ABSTRACT. Sreekanth H. Chalasani1, Sarah G. Leinwand1, and Laura A. Hale1

1 Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037.

A central question in neuroscience is to understand how neural circuits extract relevant sensory information from their environment and use that information to drive appropriate responses. We use the small, well-defined nematode C. elegans to reveal how sensory neurons encode both stimulus identity and concentration.

We will present our data suggesting three principles of neural circuit coding. First, we find that odor coding is sparse in C. elegans, similar to other species. Using calcium imaging, we find that a small subset of neurons responds to varying concentrations of food-like odors. Second, we identify that chemosensory circuits are unexpectedly distributed and comprise of primary sensory neurons that directly detect stimuli and recruit secondary neurons to represent stimulus identify and concentration. Finally, we identify a novel form of plasticity in these sensory circuits. In response to particular sensory environments or animal age, neuropeptides and neurotransmitters select one of several configurations for the active circuit. Consistent with these results, disruptions to the communication between sensory neurons cause significant deficits in odor-guided behaviors. This suggests that these sparse and flexible odor representations are essential for behavior. Furthermore, these flexible circuit configurations might represent alternate paths for information processing and are vital to an animal’s ability to respond to the changing environment.

ABSTRACT. Hui Hong1, Xiaoyu Wang4,5, Ting Lu1, Diego A.R. Zorio4,5, Yuan Wang4,5, and Jason Tait Sanchez1,2,3,

Topography in the vertebrate brain represents an orderly organization of neuronal architecture responsible for encoding sensory information. Topography in the auditory system originates as a gradual change in frequency representation across the peripheral sensory epithelium and is defined by tonotopy; a process maintained in the central pathway. In the avian nucleus magnocellularis (NM) – an auditory brainstem structure analogous to the mammalian cochlear nucleus – tonotopy is maintained along a caudolateral-to-rostromedial axis. In this presentation, I describe novel organization and neuronal biophysics of the caudolateral NM (NMc), a region representing extreme low-frequency sound processing. Examination of neuronal and dendritic properties revealed that NMc neurons contain small somas and extensive dendritic architecture. This is in stark contrast to the adendritic, large neurons located rostromedially that encode higher-frequency sounds. Axonal tract tracing studies confirmed that NMc neurons receive afferent inputs from the auditory nerve, similar to adendritic NM. However, auditory axons synapse onto NMc neurons via small bouton-like terminals, unlike the large Endbulb of Held synapses on adendritic NM neurons. Whole-cell recordings revealed that NMc neurons are significantly more excitable than NM neurons. They generate multiple action potentials to sustained depolarization and rapidly burst fire to low-frequency sinusoidal inputs. Pharmacological and immunohistochemical experiments revealed that this functional phenotype is due to distinct ion channel properties, namely voltage-dependent potassium channels and resurgent sodium currents. Taken together, NMc neurons are structurally, connectively, and physiologically unique from traditionally defined NM neurons, emphasizing specialized neuronal properties for processing sounds of varying frequencies.

1Roxelyn and Richard Pepper Department of Communication Sciences and Disorders, 2Department of Neurobiology, 3The Hugh Knowles Hearing Research Center, Northwestern University, Evanston, IL, USA, 4Department of Biomedical Sciences, 5Program in Neuroscience Florida State University College of Medicine, Florida State University, Tallahassee

ABSTRACT. Teresa Nicolson Oregon Hearing Research Center and the Vollum Institute Oregon Health and Science University Portland, Oregon Recent genetic studies have identified essential components of the mechanotransduction complex in sensory hair cells. Although controversial, the Transmembrane channel-like proteins 1 and 2 (Tmc1/2) are promising candidates for the pore forming subunits of the transduction channel in auditory/vestibular hair cells. How the Tmcs are assembled with other members of the mechanotransduction complex and transported to the site of mechanotransduction within the stereocilia of hair bundles is not known. Our studies show that mutations in zebrafish transmembrane o-methyltransferase (tomt), the orthologue of which is required for hearing in humans, abolish localization of Tmc1/2 to hair bundles. We find that tomt is exclusively expressed in hair cells and is enriched in the Golgi compartment. Mutations in tomt cause a complete loss of mechanotransduction in zebrafish hair cells. The phenotype is not due to a developmental defect because acute expression of Tomt expression in mature mutant hair cells is sufficient to rescue the transduction defect. Furthermore, expression of Tomt can restore the localization of Tmc1/2 to stereocilia in mutant hair cells. The loss of mechanotransduction along with normal morphology of hair bundles in tomt mutants is unusual among mechanotransduction mutants. These observations prompted us to take a closer look at another mutant, tmieru1000, with a similar phenotype. Mutations in transmembrane inner ear protein (tmie) are also associated with human deafness and result in functional but not morphological defects in hair cells. We found that localization of Tmie to the hair bundle is a conserved feature among vertebrates. Like tomt mutants, we discovered that the Tmc1/2 proteins are absent from the hair bundles in mutant tmie hair cells. However, in contrast to Tomt, overexpression of Tmie greatly enhances localization of the Tmcs to the hair bundles, suggesting an additional role of stabilizing protein levels of the Tmc subunits. We propose that Tomt directly or indirectly modifies Tmc1/2 proteins in the Golgi compartment, and Tmie acts in part as a chaperone, enabling the transport of the Tmcs to the site of mechanotransduction in hair cells.

