Markus Heilig - US grants

MP currently utilizes two types of animal models: animals that escalate their alcohol intake following a prolonged history of intermittent brain alcohol exposure (post-dependent), or animals that have interesting alcohol-related phenotypes as a function of genetic factors, such as rats selectively bred for high alcohol preference, or mice or rats that have been genetically engineered. These different models may tap into different biological mechanisms, and are therefore complementary. Voluntary alcohol consumption and its escalation over time are studied in several models. Relapse-like behavior is studied in animals using reinstatement models, in which lever-pressing for alcohol is first established, and then extinguished by removing the alcohol. Presentation of drug-associated cues or exposure to stress will lead to resumption of lever-pressing on the previously alcohol-delivering lever (reinstatement). Reinstatement induced by cue or stress is differentially sensitive to pharmacological manipulations, and evaluating candidate drugs for their ability to suppress reinstatement induced by the two different types of stimuli guides optimal selection of target populations in initial clinical trials. Information may also be gained on how treatments might be combined to obtain additive effects. Mu-opioid receptor gene (OPRM1) The opioid receptor antagonist naltrexone (NTX) is an established treatment for alcoholism, however there is considerable heterogeneity of responses in clinical populations. Recent evidence suggests that a non-synonymous SNP, 118A→G, found in the human OPRM1 gene that encodes the mu-opioid receptor may modulate alcohol reward and therapeutic response to NTX. In an attempt to establish the mechanistic role of human OPRM1 A118G variation, and to isolate the effects of this polymorphism from those of alleles in linkage disequilibrium with it, we created two lines of humanized mice carrying the respective human allele (118AA and 118GG). We observed a markedly enhanced dopamine (DA) release in the nucleus accumbens of 118GG mice in response to alcohol. This finding was consistent with a positron emission tomography study conducted in parallel in humans, which found a more vigorous DA response to alcohol in the ventral striatum in 118G carriers (Ramchandani et al., 2011). Further work with the humanized mouse lines revealed elevated alcohol consumption in 118GG mice, and experiments are underway to investigate whether 118GG mice will be preferentially or selectively sensitive to NTX (Thorsell et al., in preparation). Substance P and its NK1 receptor Substance P mediates stress responses, and blockade of the receptor for substance P, NK1R, produces anti-stress effects. We have shown that genetic deletion of NK1R in mice leads to markedly decreased alcohol preference, a lack of conditioned place preference for alcohol, and an absence of escalated intake over time. Furthermore, pharmacological blockade of NK1 receptors using the NK1 antagonist L-703,606 suppresses alcohol consumption similar to genetic deletion of the receptor (Thorsell et al., 2010). We recently collaborated with Dr. Kenner Rice to resynthesize a literature compound, L-822429, with high affinity for the rat NK1 receptor. L-822429 dose-dependently suppressed stress-induced reinstatement of alcohol seeking in Wistar rats, but had no effect on cue-induced reinstatement, suggesting the possibility of additive effects of an NK1R antagonist and opioid antagonists such naltrexone (Schank et al., in press). In follow-up work using genetically selected alcohol preferring P-rats, L-822429 dose-dependently suppressed self-administration rates in P-rats but was ineffective in non-dependent Wistar rats. A nuclease protection assay showed an innate upregulation of NK1R receptor expression in several brain regions in P rats, with the greatest up-regulation within the amygdala (Schank et al., in preparation). Together, these finding further support NK1R antagonism as a candidate target for the treatment of alcoholism. Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor Elevated self-administration of alcohol and an increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH1 subtype of CRH receptors in the amygdala (Sommer et al., 2008). We had previously identified a series of CRH antagonists with suitable properties for clinical development (Gehlert et al., 2007;Thorsell et al., unpublished data). Two of the candidates, pexacerfont and GSK561679, were selected to enter experimental medicine studies in our clinical program under CRADAs with Bristol Myers Squibb and GlaxoSmithKline respectively. Those studies are currently underway. Neuropeptide Y (NPY) NPY is a an endogenous anti-stress peptide that counteracts the behavioral stress effects of CRH. Activation of NPY-Y1 receptors, or blockade of presynaptic Y2 autoreceptors, suppresses escalated post-dependent drinking, while leaving basal intake of alcohol in non-dependent animals unaffected (reviewed in Thorsell et al., 