ABSTRACT. Andrew R. Ouellette1,2 a, Sarah M. Neuner1,3a, Logan Dumitrescu4,5a, Laura C. Anderson1, Daniel M. Gatti1, Emily R. Mahoney4, Jason A. Bubier1, Gary Churchill1, Luanne Peters1, Matthew J. Huentelman6, Jeremy H. Herskowitz7, Hyun-Sik Yang8,9,10, Alexandra N. Smith4, Christiane Reitz11, Brian W. Kunkle12, Charles C. White8,13, Philip L. De Jager8,13, Julie A. Schneider14, David A. Bennett14, Nicholas T. Seyfried15, Alzheimer’s Disease Genetics Consortium, Elissa J. Chesler1b, Niran Hadad1b, Timothy J. Hohman4,5b, Catherine C. Kaczorowski1,2bc

aCo-first author bCo-senior author cLead contact

At present, the genetic mechanisms that regulate cognitive decline and dementia onset during the aging process remain poorly understood. Our work utilizes the Diversity Outbred (DO) mouse population, a genetic reference panel recapitulating the genetic diversity present within the human population. Using age-interactive quantitative trait loci mapping of an aged DO population, we identified Dlgap2 as a potential modifier of working memory decline. To evaluate the translational relevance of this finding, we utilize longitudinal cognitive measures from human patients, RNA expression from post-mortem brain tissue, data from a genome-wide association study (GWAS) of Alzheimer’s dementia (AD), and new GWAS results in African Americans. We detected significant associations between DLGAP2 and AD phenotypes at the variant, gene expression, and methylation levels. Specifically, lower cortical DLGAP2 expression was observed in AD patients in addition to an association with more plaques and tangles at autopsy. Additionally, decreased DLGAP2 expression associated with faster cognitive decline. Our results highlight the benefit of using genetically diverse mice to prioritize novel candidates and will inform future studies aimed at investigating the cross-species role of Dlgap2 in regulating cognitive decline.

The Jackson Laboratory, Bar Harbor, ME, 04609, USA The University of Maine, Graduate School of Biomedical Science and Engineering, Orono, ME, 04469, USA

University of Tennessee Health Science Center, Memphis, TN, 38163, USA Vanderbilt Memory and Alzheimer’s Center, Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, 37240, USA

Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN, 37240, USA

Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, 85004, USA

Center for Neurodegeneration and Experimental Therapeutics and Department of Neurology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

Cell Circuits and Epigenomics Program, Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA, 02142, USA

Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA

Center for Alzheimer Research and Treatment, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02114, USA

Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Gertrude H. Sergievsky Center, and Departments of Neurology and Epidemiology, College of Physicians and Surgeons, Columbia University, New York, NY, 10032, USA

University of Miami, Miller School of Medicine, Miami, FL, 33136, USA

Center for Translational and Computational Neuroimmunology, Department of Neurology, Columbia University Medical Center, New York, NY, 10032, USA

Rush Alzheimer's Disease Center, Rush University Medical Center, Chicago, IL, 60612, USA

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322, USA.

ABSTRACT. Functional Genomics of the Lysosome in Parkinson’s disease Ye, H1, Yu, M2, Lee, T1, Robak, L3, Shulman, JM1,2,3,4

The endolysomal membrane system is responsible for trafficking and turnover of internalized proteins, and its dysfunction has been linked to human disease. Retromer is an evolutionarily conserved complex responsible for recycling proteins and lipids within the endosomal pathway; mutations in the VPS35 gene causes autosomal dominant Parkinson’s disease (PD). Nevertheless, the requirements of retromer in neurons and particularly within the aging nervous system remain poorly understood. In Drosophila, we show that retromer is required in the adult CNS, including for synaptic transmission, survival, and locomotion. With aging, retromer insufficiency triggers progressive endolysosomal dysfunction, impaired substrate clearance and lysosomal stress. GBA, which causes the lysosomal storage disorder (LSD), Gaucher’s, is also the most common risk factor for PD. We have discovered that genetic variants in many other LSD genes are broadly associated with PD susceptibility. Using a Drosophila model, we screened homologs of 44 LSD genes, identifying 15 enhancers of alpha-synuclein mediated neurodegeneration. In several cases, including NPC1, DNAJC5, SCARB2, and SMPD1, introducing a single mutant allele in the fly homolog induced dose-sensitive enhancement of progressive locomotor impairment. Overall, our results are consistent with an oligogenic inheritance model of PD risk converging on the lysosome in brain health and age-related disease.