ABSTRACT. Pavel Mašek1, John M. Tauber2, Elizabeth B. Brown2, Yuanyuan Li1, Maria E. Yurgel2, and Alex C. Keene2 1. Department of Biological Sciences, Binghamton University, Binghamton, NY, 13902, USA. 2. Department of Biological Sciences, Florida Atlantic University, Jupiter, FL 33458, USA. Fat represents a calorically potent food source for many animals. The taste of fat is represented by response to free fatty acids (FAs), one of the building blocks of fat. Previously, we showed that a broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (Gr64f), is required for reflexive feeding responses to both FAs and sugars but functional phospholipase C in these neurons is necessary only for FAs perception. Here, we describe specific populations of taste neurons mediating FA response that are identified by expression of Ionotropic Receptor 56d (IR56d). IR56d forms, together with IR76b and IR25a, a receptor for medium-chain FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes response to medium-chain FAs, and their activation is sufficient for feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by short- and medium-chain FAs and are sufficient for reflexive feeding responses. Behaviorally, we show that flies display different response to FAs and sugars relative to their intensity. They also discriminate between sugars and FAs in an aversive taste memory assay, further supporting the notion that FA taste is a unique modality. In addition, we test the discrimination ability between multiple classes of FAs and sweet stimuli that allows us to categorize appetitive taste stimuli into functional groups and help us to understand the principles of taste perception and coding.

ABSTRACT. To guide behavior, sensory systems detect the onset and offset of stimuli and process these distinct inputs via parallel pathways. In the retina, this strategy is implemented by splitting neural signals for light onset and offset via distinct synapses connecting photoreceptors to ON and OFF bipolar cells, respectively. It remains poorly understood which molecular cues establish the architecture of this synaptic configuration to split light onset and offset signals. A mutant with reduced synapses between photoreceptors and one bipolar cell type, but not the other, could reveal a critical cue. From this approach, we report a novel synaptic role for pregnancy associated plasma protein aa (pappaa) in promoting the structure and function of cone synapses that transmit light offset information. Electrophysiological and behavioral analyses indicated pappaa mutant zebrafish have dysfunctional cone to OFF bipolar cell synapses and impaired responses to light offset, but intact cone to ON bipolar cell synapses and light onset responses. Ultrastructural analyses of pappaa mutant cones showed a lack of presynaptic domains at synapses with OFF bipolar cells. pappaa is expressed postsynaptically to the cones during retinal synaptogenesis and encodes a secreted metalloprotease known to stimulate insulin-like growth factor 1 (IGF1) signaling. Induction of dominant negative IGF1 receptor expression during synaptogenesis reduced light offset responses. Conversely, stimulating IGF1 signaling at this time improved pappaa mutants’ light offset responses and cone presynaptic structures. Together, our results indicate Pappaa-regulated IGF1 signaling as a novel pathway that establishes how cone synapses convey light offset signals to guide behavior.

ABSTRACT. It is unclear if sex differences in immune response can influence alcohol drinking behavior. We tested the possibility that toll-like receptor 3 (TLR3) activation regulates alcohol consumption in a sex-dependent manner. Male and female mice were injected with the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C; 5 mg/kg) and brain proinflammatory responses and alcohol consumption (every-other-day two-bottle choice) were measured. In males, poly I:C produced a peak cytokine response at 3 hours post-injection. In contrast, poly I:C injection resulted in a delayed and prolonged cytokine response lasting up to 72 hours post-injection in females. Access to ethanol at peak cytokine activation decreased ethanol consumption in both sexes; whereas, access to ethanol after peak cytokine activation resulted in an increase in ethanol consumption in males, with no change in ethanol consumption in females. Repeated poly I:C and ethanol exposures altered innate immune transcript abundances. Decreased levels of the Myeloid Differentiation response gene 88 (MyD88)-dependent pathway correlated with decreased alcohol intake in females; whereas, increased levels of the TIR-domain-containing adapter-inducing interferon β (TRIF)-dependent pathway correlated with increased alcohol intake in males. We validated that the effect of poly I:C was mediated through MyD88-dependent signaling in females by testing in knockout female mice lacking Myd88. Poly I:C did not alter alcohol intake in these animals. Our results provide novel evidence that there are sex-dependent differences in TLR3 activation and that enhanced TRIF-dependent pathway expression may regulate escalation of alcohol intake. Supported by: U01 AA020926, P01 AA020683, AA013520, AA006399, F31 AA025499-02.

1Waggoner Center for Alcoholism and Addiction Research, University of Texas at Austin, Austin, TX 78712, USA 2Institute for Neuroscience, University of Texas at Austin, Austin, TX 78712, USA 3Department of Neurology, Dell Medical School, University of Texas at Austin, Austin TX 78712, USA.

ABSTRACT. Recent work has shown that the effects of nicotine use may be transgenerationally transmitted through epigenetic modifications. For the present study, we examined the effects of paternal nicotine exposure on fear learning in subsequent generations. Male adult C57BL6/J mice received either chronic nicotine or saline exposure and were crossed to naïve female C57BL/6J mice. The offspring of nicotine (Nic-Sired) and saline (Sal-Sired) exposed mice were tested in contextual and cued fear conditioning. Results indicated that paternal nicotine exposure resulted in enhanced cued and contextual fear learning in F1 and F2 generations compared to Sal-Sired mice, and this effect was reversed when F1 generation, but not F2 generation, mice received acute nicotine injections. Furthermore, Nic-Sired mice also showed more pronounced spontaneous recovery of fear when re-tested following extinction. We additionally examined general cholinergic activity in the Nic-Sired mice using nicotinic acetylcholine receptor (nAChR) binding and potassium and nicotine-evoked acetylcholine release in the dorsal and ventral hippocampus. Reduced cholinergic functioning was found in ventral, but not dorsal, hippocampus in the Nic-Sired mice along with global hippocampal increases in nAChR binding. In parallel, we also found increased DNA methylation in the ventral hippocampi of Nic-Sired mice. Finally, RNA-sequencing indicated differential expression of cholinergic synapse associated genes in Sal-Sired and Nic-Sired mice. Together, our results suggest that paternal nicotine exposure may result in alterations in the epigenome, which, in turn, leads to exaggerated fear learning and abnormal cholinergic function in subsequent generations.