2006). NPY also potently suppresses reinstatement of alcohol seeking induced by the pharmacological stressor yohimbine (Cippitelli et al., 2010). Because orally available, brain penetrant Y1 agonists are currently unavailable, we evaluated a non-peptide compound under a CRADA with Johnson and Johnson Pharmaceuticals, JNJ-31020028, targeting the Y2 receptor to determine its potential for clinical development. Although showing some of the expected activity, the effects of JNJ-31020028 followed an inverted-U-shaped dose-response curve, possibly reflecting activity at Y2 heteroceptors on glutamatergic terminals at higher doses (Cippitelli et al., 2011). Thus, despite the scientific appeal of targeting the NPY system, this line of research is no longer of high priority in our program. Neuropeptide S (NPS) NPS suppresses anxiety and appetite in experimental animals, and initial work in collaboration with the Ciccocioppo laboratory indicated that NPS signaling may play a role in relapse to alcohol seeking (Canella et al., 2009). We developed a screenable assay for the NPS receptor, and in collaboration with the NIH Chemical Genomics Center (NCGC) identified a lead molecule, NCG001865684, that was determined to be brain penetrant upon peripheral administration in rats. Pretreatment with NCG001865684 was found to prevents NPS-induced suppression of feeding, and to suppress operant responding for alcohol. However, no effect was found on cue- or stress-induced reinstatement of alcohol seeking. Optimization is currently ongoing at NCGC, and is focused on engineering out off-target activity discovered in the course of a standard CEREP screen of common drug targets. Gene expression profiling and epigenetic modifications A series of projects are being carried out looking at changes in gene expression and epigenetic modifications in the post-dependent rat model to identify potential pharmacological targets. Bioinformatic analysis of the initial runs applying RNAseq, CHIPseq and microRNA (miR) has identified several interesting, differentially expressed transcripts as well as several cases of miR-mediated changes in gene expression (Tapocik et al., in preparation). Investigation of epigenetic modifications revealed increased DNA methyltransferase activity in the hippocampus of post-dependent rats, and hypermethylation induced in the brain by L-methionine injection was found to increase alcohol self-administration as well as the motivation to consume alcohol (Barbier et al., in preparation).

Effects of Early Stress on Central Serotonin System Functioning Traumatic experiences in childhood are associated with increased risk for developing mood and anxiety disorders. Low serotonin(1A) receptor (5-HT(1A)R) density during development has been proposed as a trait-like characteristic leading to increased vulnerability to neuropsychiatric disorders. To assess the relationship between early adversity and alterations in the serotonin system, we used PET to measure in vivo 5-HT(1A)R density and dissociation constant (K(D)(app)) in the brains of juvenile rhesus monkeys with and without histories of stress in the form of peer rearing (PR vs. mother-reared, MR). We found that 5-HT(1A)R density and K(D)(app) were decreased in PR animals. We also observed an increase in receptor density in the dorsomedial prefrontal cortex of PR females. These findings demonstrate that early adversity is associated with long-term alterations in the serotonin system and support human studies suggesting that reduced 5-HT(1A)R density during development might increase vulnerability to stress-related neuropsychiatric disorders. As alterations in the serotonin system were both gender- and region-specific, this may suggest a biological basis for the higher prevalence of affective disorders in women. Early Life Stress and GxE Interactions in Nonhuman Primates Genetic factors interact with environmental stressors to moderate risk for human psychopathology. The first genetic variants to have been shown to interact with early experience in humans were repeat variants present in regulatory regions for the Monoamine Oxidase A and Serotonin Transporter genes (MAOA-VNTR and HTT-LPR, respectively). These variants occur across the catarrhine primates, providing an opportunity to examine effects of MAOA-VNTR or HTT-LPR genotype in laboratory and free-ranging primates and to determine whether these variants predict differences in behavior as a function of stress. MAOA-VNTR In humans, the promoter of the MAOA gene is sensitive to both glucocorticoids and androgens, and interactions between MAOA genotype and both early stress and testosterone levels increase levels of aggression. We have found that the orthologue of the human MAOA-VNTR low activity allele predicts highly aggressive responses to unfamiliar conspecfics, particularly in adolescent/adult males. The interaction between genotype and age might suggest that testosterone levels influence aggressive responding as a function of MAOA-VNTR variation and, consistent with