking, Dr. Grant offered examples of how the consortium integrates data. The experimental design involves induction and then maintenance of ethanol self-administration by two monkey species. The monkeys first learn to drink and experience intoxication, and then to associate drinking with intoxication. Over the past decade about 150 monkeys have participated in the study, and 60 peripheral tissues and tissue from 70 brain areas from each monkey have been saved in a tissue bank available for the consortium and other research groups to study. The tissue bank also has blood alcohol measures were taken from the monkeys every 5 days over the course of a year of drinking. The protocol has captured the phenotype of an alcoholic drinker; but individual and gender differences in risk for heavy drinking were apparent. The most important predictive phenotype represents when an animal learns to associate intoxication with drinking. Dr. Grant showed a graphic example of drinking patterns in which both animals at risk for heavy drinking and animals not at risk for heavy drinking can gulp water, but the animal at risk for heavy drinking also gulps alcohol and gets drunk whereas the animal at low risk for heavy drinking learned to sip its alcohol and drink in a manner that avoids intoxication.
Dr. Howard Becker, Scientific Director, INIAstress, and Director, Alcohol Research Center on Treatment and Implications, Medical University of South Carolina, described similar chronic alcohol exposure cycles using the mouse model of dependence and relapse drinking developed by INIAstress. In this model, all animals can drink water at any time; both control and experimental groups have access to alcohol for 2 hours a day, but controls never receive chronic ethanol exposure. Periods of withdrawal follow the periods of chronic ethanol exposure for several cycles. The procedure allows comparisons in alcohol consumption between dependent and nondependent animals, and of dependent animals at baseline and then following the alcohol exposure. These studies found that nondependent animals maintain a stable intake level; they can regulate their consumption, in contrast to alcohol-dependent animals, which consume significantly more alcohol. Dependent animals drink more over the entire 2-hour session, but they “load up” in the first 40 minutes, which the investigators believe generates significantly higher blood alcohol levels, levels above the legal limit. Nondependent animals can regulate consumption but achieve intoxication below the legal limit. Nondependent animals experience a slow increase in blood alcohol levels, which resolves soon after a 2-hour drinking session. But dependent animals exhibit a higher peak brain ethanol concentration, which remains high for more than an hour after access to alcohol ends.
Repeated cycles of chronic ethanol exposure and withdrawal produce escalation of ethanol self-administration, faster ethanol consumption, elevated blood and brain ethanol levels, increased rewarding effect of ethanol, reduced sensitivity to aversive effects of ethanol, and increased relapse susceptibility. Dr. Grant explained that by using the two excessive alcohol drinking models—repeated chronic intermittent ethanol exposure in mice and SIP induction and open access self-administration in monkeys—INIAstress hopes to provide an endocrinological characterization of chronic ethanol intoxication as a chronic state of disequilibrium, a basis for the neural circuitry group to focus on mechanisms, a basis for the genetics group to inform both circuitry and behavioral/endocrine groups, and a basis for the behavioral group for identifying anxiety-like behaviors in these animals.
Dr. Grant stated that ethanol creates a constant state of disequilibrium, and INIAstress investigators found in monkeys with chronic ethanol self-administration a profound physiological change in the HPA axis that is similar to adaptations seen chronic alcoholics. INIAstress’s work with monkeys permits examining multiple hormones over time and achieving a comprehensive view. Changes from baseline of ACTH, deoxycorticosterone, and aldosterone were found to be associated with mean ethanol intake and are highly correlated. This is evidence that chronic alcohol imposes a unique chronic stressor to the system.
Dr. Becker explained that INIAstress’s circuitry analysis, using single-cell electrophysiology and synaptic electrophysiology in conjunction with structural imaging and resting state connectivity (fcMRI) imaging, focuses on many structures in the brain. After inducing chronic heavy intoxication in both the mouse and monkey, investigators have found reduced dopaminergic tone; downregulation of GABAergic inhibition and GABA-mediated inhibition; upregulation in excitatory neurotransmission mediated by glutamate in multiple different brain regions and circuits; and upregulation of corticotrophin releasing factor in the cortex and other brain structures.
Dr. Becker stated that brain-derived neurotrophic factor (BDNF), which is expressed throughout the brain and plays an important role in neurodevelopment, neuroprotection, and addiction, has been shown to be altered as a function of chronic intermittent alcohol exposure. INIAstress discovered downregulation of BDNF in the prefrontal cortex in three replicated studies. Investigators found reduced messenger RNA for BDMF, as well as reduced protein in the PFC. Investigators injected BDNF into mice to replenish the apparent downregulation of BDNF in the PFC and to investigate the effects on drinking patterns. BDNF does not alter drinking behavior in nondependent subjects, but dependent animals showed a significant reduction in drinking levels. Dr. Becker noted that INIA-West’s multiple laboratories engaged collaboratively in genetic and genomic work in circuitry groups to demonstrate in an animal model with implications for this and other targets.