1Department of Neurology, 2Department of Neuroscience, 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 4Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA

ABSTRACT. Ash, PEA1,*, Phanse, S2,*, Pourhaghighi, R2,*, Goebels, F2, Malolepsza, E3,4, Tsafou, K3,4, Nathan, A3,4, Chen, S5, Zhang, Y5, Wierbowski, SD5, Boudeau, S1, Hu, LZM2, Cromar, G6, Guo, H2, Becker, LA7, Gitler, AD7, Youssef, A8, Ratti, A9, Parkinson, J6, Lage, K3,4, Yu, H5, Bader, GD2, Wolozin, B1,10 and Andrew Emili2,8,11,12

We defined a global ‘interactome’ of multi-protein complexes in the mammalian brain. Multi-dimensional biochemical fractionation with mass spectrometry and machine learning was used to survey endogenous macromolecules in adult mouse brain. These assemblies have distinct physical and functional attributes and show regional- and cell-type specificity. We identified an RNA-binding protein complex associated with amyotrophic lateral sclerosis, which includes Tdp-43, Fus, Tia1 and Atxn2. The fidelity of this RBP complex is responsive to neuronal disease state in a TDP-43WT/WT transgenic mouse model of ALS. Whereas complexed RBPs are co-immunoprecipitated with human TDP-43 from the cortices of transgenic mice, depletion of Atxn2 confers neuroprotection and reduces the interaction of complexed RBPs with the exogenous TDP-43. By immunofluorescent microscopy, cortical neurons showing cytoplasmic distribution of TDP-43 also showed errant cytoplasmic redistribution of other RBP complex components. Our discovery that ALS-associated RBPs natively assemble as a functional splicing module raises the possibility that a more accurate descriptor of ALS/FTD is as an RBP ‘complexopathy’ that results in part from splicing defects due to insolubility of a subnetwork of RBPs. Thus, this Brain Interaction Map – or BraInMap – resource facilitates mechanistic exploration of the molecular machinery driving core processes and diseases of the central nervous system.

1. Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine 2. Donnelly Center for Cellular and Biomolecular Research, University of Toronto 3. Department of Surgery, Massachusetts General Hospital, Harvard Medical School 4. Broad Institute of Massachusetts Institute of Technology and Harvard University 5. Department of Biological Statistics and Computational Biology, Cornell University 6. Program in Molecular Medicine, Hospital for Sick Children and University of Toronto 7. Department of Genetics, Stanford University School of Medicine 8. Program in Bioinformatics, Boston University 9. Department of Neurology and Laboratory of Neuroscience, IRCCS Italy. 10. Department of Neurology, Boston University School of Medicine 11. Departments of Biochemistry and Biology, Boston University 12. Center for Network System Biology, Boston University * These authors contributed equally

ABSTRACT. Zhiqiang Sha1, Dick Schijven1, Clyde Francks1,2*

1 Language and Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, The Netherlands 2 Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands

Autism spectrum disorder (ASD) and schizophrenia have been conceived as partly opposing disorders in terms of systemizing versus empathizing cognitive styles, with resemblances to male versus female average sex differences. Left-right asymmetry of the brain is an important aspect of its organization that shows average differences between the sexes, and can be altered in both ASD and schizophrenia. Here we mapped multivariate associations of polygenic risk scores (PRS) for ASD and schizophrenia with asymmetries of regional cerebral cortical surface area, thickness and subcortical volume measures in 32,256 participants from the UK Biobank. PRS for the two disorders were positively correlated (r=0.08, p=7.13×10-50), and both were higher in females compared to males, consistent with biased participation against higher-risk males. Each PRS was associated with multivariate brain asymmetry after adjusting for sex, ASD PRS r=0.03, p=2.17×10-9, schizophrenia PRS r=0.04, p=2.61×10-11, but the multivariate patterns were mostly distinct for the two PRS, and neither resembled average sex differences. Annotation based on meta-analyzed functional imaging data showed that both PRS were associated with asymmetries of regions important for language and executive functions, consistent with behavioural associations that arose in phenome-wide association analysis. Overall, the results indicate that distinct patterns of subtly altered brain asymmetry may be functionally relevant manifestations of polygenic risk for ASD and schizophrenia, but do not support brain masculinization or feminization in their etiologies.

ABSTRACT. M Dai1, AR Dunn1, J-G Zhang1, VM Philip1, KMS O’Connell1, CC Kaczorowski1

Alzheimer’s disease (AD) is the most common form of dementia that leads to progressive decrease in memory, cognitive function, and eventually ability to perform daily tasks. Cognitive dysfunction in AD is often accompanied (and preceded) by metabolic dysfunction, which may worsen neurological symptoms and quality of life in patients. It has been difficult to identify gene modifiers of AD that may only make a relatively small contribution to the overall disease risk. However, we recently developed a panel of AD mouse strains (AD-BXDs; Neuner, et al. 2019) that harbors the genetic and phenotypic heterogeneity similar to those observed in human AD patients. The AD-BXDs are an ideal resource to identify genetic modifiers of late-onset AD that manifest through molecular, neurological and metabolic phenotypes. Using this model, we performed weighted gene coexpression network analysis (WGCNA) in hypothalamic bulk RNA sequencing data. We identified several gene modules whose expression levels significantly correlate with cognitive and metabolic traits. Gene ontology (GO) enrichment search showed that functions of these modules included immune response, neuronal myelination, cell metabolic process, etc. We identified Laptm5 as the hub gene of the immune response module, and showed it has higher expression in aged and AD mice. Multiple previous studies have proposed Laptm5 as a target associated with amyloid-beta load in both human and mouse data (Salih et al. 2019, Chang et al. 2017, Bonham et al. 2019, Patel et al. 2020). In conclusion, we hypothesize that Laptm5 modifies vulnerability to both cognitive and noncognitive symptoms in AD.

1. The Jackson Laboratory, Bar Harbor, ME, USA 04609

ABSTRACT. Elizabeth B. Brown1, Jose Martin-Peña1,2, Samuel McFarlane1, Diego E. Rincon-Limas2, and Alex C. Keene1,3

Deficits in chemosensory processing are associated with healthy aging as well as numerous neurodegenerative disorders, including Alzheimer’s Disease (AD). In many cases, chemosensory deficits are harbingers of neurodegenerative disease, and understanding the mechanistic basis for these changes may provide insight into fundamental dysfunction associated with aging and neurodegeneration. The fruit fly, Drosophila melanogaster, is a powerful model for studying chemosensation, aging, and aging-related pathologies, yet the effects of aging on chemosensation remain largely unexplored in this model, particularly with respect to taste. To determine whether the effects of aging on taste are conserved in flies, we measured the response of flies to appetitive tastants. Aged flies exhibit an impaired response to sugars, but not fatty acids that are sensed by a shared population of neurons, revealing modality-specific deficits in taste. Expression of a human variant of Aβ, Arctic, selectively in the neurons that sense sugars and fatty acids phenocopy the effects of aging, suggesting that the age-related decline in response is autonomous to gustatory neurons. The axon terminals of gustatory neurons are retained in aged and Arctic-expressing flies, suggesting that the taste deficits are not due to neuronal death or loss of axon innervation. Conversely, functional imaging of the axon terminals revealed reduced response to sugar, but not fatty acid. Together, these findings reveal deficits in detection of taste are due to signaling pathway-specific changes and provide a model to examine cellular deficits in taste neurons associated with aging and Alzheimer’s Disease.