this, we find that free-ranging macaque males with low activity allele that have high age-adjusted levels of testosterone are more likely to emigrate at a young age from the natal troop. In macaques, highly aggressive males are often peripheralized from the natal troop, forcing early emigration. This can result in early mortality, but is thought to be balanced by the potential increase in reproductive output for these individuals. As stated above, the low activity MAOA-VNTR allele predicts antisocial behavior in humans exposed to early adversity. We wanted to investigate the effects of the orthologous rhesus variant on high-risk aggression and alcohol consumption in rhesus macaques that were either MR or PR. We found that, when controlling for age, MAOA-VNTR genotype interacts with early stress to predict high risk aggression towards an unfamiliar conspecific and, further, that under conditions that involve availability of alcohol, PR monkeys that are carriers of the low activity MAOA-VNTR allele exhibit higher levels of alcohol consumption. Our results demonstrate that macaques carrying the low-activity MAOA-VNTR allele and who are exposed to early life stress are more prone to high risk behaviors, such as aggression and alcohol consumption, and provide further evidence that MAOA-VNTR may be a risk allele for early onset alcohol dependence in human subjects. HTT-LPR The short (s) HTT-LPR allele has been shown to moderate the effects of stress on vulnerability to mood and anxiety disorders. The mechanism by which this occurs may relate to differential sensitivity to stressful life events. We have previously demonstrated that the effects early stress in the form of peer rearing is moderated by HTT-LPR genotype, and these findings have been shown to translate to the human condition. However, whether genotype predicts responses to stress in normally-reared primates and across step-wise increases in cumulative stress exposures has not yet been examined. Such an approach could be important because of its potential to be highly sensitive for detecting moderating effects of genotype on depression-related behavior. It could also inform us of how genotype predicts tendencies toward habituation and/or sensitization to stress, which could potentially be more easily translated to the human condition. We wanted to explore whether behavioral responses to repeated maternal separation stress would be influenced by HTT-LPR genotype in infant rhesus macaques and whether these responses differed as a function of duration and repetition (number of exposures) of the stressor. We found that, across successive exposures to stress and as a function of increased duration of the stressor, infants carrying the HTT-LPR s allele were more likely to develop signs of distress and behavioral pathology. Our data in nonhuman primates suggest that in children, the s allele may increase sensitivity to repeated, chronic stressors, leaving them more vulnerable to affective psychopathology. This may make such individuals more reactive during early development, may influence the trajectory of responses over periods of adjustment or stress, and may predict vulnerability to psychopathology later in life. Effects of Early Adversity on Alcohol Consumption Patterns in Humans Over the years, we have been examining persistent effects of early adversity on endocrine and behavioral responses to stress and alcohol consumption using a rhesus macaque model. The advantage of this model is that it is tightly controlled and that potential confounds are reduced or eliminated. The ultimate goal of these studies has been to identify effects of stress in macaques that may increase risk for human psychopathology. We recently performed analyses on a clinical sample to determine whether our primate findings would translate to the human condition. We wanted to determine the prevalence of early life trauma and stress in a sample of detoxified alcoholics to determine whether early stress increased risk for alcohol problems in our sample. We then wanted to evaluate the relationship between childhood adversity and alcohol-related outcome measures, including co-morbid psychiatric diagnoses. We found that adverse childhood experiences were highly prevalent in our sample of recently detoxified alcoholics. While patients who experienced early life stress or trauma did not differ from patients lacking these experiences in their recent drinking history, they did exhibit earlier onset of alcohol use as well as a higher level of alcohol dependence severity. In addition, early adversity was associated with a higher risk of co-morbid anxiety, mood, and substance use disorders. Our findings are consistent with data derived from nonhuman primate studies and with previous research demonstrating the impact of adverse childhood experiences on adult alcohol-related and behavioral health outcomes. Future investigations will examine the moderating role of sex and of genetic factors on the effects of early life stress exposure in this population.