In a similar approach with monkeys, Dr. Grant explained that imposing repeated periods of abstinence from alcohol resulted in a more defended daily intake rather than an erratic day-to-day drinking pattern. This appeared to be a learned phenomenon as alcohol intakes were still highly variable after the first interval of abstinence, but after the second abstinent period, animals became heavier and more highly regulated drinkers. Therefore, abstinence in both the mouse and monkey changes behavior towards alcohol. Bout-like drinking, with increased duration and volume ethanol consumed by gulping down alcohol, leads to high blood ethanol concentration levels. INIAstress’s electrophysiological group found that repeated abstinence periods increase the excitatory post-synaptic firing in the putamen, but not the caudate, of ethanol animals. Thus INIAstress believes that this type of habitual behavior has a neurobiological correlates that can serve as targets to understand who an organism can regulate the daily excessive intake of alcohol. For example, chronic ethanol self-administration with repeated abstinence results in a selective increase in putamen dendritic spine density and suggests an increase in the number of glutamatergic excitatory synapses. How this altered activity in the putamen is correlated with adaptations in the HPA axis is the next step in understanding the feed-forward cycle of stress and chronic alcohol consumption.
INIAstress’s collaboration with INIA-West on structural MRI imaging has found that just 6 months of open access drinking produces a decrease in monkeys’ cerebral cortical volume that is highly related to alcohol consumption. Noting with excitement that this is a translational effect, Dr. Grant stated that this type of experiment can reveal the threshold dose for a decrease in cortical volume and can estimate a unit dose related to percentage decrease in cortical volume. Investigators found that not all cortical areas change in concert; for example, the prefrontal area decreased less than other areas in the brain. INIA-West conducts human and mouse brain research on resting state conductivity and INIAstress is collaborating on monkey brain research. The investigators’ hypothesis testing will involve areas that start to decrease their connectivity with chronic heavy drinking.
Dr. Grant observed that the Oregon National Primate Center’s pedigreed population enables choosing subjects for ethanol studies that have no common parents or grandparents. This longitudinal study’s goal is to compare within-brain changes and changes between blood and brain in terms of methylation. Epigenetic changes in genomic DNA—genes related to inflammation, serotonin, dopamine, cortisols, GABA, melanocorticoid, and neurotrophins—have been found in response to chronic consumption of alcohol. In studying a combination of recombinant mice and individual rhesus monkeys, INIAstress has identified 51 genes that are highly coregulated between mouse and monkey models, establishing a good platform for the translation from mouse to monkey and then from monkey to humans.
Dr. Grant identified INIAstress’s major findings in its work on alcohol and stress comorbidity. The monkey model developed in the consortium is the first model where animals drink voluntarily to physical dependence and show associated brain and multi-organ pathology. The bioinformatics developed in the consortium has identified key nodes in oxidative and metabolic stress response that can be targeted for pharmacotherapeutic development. CNS imbalance in excitatory/inhibitory tone underlies alcohol dependence with an upregulation of excitatory tone and a downregulation of inhibitory tone. The in vivo imaging studies provide a translational link in human and nonhuman primates; decreases in cortical volume are apparent after heavy drinking.
INIAstress is working on a new novel target, apamin, a small potassium channel blocker that in control animals increases ethanol drinking in a dose-related manner, but not in already heavy-drinking animals. This information likely can be used to understand neuroadaptations related to ethanol. Alcohol has both pro-inflammatory and anti-inflammatory qualities; NF-kappaB is a main target in pro-inflammatory work.
The INIAstress Consortium is a unique translational approach to discovering drug-able targets and addressing mechanisms of chronic ethanol consumption and dysfunctional homeostasis indicative of disease. The approach represents a translational bridge to human research.
Discussion. In response to a question from Dr. de la Monte, Dr. Grant stated that INIAstress has observed profound age–dependency on risk for heavy drinking. Young adult male monkeys are most at risk but just slightly more at risk than the later adolescent. Both groups are at much higher risk than monkeys with adult onset drinking. In contrast, female rhesus monkeys are at greater risk for future heavy drinking if they begin drinking in late adolescence. The risk for males correlates with testosterone. Dr. Crews encouraged INIA-West to investigate whether adolescent alcohol exposure might change the way mice eventually respond to alcohol in the current model. Dr. Grant responded to Dr. de la Monte that INIAstress currently is conducting DTI analysis for tracking white matter. Dr. Barthwell observed that a 28-day period of abstinence is the current model of care for humans and inquired about how the model might affect humans. Dr. Grant responded that humans would respond differently whether the abstinence were voluntary or involuntary, and Dr. Barthwell commented that most voluntary treatment is imposed. Dr. Becker added that multiple detoxifications tend to have more medically complicated and more severe withdrawal responses in both human and animal models, demonstrating the need to develop new therapeutics.”
Time was set aside for public comment, but no one stepped forward to speak.
Dr. Warren adjourned the meeting at 12:35 p.m.
I hereby certify that, to the best of my knowledge, the foregoing minutes are accurate and complete.
Kenneth R Warren, Ph.D.
Acting Director, National Institute on Alcohol Abuse and Alcoholism
Chairperson, National Advisory Council on Alcohol Abuse and Alcoholism
Abraham P. Bautista, Ph.D.
Director, Office of Extramural Activities
Executive Secretary, National Advisory Council on Alcohol Abuse and Alcoholism