1Department of Biological Sciences, Florida Atlantic University, Jupiter, FL, United States of America; 2Department of Neurology, McKnight Brain Institute, University of Florida, Gainesville, FL, United States of America; 3Wilkes Honors College, Florida Atlantic University, Jupiter, FL, United States of America.

ABSTRACT. RW Logan1,2, ML Seney3, SM Kim3, JR Glausier3, MC Gamble1, MA Hildebrand3, X Xue4, W Zong4, J Wang4, MA Shelton3, BN Phan5, C Srinivasan5, AR Pfenning5, WE Johnson2,6, OA Olayinka2, GC Tseng4, DA Lewis3, A Watson7, Z Freyberg3,7, ML MacDonald3

Sleep and circadian disruptions are implicated in vulnerability to opioid dependence and the risk for relapse. However, few studies have determined the precise mechanisms at the intersection of sleep, circadian rhythms, and opioid addiction in human brain. We investigated the molecular alterations in key reward nodes, dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), in human postmortem brains from unaffected subjects and subjects with opioid use disorder (OUD). We discovered a high degree of overlap in transcripts altered in subjects with OUD between the DLPFC and NAc. Pathway analyses revealed enrichment for inflammation, synapse, and circadian rhythm signaling. To explore circadian rhythm pathways further, we used time of death (TOD) analysis to derive transcriptional rhythms in human postmortem brains. Using TOD analysis, we found striking differences in the patterns of rhythmicity in transcripts between unaffected subjects and subjects with OUD. Further analyses of the transcripts which specifically lost rhythmicity in OUD revealed upstream regulators related to opioids and circadian rhythms in DLPFC and NAc. The circadian transcription factor, NPAS2, was an upstream regulator in the NAc of subjects with OUD. We further explored the role of NPAS2 in opioid tolerance, withdrawal, and reward-related behaviors in male and female mice. We found sex-specific effects of NPAS2-deficiency on fentanyl self-administration, conditioned place preference, and the development of tolerance and withdrawal. Additional evidence from mice suggests NPAS2 modulates opioid-related behaviors via action in NAc. Our results begin to uncover the cellular and molecular mechanisms of OUD that involve sleep and circadian rhythm pathways.

1Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, 2Graduate Program in Bioinformatics, Boston University, 3Department of Psychiatry, University of Pittsburgh School of Medicine, 4Department of Biostatistics, University of Pittsburgh, 5Department of Computational Biology, Carnegie Mellon University, 6Department of Medicine: Computational Biomedicine, Boston University School of Medicine, 7Department of Cell Biology, University of Pittsburgh

Funding Support: NIDA R21DA052419, NHLBI R01HL150432, NHLBI R01HL150432-S1, NIDA R33DA041872, NIDA R01DA051390, NIDA R21DA041872

ABSTRACT. Chelcie Heaney1, Sara Wolfe2, Sean Farris3, R. Dayne Mayfield4, R. Adron Harris4, and Kimberly Raab-Graham1*

Alcohol use disorder (AUD) and major depressive disorder (MDD) are prevalent, debilitating, and highly comorbid disorders. Herein, we provide evidence that acute ethanol exposure produces rapid antidepressant-like responses at the biochemical and behavioral levels in naïve and ethanol-dependent animals. Both ethanol and fast-acting antidepressants block N-methyl-D-aspartate receptor (NMDAR) activity, leading to synaptic changes and long-lasting antidepressant-like behaviors. Using RNA sequencing, we analyzed the changes in the synaptic transcriptome required for the antidepressant efficacy of ethanol. Surprisingly, we found a striking overlap of differentially expressed exons (DEE) between ethanol- and Ro 25-6981 (fast acting antidepressant) -treated animals, with few differentially regulated genes. These data suggest that changes in exon usage mediates antidepressant efficacy. To better understand how these DEE are delivered and translated at the synapse, we sought to identify a master translation regulator of these DEEs. We noted that several of the common DEEs have binding sites for Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein. Importantly, we show that FMRP is required for new synapse formation and antidepressant-like behaviors induced by ethanol and Ro 25-6981. Altogether, these findings indicate that the rapid antidepressant effects of ethanol and NMDAR antagonists that have been previously reported may depend on FMRP-mediated synaptic exon usage, rather than gene expression.