The following projects have been or are currently being conducted by CATE. 1) Modulation of central glutamate by acamprosate A prevalent theory states that progressive emergence of a hyperglutamatergic state is key to the pathophysiology of alcoholism, and is associated both with emotional dysregulation (leading to craving and relapse), and neurotoxicity (leading to loss of grey matter and cognitive impairment). Acamprosate is a medication thought to target the hyperglutamatergic state. Here we used magnetic resonance spectroscopy (MRS) as a translational biomarker to obtain a measure of central glutamate using single-voxel 1H-MRS at 3T in the anterior cingulate cortex. We are currently performing secondary analyses on biospecimens to assess the effects of treatment on cytokine levels in the CSF and plasma. 2) Effect of naltrexone on craving and ethanol-induced brain activity Findings from animal and human studies indicate that the rewarding properties of ethanol arise in part from a complex interaction between alcohol, endogenous opioids, and dopamine systems. Naltrexone (NTX), an opioid receptor antagonist, has been studied widely in both preclinical and clinical research for the treatment of alcoholism. This protocol is the first study which involves administering IV alcohol to treatment-seeking alcohol dependent patients. Following detoxification, patients are randomized, using a double-blind design, to receive NTX or placebo throughout the course of their hospitalization. This study examines interactions between brain alcohol exposure and naltrexone treatment on the processing of positive and negative emotional stimuli using fMRI. Data analysis reveals that at a BAL of approximately 0.8 g% there is a lack of ethanol-induced BOLD response in the ventral striatum (VS). This result is in contrast to our previous findings in social drinkers that showed activation of the VS. Results have been submitted for publication. 3) NK1R antagonism in alcoholism co-morbid with PTSD Alcoholism is the most common co-morbidity in men with PTSD and the second most common in women with this disorder. It is theorized that stress associated with PTSD (e.g., fear/anxiety, anger, hyper vigilance) promotes negatively reinforced use of alcohol to cope with negative affect. Therefore, we hypothesized that Substance P (SP)/NK1R signaling may be an attractive target for treatment of co-morbid alcoholism and PTSD. We asked whether NK1R blockade would suppress alcohol craving induced by stress or alcohol-associated stimuli, and whether it will improve PTSD symptoms in patients with co-morbid alcoholism and PTSD. Patient accrual for this protocol is complete. Results have been submitted for publication. 4) Corticotropin-releasing hormone receptor 1 (CRH1) antagonism and stress-induced craving in alcohol dependent women with high anxiety. As alcoholism evolves, a shift occurs from positively to negatively reinforced alcohol use. Along the way, stress becomes a major trigger of relapse and excessive alcohol intake. Corticotrophin-releasing hormone (CRH) plays a major role in this state. We have negotiated a CRADA with GlaxoSmithKline, obtained an IND, and initiated a protocol that uses GSK561679 (CRH antagonist) as a translational tool in anxious female alcoholics (preclinical toxicology studies show that its use leads to a suppression of spermatogenesis, thus precluding its use in males). The hypothesis is that GSK561679 will reduce alcohol craving in response to the combined challenges of the Trier Social Stress Task, alcohol-cue exposure, and guided imagery scripts. We will also examine whether GSK561679 will reduce spontaneous craving, reduce fMRI BOLD responses to negative affective stimuli within the ventral visual stream, medial temporal lobe and/or the anterior insula, and modulate blood markers of endocrine function in a manner indicative of anti-stress effects. Thirty-seven subjects have completed. 5) CRH1 antagonism in anxious alcoholics This study uses another CRH1 antagonist, pexacerfont, obtained through a CRADA with Bristol Meyers Squibb. Pexacerfont is also an orally available, brain penetrant selective CRH1 antagonist. It has the advantage that safety has been established for the administration in both males and females. Because of the broader utility of pexacerfont in a therapeutic area where the majority of patients are male, we chose pexacerfont as the tool for a consortium of NIAAA/NIDA investigators who will use it in a cluster of related protocols. In the pexacerfont alcohol protocol, we will examine whether the CRH1 antagonist will reduce alcohol craving in response to the challenges as previously described for the GSK561679 CRH antagonist. We will also examine whether pexacerfont will reduce fMRI BOLD responses to a novel social stress challenge developed jointly with our NIDA collaborators. Sixty subjects have been enrolled and completed. In addition 8 subjects have been enrolled in a sub-study involving a lumbar puncture to investigate the concentration of the drug in the brain. 