1Department of Biobehavioral Health, Penn State University, 2Department of Pharmacology, Vanderbilt University, 3Department of Psychology, Temple University Funding: DA017949; 1U01DA041632 (NIDA, T.G.), DA037421 (NIDA, V.P.)

ABSTRACT. Alzheimer's disease (AD) is complex, with both genetic (G) and environmental (E) factors regulating disease progression. Identification of GxE interactions that modulate AD pathogenesis is critical to developing novel and personalized treatments. However, extracting GxE effects is challenging in humans due to human genome complexity and difficulty controlling environmental factors. To overcome these barriers, we developed a panel of genetically diverse mice carrying human familial AD mutations (AD-BXDs). Because the AD-BXDs model some genetic heterogeneity of humans, they are ideally suited to investigate translationally-relevant GxE interactions. Here, we used AD-BXDs to determine how genetics and diet interact to modify AD-related pathogenesis. We fed a chronic high fat diet (HFD) to 10 strains of AD-BXDs and monitored metabolic and cognitive function before and after HFD. Control groups included AD-BXDs on chow, and nontransgenic BXDs on chow and HFD. We observed accelerated working memory decline in AD-BXDs on HFD compared to controls. However, this was dependent on genetic background, with some AD-BXD strains maintaining cognitive function on HFD. Subsequent analyses indicated gene-by-diet interactions accounted for 18% of individual variation in memory decline. Higher body weight and adiposity were protective against working memory decline in nontransgenic BXDs, but not in AD-BXDs. Our results suggest that diet and genetic background interact to mediate vulnerability to AD pathogenesis, and that metabolic factors may contribute to cognitive decline differentially in normal aging versus AD. Future analyses will identify genetic and molecular targets contributing to AD pathogenesis that may be exploited to delay, prevent or treat AD.

(1) The Jackson Laboratory, Bar Harbor, ME, USA, (2) The University of Tennessee Health Science Center, Memphis, TN, USA Funding support: NIA 1 R01 AG057914-01 to C.C.K.; NIA 1 R01 AG054180-01A1 to C.C.K; JAX Director’s Innovation Fund to K.M.S.O; Alzheimer’s Association AARF-18-565506 to A.R.D.

ABSTRACT. Sarah E. Bergen, PhD1,2; Alexander Ploner, PhD1; Daniel Howrigan, PhD3; CNV Analysis Group and the Schizophrenia Working Group of the Psychiatric Genomics Consortium; Michael C. O’Donovan4, MD, PhD; Jordan W. Smoller, MD, ScD5; Patrick F. Sullivan, MD, FRANZCP1,6; Jonathan Sebat, PhD7; Benjamin Neale, PhD3; Kenneth S. Kendler, MD8

Both rare copy number variants (CNVs) and common single nucleotide polymorphisms (SNPs) contribute to liability to schizophrenia, but their etiological relationship has not been fully elucidated. We evaluated an additive model, whereby risk of schizophrenia requires less contribution from common genetic variation in the presence of a rare CNV and test for interactions. Individual genetic data from 21,094 schizophrenia cases and 20,227 controls from the Psychiatric Genomics Consortium were used. We assessed three classes of rare CNVs: those previously associated with schizophrenia, large deletions ≥500kb, and total CNV burden. We compared mean polygenic risk scores (PRS) between subjects with and without rare CNVs and modeled the joint effects of PRS and CNVs on schizophrenia liability using logistic regression. Schizophrenia cases carrying risk CNVs have lower polygenic risk than without CNVs, but still higher than controls. For cases carrying known risk CNVs, the PRS was diminished proportional to the effect size of the CNV. The strongly-associated 22q11.2 deletion required little added PRS to produce schizophrenia. Large deletions and increased CNV burden were also associated with lower polygenic risk in cases. However, the results for controls suggested more complex relationships. We found evidence for interactive effects of PRS and previously associated CNVs for risk for schizophrenia, while our results for large deletions and total CNV burden support an additive model. These findings offer insights into the genetic architecture of schizophrenia by illuminating how different established genetic risk factors act and interact to influence liability to schizophrenia.