1Wake Forest Translational Alcohol Research Center, Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC, 27157-1083, United States.

2Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA, 92037, United States.

3Department of Anesthesiology and Perioperative Medicine, University of Pittsburgh, 6068 4A Biomedical Science Tower 3, Pittsburgh, PA 15213, United States.

4Waggoner Center for Alcohol and Addiction Research, Department of Neuroscience, University of Texas at Austin, 2500 Speedway, Austin, TX, 78712, United States.

*Speaker, Corresponding Author, kraabgra@wakehealth.edu

ABSTRACT. HM Cates1, I Purushothaman2, CK Lardner2, RL Neve3, CJ Peña4, DM Walker5, RC Bagot6, EA Heller7, L Shen2, EJ Nestler2

Lasting changes in gene expression in brain reward regions, including prefrontal cortex (PFC) and nucleus accumbens (NAc), contribute to persistent functional changes in the addicted brain. We and others have demonstrated that altered expression of several candidate transcription factors in NAc regulates drug responses. A recent large-scale genome-wide study from our group predicted transcription factor E2F3 (E2F3) as a prominent upstream regulator of cocaine-induced changes in gene expression and alternative splicing. We studied expression of two E2F3 isoforms—E2F3a and E2F3b—in mouse NAc and PFC. We showed that E2f3a, but not E2f3b, overexpression or knockdown in mouse NAc regulates cocaine-induced locomotor and place conditioning behavior. Furthermore, we demonstrated that E2f3a overexpression substantially recapitulates genome-wide transcriptional profiles and alternative splicing induced by cocaine in NAc. We further validated direct binding of E2F3a at key target genes in NAc following cocaine exposure. In PFC, E2F3b, but not E2F3a, regulates cocaine-induced behavior. Interestingly, RNA-seq of PFC following E2f3b overexpression or I.P. cocaine exposure showed very different effects on expression levels of differentially expressed genes. However, we found that E2F3b drives a similar transcriptomic pattern to that of cocaine SA with overlapping upstream regulators and downstream pathways predicted. These findings reveal novel transcriptional mechanisms in NAc and PFC that control behavioral and molecular responses to cocaine.

1Department of Biology, Adelphi University, Garden City, NY, 2Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 3Massachusetts General Hospital, Boston, MA, 4Princeton Neuroscience Institute, Princeton University, Princeton, NJ, 5 Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR, 6Department of Psychology, McGill University, Montreal, QC, 7Department of Pharmacology, Perelman School of Medicine at University of Pennsylvania, Philadelphia, PA

ABSTRACT. AW Lasek1, L Martins de Carvalho1, WY Chen1, H Zhang1, H Chen1, HR Krishnan1, DR Grayson1, and SC Pandey1,2

Maladaptive changes in gene expression occur in the brain after chronic alcohol drinking and withdrawal. These alterations can cause negative mood states and promote relapse to alcohol abuse. In order to discover novel gene expression changes induced by alcohol withdrawal, we performed analysis of the transcriptome in the rat hippocampus, a brain region that regulates mood and cognition, after chronic alcohol drinking and withdrawal using RNA sequencing (RNA-Seq). Male adult Sprague Dawley rats were fed an ethanol or control liquid diet (Lieber-DeCarli) for 15 days and then withdrawn from ethanol for 24 hours. Hippocampal RNA was isolated and subjected to RNA-Seq. Data was further analyzed by weighted gene co-expression network analysis (WGCNA) to identify modules of co-expressed genes. Expression of genes in module 1 were elevated in rat hippocampus during alcohol withdrawal and enriched in components of the splicing machinery (spliceosome). These genes included Sf3a2, Puf60, Alyref, Pcbp1, Pcbp2, Ptpb1, Eif4a3, Dhx9, Dhx38, Lsm4, Snrpa, and Snrpb. We reasoned that increased expression of splicing machinery components would alter mRNA splicing patterns, so we analyzed the RNA-Seq data for differentially expressed (DE) splice junctions in the withdrawal vs. control groups and found 108 DE junctions (81 increased and 27 decreased). These junctions mapped to 53 annotated unique genes. One of these genes, Hapln2, had 3 significantly DE junctions, one of which was not annotated in the rat genome and was predicted to alter protein coding sequence. Validations of the DE spliced junctions were done by qPCR, confirming the RNA-Seq data.