6) Collaborative project with NIMH: Imaging cannabinoid CB1 receptors in patients with alcohol dependence The brain endocannabinoid (EC) system involves endogenous cannabinoid agents (ECs) that act upon specific receptors (CB1 and CB2). ECs and CB1 receptors appear to modulate the brain reward system. During chronic alcohol exposure, EC levels in the brain are elevated and CB1 receptor levels are consequently reduced; this appears to be reversible following withdrawal. To what extent ECs and CB1 receptors are involved in the pathophysiology of alcohol dependence in humans is currently unknown. In this protocol, we utilize positron emission tomography to explore CB1 receptor abnormalities at various stages of alcohol withdrawal in humans. Results show that alcohol dependence is associated with a widespread reduction of cannabinoid CB1 receptor binding in the human brain, and this reduction persists at least 2-4 weeks into abstinence. The correlation of reduced binding with years of alcohol abuse suggests an involvement of CB1 receptors in alcohol dependence. Initial results were published in Molecular Psychiatry (2012). Enrollment continues to study individuals with other addictions. 7) Pilot study of deep brain stimulation (DBS) for the treatment of alcoholism Case reports of beneficial effects from DBS of the ventral striatum in alcoholism have been published, but no controlled data are available. In collaboration with NINDS and Medtronic, a DBS protocol that uses a controlled design has been approved by the IRB and the FDA. The review process of this protocol has been extensive. Patient accrual is in progress. 8) Role of proinflammatory signaling in alcohol craving Ethanol-induced activation of innate immunity has emerged as a potential mechanism for understanding the pathophysiology of alcoholism. The objective of the present study is to evaluate the role of proinflammatory signaling in alcohol craving. The peroxisome proliferator-activator receptor gamma agonist pioglitazone, which modulates glial activity, will be used as an experimental treatment. Guided imagery scripts will be used as an established set of stimuli to induce craving. Low dose lipopolysaccharide (LPS) administration, which activates proinflammatory signaling, will be used as a novel challenge and evaluated for its ability to provoke alcohol craving. If LPS in fact induces alcohol craving, the present design will allow evaluation of whether pioglitazone can inhibit this response. Nine patients have completed.

The Office of the Clinical Director (OCD), Intramural Research Program (IRP), National Institute on Drug Abuse (NIDA), NIH provides research support to two clinical research branches at the IRP. The scope of the research program is broad and is strongly represented in neuroimaging, while also incorporating both behavioral and pharmacological treatments for substance abuse disorders. The Office of the Clinical Director has a staff of an Administrative Assistant who provides primary support to the Director and Deputy Director. This Administrative Assistant also coordinates with the IRP Pharmacy, Matthews Media Group, Inc. (MMG), Medical Records Department, the Mid-Level Providers and Nursing. The three full time Mid-Level Providers (Federal) medically screen potential participants as well as assist in the day to day running of various protocols. Four full time RNs provide the staffing for running the protocols. Additional Clinical staff supporting the NDA/IRP programs include 5 full time Research Associates. These positions are part of the clinical services provided through the Johns Hopkins Bayview Medical Contract with NIDA/IRP. This contract also provides essential infrastructure and services including professional physician consultations, laboratory medicine and staff support as well as overnights for subjects at Behavioral Pharmacological Research Unit (BPRU) located on the Bayview campus. The NIDA IRB office is under the direction of the OCD and is staffed by an IRB Administrator (Federal). The office handles approximately 400 IRB related actions a year, from 40 active protocols. This year the IRB office has joined with the NIAAA forming the Addictions IRB which continues to answer to the OHRP. The OCD oversees a fully funded contract with MMG, an outside organization. MMG represents NIDA/IRP, recruiting research participants in the Baltimore Washington area. At this time MMG employs 13 screening specialists, 2 patient counselors and a 3 member management team. There is a yearly budget of $225,000 for print, web, radio and television advertising and $50,000 for community outreach. The Medical Records Department at NIDA/IRP is designed to maintain department compliance of Policy and Procedures while safekeeping the Privacy of over 6300 electronic (HuRIS System) medical records annually. Both microfilm and/or DVD as well as hardcopy versions of the medical records are accessed for both in-house and outside sources. This is done according to the National Institute of Health, Federal and State Rules and Regulations (Including the Privacy Act of 1974 and HIPPA). The IRP Pharmacy employs three full time pharmacists. The IRP pharmacy currently supports about 20 clinical studies including 5 Archway studies and 70 researchers/labs for nonclinical studies. One pharmacist devotes about 80% of her time supporting the Archway studies, including IND support. The other two pharmacist spends about 60 % effort on clinical and 40 % effort on nonclinical studies. Clinical research support includes reviewing, preparing, compounding, and dispensing the study medications. Nonclinical support includes ordering, compounding, dispensing, and laboratory auditing. In addition, the pharmacy monitors drug inventories, and meets all DEA and FDA regulatory requirements including licensing and IND reporting. Recently the OCD established the Office of Regulatory Affairs. The Regulatory Affairs Officer oversees the compliance of all research for local, federal and NIH guidelines. That full time Federal employee works with the IRB, DSMB (Data Safety and Monitoring Board)and outside contractors to fulfill monitoring needs.