1 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 2 Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA 3 Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA 4 MRC Centre for Neuropsychiatric Genetics and Genomics, Division of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, UK 5 Harvard Medical School, Department of Psychiatry, Massachusetts General Hospital, Boston, MA 02114, USA; Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetics Research, Massachusetts General Hospital, Boston, MA 02114, USA 6 Departments of Genetics and Psychiatry, University of North Carolina, Chapel Hill, NC, USA 7 Departments of Psychiatry and Cellular and Molecular Medicine, University of California, San Diego, CA, USA 8 Virginia Institute for Psychiatric and Behavioral Genetics, Department of Psychiatry, Virginia Commonwealth University, Richmond, VA, USA

ABSTRACT. S.P. Farris1, CM Borghese1, E.A Osterndorff-Kahanek1, Y.A. Blednov1, G.E. Homanics2, R.D. Mayfield1, and R.A. Harris11Waggoner Center for Alcohol and Addiction Research The University of Texas at Austin, Austin, TX, 78712-1095, U.S.A.; 2 Departments of Anesthesiology and Pharmacology and Chemical Biology, University of Pittsburgh PA, 15261

Transcriptional regulation of gene expression is an important aspect of human health and diseases. The human transcriptome contains both protein-coding and non-coding RNAs (ncRNAs), with ncRNAs greatly outnumbering protein-coding transcripts. Despite the large number, few ncRNAs have been assigned names, biologically characterized, or implicated in disease. A large fraction of ncRNAs, particularly long non-coding RNAs (lncRNAs), are specifically expressed in brain. To test the contribution of altered lncRNA expression in alcohol use disorder (AUD) we conduced RNA-Seq profiling of multiple human postmortem brain regions (n=385 samples). Focusing on lncRNAs with evidence for evolutionary conservation, our analyses identified over 100 unique cross-validated lncRNAs associated with AUD. Using Xenopus oocytes as an expression system, we evaluated the potential biological impact of candidate human lncRNAs on known alcohol-responsive ion-channels. One lncRNA, markedly increased in AUD, decreased NMDAR subunit GRIN1 and GRIN2B expression (p < 1e-03, n=10/group). Two-electrode voltage clamp recordings showed an ~80% reduction in maximum glutmate currents, without affecting other ion-channels (e.g. GABA-A or Glycine). To test the potential role of this lncRNA in vivo we generated a knockout mouse for the predicted homolog using CRISPR/Cas9. The lncRNA KO mouse showed a significant increase for alcohol-induced loss of righting response (LORR) (p < 0.01, n=8/group). Consistent with NMDAR oocyte studies, lncRNA KOs also showed enchanced ketamine-induced LORR; however, no differences were observed for the GABAergic sedative gaboxadol. Overall, our work supports the functional importance of lncRNAs, and validates a novel candidate for an alcohol-related behaivor affecting NMDARs. Supported NIAAA (K99AA024836 and U01AA020926).

ABSTRACT. Sa-Ik Hong1, Seungwoo Kang1, and Doo-Sup Choi1, 2, 3

Top-down control facilitates cortico-limbic circuits for the decision-making process. Dysregulation of this process is implicated in addiction including alcohol use disorder (AUD). Activation of striatopallidal neurons is known to suppress hierarchical response in reward-seeking behavior. Adenosine A2A receptor (A2AR) is expressed in the indirect inhibitory circuit. Although A2AR is co-expressed with dopamine D2 receptor (D2R) in the medium spiny neurons (MSN) in the striatum, the precise role of A2AR in the dorsomedial striatum (DMS) in reward-seeking behaviors through top-down inhibitory pathway has not been investigated. In our study, to investigate a DMS-specific role of A2AR in top-down ethanol-seeking behavior, first, we trained mice to voluntarily seek ethanol in the nose-poke operant chambers. Then, to examine the effect of A2AR, we measured neuronal activity in the DMS and a major output targeting neurons in the external part of globus pallidus (GPe). Using in vivo pharmacologic and optogenetic techniques, we manipulated neural activities in the A2AR expressing DMS neurons to GPe. We found that mice pre-exposed to ethanol exhibited increased top-down reward-seeking behavior. Pharmacological activation of A2AR and optogenetic stimulation to A2AR-containing indirect pathway suppressed top-down response. In contrast, A2AR-inhibition and optogenetic suppression of DMS-GPe circuit reversed top-down inhibitory response. Taken together, activation of striatopallidal A2AR and indirect pathway dampens top-down ethanol-seeking behavior, indicating that activation of A2AR signaling in the striatal indirect pathway could be a potential therapeutic target for AUD.

1Department of Molecular Pharmacology and Experimental Therapeutics, 2Neurobiology of Disease Program, 3Department of Psychiatry and Psychology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA. Funding Support: NIH AA018779, Samuel C. Johnson for Genomics of Addiction Program at Mayo Clinic, Ulm Foundation, Godby Foundation, David Lehr Research Award from American Society for Pharmacology and Experimental Therapeutics

ABSTRACT. Spencer B. Huggett1,2 & Michael C. Stallings1,2

1Department of Psychology and Neuroscience, University of Colorado 2Institute for Behavioral Genetics, University of Colorado Supported in part by: NIDA P60 DA11015

We investigated the genetic and transcriptional landscape of cocaine dependence (CD) and chronic cocaine use. We performed and integrated popular genome-wide and transcriptome-wide analyses using data from the largest genome wide association study (GWAS) on CD to date (Gelernter et al. 2014), 3,176 European Americans (EAs), and human post-mortem brain tissue from seven cocaine users and eight drug free controls. First, linkage disequilibrium (LD) score regression analyses was performed and detected a significant genomic heritability of 28% (s.e = 0.14) for CD and gene-based association tests found three novel genes underlying this heritability: the C1QL2, KCTD20 and STK38 genes. Tissue specificity analyses indicated robust enrichment in numerous brain regions, including the hippocampus, padj = 2.02e-06. Therefore using RNA-sequencing (RNA-seq) analyses we performed differential expression and weighted gene covariance network analyses (WGCNA) on post-mortem hippocampal brain tissue from Zhou et al. 2011. Differentially expressed genes or transcripts between chronic cocaine users versus drug free controls were enriched for genes associated with CD (p < 0.05), OR = 1.34, p = 0.031, and were used to find various potential therapeutic compounds for cocaine use/toxicity. Lastly, we found that KCTD20 was a central part of a hippocampal gene network strongly associated with cocaine use and thus, might be contributing to the genetic liability of CD by disrupting intricate gene networks in the brain. Overall, our study elucidates the biological architecture of cocaine use/dependence, proposes various novel therapeutic compounds for cocaine use and includes an alternative framework to validate/provide biological meaning to genome-wide findings.