1Center for Alcohol Research in Epigenetics and Department of Psychiatry, University of Illinois at Chicago and 2Jesse Brown VA Medical Center, Chicago, IL 60612 USA

Funding supported: NIAAA P50 AA022538 and U01 AA020912

ABSTRACT. Tariq Brown1, Emily Petruccelli1,2, Amanda Waterman1,2, Karla Kaun1,2,3

Repeated alcohol experiences can produce long-lasting memories for sensory cues associated with intoxication. These memories can problematically trigger relapse in individuals recovering from alcohol use disorder (AUD). The molecular mechanisms by which ethanol changes memories to become long-lasting and inflexible remain unclear. We recently demonstrated that formation of these memories results in expression of alternative transcript isoforms in memory-encoding neurons in Drosophila melanogaster. Drosophila rely on mushroom body (MB) neurons to make associative memories, including memories of ethanol-associated sensory cues. Decreasing expression of genes that play a role in splicing in adult MB neurons reduces formation of these memories, demonstrating the necessity of RNA processing in ethanol memory formation. Moreover, decreasing expression of genes that are alternatively spliced, like Dop2R and Stat92E in adult MB neurons reduces ethanol memory formation. This suggests that the splicing changes in these genes has functional implications for future memory formation. To test this, we generated mutant Drosophila that have forced expression of the naïve isoform expressed in control flies, and the spliced isoform expressed in trained animals. To test whether the serine excluded in the spliced isoform has functional consequences, we also generated mutants that have the serine swapped with an amino acid that is phosphomimetic (aspartate), or a phosphoinactive amino acid (alanine). Our data provide causal evidence suggesting that splicing induced by ethanol memory formation has functional consequences on subsequent behaviors. These findings highlight the dynamic, context-specific regulation of transcription in cue-encoding neurons, and the lasting impact of ethanol on transcript usage during memory formation.

1Neuroscience Graduate Program, Brown University, Providence, Rhode Island, USA Funding 1NIAAA (R01AA024434)

ABSTRACT. Rebecca M. Sena & David N. Linsenbardt

Department of Neurosciences and New Mexico Alcohol Research Center, University of New Mexico Health Science Center, Albuquerque, NM 87131

Approximately 50% of an individual’s risk for developing an alcohol use disorder (AUD) is attributable to their genes, with increased genetic risk associated with alcohol-induced changes in corticostriatal brain function. Despite this, we know very little about the molecular genetic mechanisms regulating alcohol-induced plasticity in the corticostriatal circuit. To assess the acute effects of alcohol on corticostriatal gene expression following many daily binge experiences, mice were given access to water or alcohol using drinking-in-the-dark (DID) procedures, and cortical (mPFC) and striatal (Acb) brain tissue was collected, sequenced, and evaluated for alternative splicing and polyadenylation. A total of 8 genes were found to be alternatively spliced by ethanol exposure in both brain regions, while polyadenylation differences were unique to each brain area. However, alternative splicing and alternative polyadenylation of genes involved in chromatin remodeling were observed. For example, ethanol-induced skipping of exon 5 of the HdC4 histone deacetylase gene was observed, as was poly(A) lengthening of H3f3a histone protein gene. Collectively these findings identify ethanol-induced co-or post-transcriptional modifications that may be involved in functional alterations associated with repeated binge ethanol exposure and point to chromatin biology as a potential upstream pathway for investigation.

Acknowledgments: This work was supported in part by grant #s: AA025120 (DNL), and the New Mexico Alcohol Research Center P50-AA022534.