MR Volumetric Measurements: We have collected full, volumetric T-1 weighted MR images to measure intracranial volumes several hundred alcoholics and healthy, non-alcoholic subjects. Alcoholics show greater brain degeneration than non-alcoholics. In addition, alcoholics have smaller ICVs than controls suggesting that pre-morbid differences in brain size may contribute to the risk for alcoholism. Despite the significant difference in ICV, degeneration accounts for a greater amount of the difference in brain volume between alcoholics and controls than brain growth does. We have examined how a family history (FH) of heavy drinking affects both brain shrinkage as measured by the ratio of brain volumes to ICV as well as maximal brain growth as measured by ICV in early-onset and late-onset alcoholics. FH positive alcoholic patients have significantly smaller ICVs than FH negative patients, suggesting smaller premorbid brain growth among alcoholics with a heavy drinking mother or father. Brain shrinkage was not affected by FH. Late-onset alcoholics show a greater difference in ICV between FH positive and FH negative patients than early-onset alcoholics. These data provide evidence that heavy parental alcohol use may increase risk for alcoholism in offspring in part by a genetic and/or environmental effect resulting in reduced brain growth. Insula Morphometry: The insula, a structure involved in higher order representation of interoceptive states, has recently been implicated in drug craving and social stress. Here, we performed brain MRI to measure volumes of the insula and subcortical regions with direct insular connections in 26 alcohol dependent patients (ADPs) and 26 healthy volunteers (HVs). In ADPs, anterior insula volumes were bilaterally reduced compared with controls. This reduction was selective, as neither posterior insula nor total brain gray matter volumes differed between groups. In ADPs, we also found increased volume of several subcortical structures that have direct afferent, efferent or reciprocal connections with the insula. Specifically, left and right amygdala, left and right thalami, and the right nucleus accumbens were larger in ADPs than HVs. The amygdala, which demonstrated the greatest volume increase in the alcoholics, has strong reciprocal connections with the insula. Postmortem studies of the anterior insula showed that decrease in the volume of the anterior insula was associated with a diminished population of Von Economo neurons in the subjects with a history of alcoholism (n=6) as compared with the subjects without a history of alcoholism (n=6). The pattern of neuroanatomical change observed in our alcohol dependent patients might result in a loss of top-down control of amygdala function, potentially contributing to an inability to control negatively reinforced alcohol seeking and use (Senatorov et al., manuscript in preparation). MR Diffusion Tensor Measurements: Many brain imaging studies have demonstrated reductions in gray and white matter volumes in alcoholism, with fewer investigators using diffusion tensor imaging (DTI) to examine the integrity of white matter pathways. Among various medical conditions, alcoholism and post-traumatic stress disorder (PTSD) are two comorbid diseases that have similar degenerative effects on the white matter integrity. It is widely believed that these types of abnormalities in both alcoholism and PTSD are related to fronto-limbic dysfunction. DTI was used to measure white matter fractional anisotropy (FA), which provides information about tissue microstructure. We quantitatively investigated the microstructure of white matter through whole brain DTI analysis in 19 healthy volunteers (HV) and 19 alcohol dependent subjects without PTSD (ALC) and with 17 PTSD (ALC+PTSD). These data show significant differences in FA between alcoholics and non-alcoholic HVs, with no significant differences in FA between ALC and ALC+PTSD in any white matter structure. We performed a post-hoc region of interest analysis that allowed us to incorporate multiple covariates into the analysis and found similar results. Relative to all alcoholics, HV had higher FA in several areas implicated in the reward circuit, emotion, and executive functioning, suggesting that there may be microstructural abnormalities in white matter pathways that contribute to neurocognitive and executive functioning deficits observed in alcoholics. Furthermore, our data do not reveal any differences between ALC and ALC+PTSD, suggesting that the effect of alcohol on white matter microstructure may be more significant than any effect caused by PTSD (Durkee et al., manuscript in review). Morphometric Measures: Alcoholism has been associated with a widespread pattern of gray matter atrophy. We sought to investigate the spectrum of volume alterations in a population of alcoholics with only alcohol dependence, polysubstance abusing alcoholics, and a comparison population of healthy controls. Thirty-seven pure alcoholics, 93 polysubstance abusing alcoholics, and 69 healthy controls underwent structural T1 MRI scans. Voxel-based morphometry was performed to investigate gray matter alterations. Alcoholic dependent inpatients (both with and