ABSTRACT. WY Chen1, H Chen1, Y Chen1, H Zhang1, HR Krishnan1, C Liu1, DR Grayson1, SC Pandey1,2, & AW Lasek1 Chronic alcohol drinking and withdrawal causes epigenetic and transcriptional changes in the brain that may contribute to relapse. In order to find novel genes altered during withdrawal, we performed a genome-wide analysis of transcripts in the rat hippocampus. Male adult Sprague Dawley rats were fed an ethanol or control liquid diet for 15 days and withdrawn for 24 hours. Hippocampal RNA was isolated and subjected to RNA-Seq. Weighted gene co-expression network analysis (WGCNA) was used to identify modules of co-expressed genes and demonstrated that the genes fit into 53 co-expression modules. Genes in module 1 were significantly higher during withdrawal and were enriched in the “TNF signaling pathway” and “Epstein Barr virus Infection” by gene ontology analysis, indicating disruptions in neuroimmune signaling during withdrawal. To validate the RNA-Seq findings, hippocampal RNA and chromatin were isolated for qPCR and chromatin immunoprecipitation (ChIP) analyses, respectively. Expression of Tnfrsf1a, Stat3, Relb, Plat, Serpine1, and Timp1 were increased during withdrawal, similar to the RNA-Seq findings. Decreased acetylation of histone H3 lysine 9/14 was also observed at the promoters of these genes. Notably, treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) during withdrawal normalized expression and histone acetylation at the promoters of the Tnfrsf1a, Stat3, and Relb genes. Our results demonstrate that transcriptional changes in neuroimmune genes occur in the hippocampus during withdrawal from chronic ethanol drinking and that some of these changes can be reversed by SAHA treatment, suggesting epigenetic regulation of neuroimmune genes and possible involvement in pathophysiology of alcoholism.

1University of Illinois at Chicago, Center for Alcohol Research in Epigenetics and Department of Psychiatry & 2Jesse Brown VA Medical Center, Chicago, IL 60612 USA, NIAAA P50 AA022538 (to AWL and SCP) and U01 AA020912 (to AWL).

ABSTRACT. David N. Linsenbardt, Nicholas M. Timme, & Christopher C. Lapish

Indiana Alcohol Research Center and Department of Addiction Neuroscience - Psychology, Indiana University – Purdue University Indianapolis, Indianapolis, IN 46202.

Aberrant decision-making is both a risk factor for, and the result of, an Alcohol Use Disorder (AUD). The decision to use alcohol can be robustly influenced by exposure to stimuli associated with the drug, and these stimuli are a critical underlying factor that contributes to compulsive drinking and relapse. Additionally, the identification of risk factors for AUD’s in human epidemiological studies and animal models has led to the view that there is a strong genetic component to the disease. Therefore, identifying heritable alterations in computation that occur in brain regions that guide the decision to seek and consume alcohol is critical to develop novel intervention strategies. The prefrontal cortex plays a central role in guiding decision-making, and is altered by alcohol use and familial (genetic) risk for excessive drinking. Thus, we chose to record neural signals from the medial Prefrontal Cortex (mPFC) of animals given the opportunity to drink alcohol. We used an information theoretic statistical approach to quantify and compare the amount of information neurons provided about trial-to-trial alcohol drinking decisions. Animals without familial risk (Wistar rats) exhibited patterns of neural activity consistent with the intention to drink or abstain, whereas these patterns were blunted or absent in animals (‘P’ rats) with high risk. These data indicate that computations guiding drinking decisions are not encoded by the mPFC in populations with increased familial risk, possibly indicating a lack of control over decision-making by this otherwise well-validated supervisory brain region.

Acknowledgments: This work was supported by NIAAA grant #’s AA022268 (DNL), AA025120 (DNL), AA007462 (NMT), AA022821 (CCL), AA023786 (CCL), and the Indiana Alcohol Research Center P60AA007611 (D. Kareken). This research was supported in part by Lilly Endowment, Inc., through its support for the Indiana University Pervasive Technology Institute, and in part by the Indiana METACyt Initiative. The Indiana METACyt Initiative at IU is also supported in part by Lilly Endowment, Inc.