without a history of DSM-IV substance abuse/dependence diagnosis) displayed significant gray matter differences in the mesial region of the frontal lobe and right temporal lobe. Pure alcoholics exhibited a pattern of subcortical changes similar to that seen in Wernicke-Korsakoff Syndrome when compared to polysubstance abusing alcoholics. Pure alcoholics and polysubstance abusing alcoholics did not differ significantly in measures of cortical gray matter, liver function, or nutrition. This study calls for additional research in order to investigate the spectrum from uncomplicated alcoholism to Wernicke-Korsakoff Syndrome (Grodin et al., 2013). Cortical Thickness: Alterations of brain structures have been seen in patients suffering from drug abuse or mental disorders. Similar changes in volume of brain structures have been observed in both alcoholic men and women. We examined the thickness of gray matter in the cerebral cortex in control men and women (n=69, 47 men) and alcohol dependent subjects (n=130, 83 men) to test the hypothesis that alcoholic inpatients would have more cortical damage than controls. Covarying for age and years of education, we confirmed significant differences between alcoholics and healthy controls in cortical thickness in both the left and right hemispheres. Significant differences in cortical thickness between control men and women were also observed. These differences may reflect sexual dimorphisms in the human brain, a genetic predisposition to alcoholism and comorbid drug use, and the extent of gray matter damage in alcoholism and substance use (Momenan et al., 2013). MR Spectroscopy of acute alcohol infusion: We are collaborating with Section on Human Psychopharmacology (Dr. Ramchandani) to measure the effect of acute alcohol infusion on brain metabolites, particularly glutamate, in heavy and light drinkers. We bring human subjects to a steady state BAC of 0.08 g/dl while using MRS scans to measure the concentrations of alcohol, glutamate and other brain metabolites in the subject's brain. Our preliminary results not surprisingly indicate significant differences between brain Ethanol/NAA concentration ratio before and after infusion. However, there was no difference in pre- and post-infusion Glutamate/NAA ratio. When separating the only two Heavy Drinkers in current data set, the Light Drinkers had higher Glutamate/NAA ratio than Heavy Drinkers both before and after the infusion. Interestingly, the Light Drinkers had higher Glutamate/NAA ratio after the infusion. But, the Heavy Drinker subjects had decreased Glutamate/NAA ratio after the infusion.

Resting State and Functional Connectivity: Previous studies have shown diverse effects of alcoholism on resting state MRI (rsMRI) connectivity. Long-term alcoholics who are currently in a period of sobriety showed decreased synchrony of limbic reward regions with respect to both the anterior cingulate cortex (ACC) and the nucleus accumbens (NAcc) seeds (Camchong et al., 2013). However, few have examined task-free rsMRI connectivity with a population of alcohol-dependent subjects. We sought to investigate rsMRI connectivity with alcohol-dependent subjects (n = 22) and healthy control subjects (n = 22). Five minutes of closed-eyes rsMRI images were collected. Preliminary results indicate that compared to alcoholics, healthy controls had an increased functional connectivity in the default mode network (DMN), especially around the seed area (left posterior cingulate). Similarly, healthy controls also demonstrated stronger connectivity in the left executive control network (lECN). The regions of connectivity were, however, more diffuse in alcoholics On the other hand, alcoholics showed stronger connectivity in the visual occipital network (VON). Although in general control subjects demonstrated stronger resting state connectivity in various networks, including the default mode network (DMN), alcoholics showed more diffused connectivity. This suggests that long-term alcohol use may result in recruiting more regions of the brain in order to compensate for localized adverse effects of alcohol (Zhu et al., manuscript in preparation). Conditioned Place Preference: Classical conditioning is widely used to study motivational properties of addictive drugs in animals, but has rarely been used in humans. We established a procedure suitable for studying the neurobiology and individual determinants of conditioned place preference in humans. In a collaborative work with the University of Chicago (Harriett de Wits Lab) healthy volunteers were randomly assigned to four groups that received methamphetamine or placebo in the presence of distinctive environmental cues under paired or unpaired conditions. During each session, subjects performed tasks known to activate the ventral striatum. Tasks were performed in the presence of a distinctive context, consisting of a screen background image of a beach or mountains, accompanied by corresponding sounds. Separate groups of subjects carried out the tasks under high ($35-50) or low ($5-20) reward conditions. Within each of the two reward conditions, one group (paired), received methamphetamine (20 mg, oral) or placebo consistently associated with one of the contexts, while the other (unpaired) received drug or placebo unrelated to context. A fifth group (paired) performed the tasks with contextual cues but in the absence of monetary incentives. Before and after conditioning, participants carried out a series of forced choice tasks for the contextual cues and change of preference over time was analyzed. All paired groups showed a significant increase in preference for the drug-associated context, with a linear trend for increase in preference across the levels of reward. Preference was unrelated to subjective drug effects and did not change in the unpaired group. These data support the translational utility of our conditioning procedure for studies of reward mechanisms in humans (Mayo, et al., 2013) Medication Studies: a) Naltrexone Our section has designed fMRI tasks and analyzed the imaging data of Effect of Naltrexone on Craving and Ethanol-Induced Brain Activity, by the CATE (Dr. George) section. Positively reinforcing properties of alcohol are in part mediated by activation of the ventral striatum (VS). Alcohol-induced release of endogenous opioids is thought to contribute to this response. Preclinical studies show that the opioid antagonist naltrexone (NTX) can block this cascade, but its ability to do so in treatment seeking alcoholics has not been examined. In order to address this question we randomized sixty-three treatment-seeking alcoholics to NTX (50 mg) or placebo (PLC) daily. On day 7, participants underwent an alcohol cue reactivity session, and craving was measured. On day 9, participants received a saline infusion followed by alcohol, and also viewed affective stimuli in an MR scanner. Irrespective of medication treatment condition, and in contrast to prior findings in social drinkers, alcohol infusion did not activate the VS in the alcohol dependent patients. Unexpectedly, and across all other conditions, VS activation was greater in NTX treated patients than in the PLC group. NTX treated patients also reported increased craving in response to alcohol cue exposure compared to PLC subjects (Spagnolo et al., submitted). b) PTSD: Our section has designed fMRI imaging tasks and analyzed the imaging data of The Effect of NK1R Antagonism on Alcohol Craving and PTSD Symptoms in Alcohol Dependent Patients with PTSD, by the CATE (Dr. George) Section. Post-traumatic stress disorder (PTSD) and alcoholism are frequently co-morbid. This study was designed to determine the neural effects of neurokinin 1 (NK1) antagonist aprepitant on alcohol-dependent subjects with PTSD. Fifty-three patients with PTSD and alcoholism were admitted for 4 weeks to this experimental medicine study. In this randomized and double-blind study subjects fMRI responses to stimuli with positive or negative emotional valence were analyzed. In this study we found treatment group robustly potentiated ventromedial prefrontal cortex (mPFC) responses to aversive visual stimuli. This region of brain is critical for extinction of fear memories and in alcohol craving and relapse risk, our finding suggests that NK1 antagonism, rather than acutely attenuating stress responses, might be a useful pharmacological treatment to enhance the effect of extinction-based cue-exposure therapies (Kwako, et al., submitted manuscript). c) Varenicline: Our section has designed fMRI imaging tasks and analyzed the imaging data of as study conducted by Section on Human Psychopharmacology (Dr. Ramchandani). In this double-blind randomized study, 36 male and female heavy drinkers, aged 21-58 years, were randomized to receive Varenicline (2 mg/day) or placebo for 3 weeks. Following 2 weeks of treatment, participants underwent an fMRI scan while performing a variation of the MID task designed to evaluate the incentive salience of alcohol cues. In this version, instead of monetary rewards, participants were presented with visual cues signaling alcohol (intravenous alcohol infusion), food (highly palatable snacks) or no rewards. Results indicated that participants in the placebo group (n=17) showed significantly higher BOLD activation in striatal regions during anticipation of working for alcohol reward (alcohol cue) than the Varenicline group (n=19). The placebo group showed robust striatal activation to alcohol cue that was attenuated in the Varenicline group. A direct contrast of treatment groups showed significantly lower activation in the caudate for Varenicline compared to placebo group. There were no significant differences in response to food cues between treatment groups. The placebo group also showed striatal activation in response to notification of alcohol reward (alcohol hits), and this response was also attenuated in the Varenicline group. A direct contrast of treatment groups showed significantly lower activation in Varenicline compared to placebo group. These results indicate significant activation of striatal regions to alcohol cues and notification of alcohol reward in the placebo but not in the Varenicline group. This suggests that Varenicline may exert its effects by modulating the neural substrates underlying motivation for and incentive salience of alcohol reward in heavy drinkers (Vatsalya, et al., manuscript in preparation).