ABSTRACT. Jacob A. Beierle 1,2,,3, Lisa R. Goldberg 1,2, Julia C. Kelliher 1, Kimberly P. Luttik 1, Julia L. Scotellaro 1, Alex M. Luong 1,4, Jiayi Wu 3,5, Eric R. Reed 6, David F. Jenkins 6,7, Qiu T. Ruan 1,2,3, Ali Al Abdullatif 8, Stacey L. Kirkpatrick 1, Cory Parks 9, Christine Watkins 9, Morgan Dickerson 9, Sufiya Khanam 9, Sydney B. Crotts 1, Timothy A. Drescher 1, Neema Yazdani 1,2,3, Robert W. Williams 9, Gregg E. Homanics 10, William E. Johnson 7, Benjamin Wolozin 8, Megan K. Mulligan 9, Camron D. Bryant 1

Murine forward genetic studies of addiction traits can identify genetic factors and biological pathways relevant to humans. Reduced Complexity Crosses (RCC) facilitate gene identification by decreasing the number of candidate variants by up to 300-fold. We used an RCC between C57BL/6J (B6J) and C57BL/6NJ (B6NJ) substrains to map opioid (oxycodone; OXY) and psychostimulant (methamphetamine; MA) behaviors in our Multi-Stage Addiction Assessment Protocol (MSAAP). We identified a major QTL on distal chr. 1 underlying OXY-induced locomotor activity and anxiety-like withdrawal. We fine mapped this locus to 167-174 Mb by backcrossing select recombinant F2 mice. Cis-expression (eQTL) combined with transcript/behavior correlation identified Pcp4l1, Atp1a2, and Cadm3 as positional/functional candidate genes. Transcriptome analysis of the chr. 1 locus identified a dual-hub network of trans-downregulated genes comprising neurodegenerative proteins APP and TAU. Mapt (TAU) knockout mice show preliminary enhanced OXY tolerance and withdrawal, suggesting TAU protects against chronic opioid-induced neurobehavioral plasticity. We also mapped a QTL on medial chr. 5 for increased MA-induced locomotor activity that contains the cis-modulated α2 subunit of the GABA-A receptor (Gabra2). The B6J allele harbors an intronic deletion resulting in decreased mRNA and protein levels. The causal role of Gabra2 on MA-induced locomotor activity was confirmed using gene editing to repair the deletion in B6J. Lower Gabra2 levels associated with the B6J allele confer a greater MA behavioral response compared to the repair allele. Studies are underway to validate Gabra2 in conditioned aversive behaviors induced by the opioid receptor antagonist naloxone that also mapped to chr 5.

1. Laboratory of Addiction Genetics (R01DA039168, R21DA038738), Departments of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine, Boston, MA USA 2. Biomolecular Pharmacology Training Program (T32GM008541), Boston University School of Medicine, Boston, MA USA 3. Transformative Training Program in Addiction Science (1011479), Burroughs Wellcome Fund 4. Masters Program in Biomedical Sciences, Boston University, Boston, MA USA 5. Genetics and Genomics, Program in Biomedical Sciences, Boston University, Boston, MA USA 6. Ph.D. Program in Bioinformatics, Boston University, Boston, MA USA 7. Computational Biomedicine, Boston University School of Medicine, Boston, MA USA 8. Laboratory of Neurodegeneration, Departments of Pharmacology and Experimental Therapeutics and Neurology, Boston University School of Medicine, Boston, MA USA 9. Departments of Genetics, Genomics, and Informatics and Anatomy and Neurobiology, University of Tennessee Health and Science Center, Memphis, TN USA 10. Departments of Anesthesiology, Neurobiology, and Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA USA

ABSTRACT. CC Parker1, VM Philip2, DM Gatti3, A Holmes4, EJ Chesler3 1Department of Psychology and Program in Neuroscience, Middlebury College, VT 05753 2Center for Computational Sciences, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 3Center for Mammalian Genetics, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 4Laboratory of Behavioral and Genomic Neuroscience, NIAAA, NIH, Rockville MD 20852

A strong predictor for the development of alcohol use disorders (AUDs) is altered sensitivity to the intoxicating effects of alcohol. Individual differences in the initial sensitivity to alcohol are controlled at least in part by genetic factors. Mice offer a powerful tool for elucidating the genetic basis of behavioral and physiological traits relevant to AUDs; but conventional experimental crosses have only been able to identify large chromosomal regions rather than specific genes. Genetically diverse, highly recombinant mouse populations allow for the opportunity to observe a wider range of phenotypic variation, offer greater mapping precision, and thus increase the potential for efficient gene identification. We have taken advantage of the Diversity Outbred (DO) mouse population to identify and map narrow quantitative trait loci (QTL) associated with ethanol sensitivity. We phenotyped 778 JAX DO mice for three measures of ethanol sensitivity: ataxia, hypothermia, and loss of the righting response. We used high density MEGAMuga and GIGAMuga arrays to obtain genotypes ranging from 77,808 – 143,259 SNPs. We also measured striatal gene expression using RNA sequencing. We identified multiple QTLs associated with ethanol sensitivity related traits. With the inclusion of RNA-Seq we are able to apply a systems genetic strategy to construct the network of correlations that exist between DNA sequence, gene expression values and ethanol-related phenotypes. This information can in turn be used to identify alleles that contribute to AUDs in humans, elucidate causative biological mechanisms, or assist in the development of putative treatment strategies.

ABSTRACT. JD Jentsch1, M Zheng2, A Arslan2, G Peltz2 Cocaine use disorder is a substantially heritable trait in humans, yet relatively few candidate genes have been associated with it. To address this issue, we examined intravenous cocaine self-administration in adult male mice derived from a panel of >70 inbred mouse strains. Most strains readily acquired operant responding reinforced by delivery of 0.5 mg/kg/infusion of cocaine. After 10 days of testing, levels of intake were relatively stable at an individual level but were highly variable across strains. The broad sense heritability of levels of intake at the end of the acquisition period was >0.8. Haplotype-based computational genetic mapping (HBCGM) was conducted, using the cocaine self-administration phenotypes of 21 inbred strains for which whole genome sequence data exists. This analysis revealed that multiple genes within a region (134-7MB) on mouse chromosome 1 show an allelic pattern strongly associated with strain differences in cocaine self-administration. Based upon the presence of associated non-synonymous coding SNPs within it and its brain expression profile, Nav1, a member of the neuron navigator gene family, was evaluated further. Its expression in striatal tissue in BxD mice was analyzed using Genenetwork.org (record ID: ILM6620129). The Nav1 eQTL mapped to the Nav1 locus. Strain level variation in Nav1 expression correlated negatively with dopamine D2 receptor mRNA expression (record ID:15186; r=-.78; p=4.23e-08) and positively with locomotor response to cocaine (record ID:10320; r=0.59, p=4.34e-03). These studies suggest that variation within Nav1 may powerfully influence the tone of dopaminergic transmission in brain, in turn producing heritable variation in cocaine-evoked behaviors.

1Department of Psychology (Behavioral Neuroscience), Binghamton University; 2Department of Anesthesia, Stanford University School of Medicine

ABSTRACT. Jeyeon Lee¹, Inyong Kim², Hoonki P. Min³, In MyungHo³, Hang Joon Jo², and Su-Youne Chang^{1, 4}

Harmaline induced tremor is the most commonly utilized disease model for essential tremor. However, underlying mechanisms of harmaline-induced tremor have not yet been fully elucidated. Therefore, understanding tremorgenesis mechanism of harmaline will be crucial to develop a novel treatment for essential tremor. In this study, we tried to define the harmaline effect at the system level. To do so, we performed whole brain imaging via fMRI (functional magnetic resonance image) in swine and analyzed an acute effect of harmaline injection in BOLD (blood-oxygen level dependent) signal. As results, we observed significant BOLD changes in inferior olivary nucleus (ION), cerebellum, and thalamus, which are known as tremor-related regions (n= 5; p <0.0005, one-sample t-test). In addition, significant increase of inter-regional correlation between cerebellum and deep cerebellar nuclei and between cerebellum and thalamus was also found (n= 5, p <0.001, Wilcoxon signed-rank test). Our results of this study suggest ph-fMRI (pharmachological fMRI) as an applicable method to evaluate novel therapeutic agents and/or neuromodulatory therapies for essential tremor and the swine model of tremor as an acute animal model for essential tremor.

¹Department of Neurologic Surgery, Mayo Clinic

²Department of Neurology, Mayo Clinic

³Department of Radiology, Mayo Clinic

⁴Department of Physiology and Biomedical Engineering, Mayo Clinic

This study was funded by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (NS 88260).

ABSTRACT. KH Flippo1,2, SO Idiga1,2, KE Claflin1,2, MC Naber1,2, MJ Potthoff1,2

In the United States alcohol use disorder (AUD) affects ~15% of adults with the prevalence of binge drinking on the rise in adolescents and young adults. AUD represents a major issue to healthcare given that chronic excessive alcohol consumption in humans is associated with cardiovascular disease, metabolic syndrome, and cancer while acute alcohol intoxication can prove lethal. Economically, AUD represents a massive burden due to loss of productivity and associated healthcare costs. Recently, the endocrine hormone fibroblast growth factor 21 (FGF21), known for its potent metabolic effects, was illustrated to significantly reduce alcohol consumption via an undescribed mechanism requiring expression of the obligate FGF21 co-receptor ß-klotho (KLB) in the brain. Importantly, single nucleotide polymorphisms (SNPs) in both FGF21 and KLB genomic loci are highly associated with increased alcohol consumption in humans. Here we extend those findings illustrating that FGF21 can reverse alcohol consumption even in mice chronically administered ethanol prior to FGF21 administration. Furthermore, excessive alcohol consumption promotes FGF21 secretion from the liver perhaps representing a homeostatic feedback loop to regulate alcohol consumption. However, the target of FGF21 in the brain mediating these effects remains unclear. Excitingly, we have observed FGF21 dependent activation of neurons in the piriform cortex. Additionally, deletion of KLB in glutamatergic neurons significantly increases alcohol consumption in mice. These findings represent a novel endocrine circuit regulating alcohol consumption in an FGF21 dependent manner. Future studies will focus on mapping this circuit and characterization of neurons expressing KLB.

1Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA, 2Fraternal Order of Eagles Diabetes Research Center, University of Iowa Carver College of Medicine, Iowa City, IA

ABSTRACT. Sarah J. Marzi1, Szi Kay Leung2, ^, Teodora Ribarska3, ^, Eilis Hannon2, Adam R. Smith2, Ehsan Pishva2,4, Jeremie Poschmann2,5, Karen Moore3, Claire Troakes1, Safa Al-Sarraj1, Stephan Beck6, Stuart Newman7, Katie Lunnon2, Leonard C. Schalkwyk7+, Jonathan Mill2+*

1 Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, UK. 2 University of Exeter Medical School, University of Exeter, Exeter, UK. 3 German Cancer Research Center (DKFZ), Heidelberg, Germany. 4 Department of Psychiatry and Neuropsychology, Maastricht University Medical Centre, Maastricht, The Netherlands.5 Centre de Recherche en Transplantation et Immunology, Inserm, Université de Nantes, Nantes, France. 6 UCL Cancer Institute, University College London, London, UK. 7 University of Essex, Colchester, UK ^ / + Equal contributions

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by the progressive accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles in the neocortex. Recent studies have implicated a role for regulatory genomic variation in AD progression, finding widespread evidence for altered DNA methylation associated with neuropathology. To date, however, no study has systematically examined other types of regulatory genomic modifications in AD. In this study, we quantified genome-wide patterns of lysine H3K27 acetylation (H3K27ac) - a robust mark of active enhancers and promoters that is strongly correlated with gene expression and transcription factor binding - in entorhinal cortex samples from AD cases and matched controls using chromatin immunoprecipitation followed by highly parallel sequencing (ChIP-seq). Across ~182,000 robustly detected H3K27ac peak regions, we found widespread acetylomic variation associated with AD neuropathology, identifying 4,162 differential peaks (FDR < 0.05) between AD cases and controls. These differentially acetylated peaks were enriched in disease-specific biological pathways and include regions annotated to multiple genes directly involved in the progression of Aβ and tau pathology (e.g. APP, PSEN1, PSEN2, and MAPT), as well as genomic regions containing variants associated with sporadic late-onset AD. Partitioned heritability analysis highlighted a highly-significant enrichment of AD risk variants in entrohinal cortex H3K27ac peak regions. Finally, targeted gene expression analysis showed that variable H3K27ac is associated with transcriptional variation at proximal genes including CR1, GPR22, KMO, PIM3, PSEN1 and RGCC. This is the first study of variable H3K27ac yet undertaken in AD and the largest study investigating this modification in the entorhinal cortex. In addition to identifying molecular pathways associated with AD neuropathology, we present a framework for genome-wide studies of histone modifications in complex disease, integrating our data with results obtained from genome-wide association studies as well as other epigenetic marks profiled on the same samples.

ABSTRACT. CM Nelson1,2, H Lee3, RG Krug3, A Kamilova1, NN Madigan4, KJ Clark3, VA Lennon2,4,5, AJ Windebank4, JR Henley1,6

Following injury, the mammalian spinal cord forms a glial scar and fails to regenerate. In contrast, spinal cord tissue of vertebrate fish regenerates and swimming movements recover. The mechanisms underlying functional regeneration are not fully understood. Here we report that the glucocorticoid pathway regulates functional neural regeneration by directly affecting ependymal glia. Cord transection in larval zebrafish (Danio rerio) causes paralysis and neural cell death, with subsequent ependymal glial proliferation, extension of bipolar glia across the lesion, and neurogenesis. Functional connectivity is restored by axons extending from spared and nascent neurons along trans-lesional glial bridges. Studies in the transgenic SR4G reporter zebrafish reveal downregulation of both the glucocorticoid receptor Nr3c1 and glucocorticoid signaling activity in ependymal glia follow injury. Functional recovery is impaired by dexamethasone (Dex) treatment, which attenuates injury-induced ependymal glial proliferation, bridging, and neural tissue regeneration, and is independent of haematopoietic-derived immune cells. Loss-of-function mutagenesis of nr3c1 reverses functional impairment by Dex. By contrast, in the adult rat, NR3C1 levels and signaling activity in ependymal glia are upregulated following spinal cord transection. The unanticipated negative regulation of neural regeneration by glucocorticoid signaling via a direct effect on ependymal glia calls into question the putative benefit of corticosteroid therapy in early management of spinal cord injury. Indeed, therapeutic down-regulation of CNS glucocorticoid receptors might improve patient outcomes.

Departments of 1Neurological Surgery, 2Laboratory Medicine and Pathology, 3Biochemistry and Molecular Biology, 4Neurology, 5Immunology, and 6Physiology and Biomedical Engineering, Mayo Clinic, College of Medicine, Rochester, MN, USA 55905. Present address, AK: Vanderbilt Brain Institute, Vanderbilt University School of Medicine, Nashville, TN, USA 37232. Funding Support: NIH-NINDS (USA) R01 NS67311.

ABSTRACT. SV Bach, D Hosein, D Williams, NV Gallus, FA Sultan, KD Bunner, KE Savell & JJ Day

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, neuronal differentiation, dendritic growth and synaptic plasticity. Rodent Bdnf gene consists of nine 5’ non-coding exons (I-IXa) and one 3’ coding exon (IX). Each non-coding exon has its own promoter region where transcription of different variants is initiated. To investigate specific roles of the activity-regulated Bdnf variants I and IV we used a CRIPSR/dCas9 – VPR system, in which a strong transcriptional activator, VPR, is targeted to Bdnf I and IV promoter regions with the help of specific guide RNAs (gRNAs). Using this system in primary rat hippocampal neurons we are able to selectively upregulate Bdnf variants I and IV from their endogenous gene loci while leaving other variants mostly unaffected. To assess functional significance of selective Bdnf variant upregulation, we used Multichannel Electrode Arrays (MEAs) to perform single-unit recordings from neurons treated with CRISPR constructs. Upregulation of select Bdnf variants causes an increase in the spike frequency as well as the number of spontaneously active neurons. To assess subcellular localization of Bdnf mRNAs, single-molecule RNA fluorescent in situ hybridization (FISH) was used to visualize individual Bdnf I, IV and IX transcripts, which occupy diverse cellular compartments upon neuronal depolarization with potassium chloride. With this work we demonstrate the unprecedented precision of the endogenous Bdnf transcript variant upregulation using CRIPSR/dCas9 tools, the functional significance of Bdnf transcript variant upregulation for neuronal physiology, and the subcellular localization of Bdnf mRNAs with unparalleled resolution.

Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

ABSTRACT. Chronic alcohol drinking and withdrawal causes epigenetic and transcriptional changes in the brain that may contribute to relapse. In order to find novel genes altered during withdrawal, we performed a genome-wide analysis of transcripts in the rat hippocampus. Male adult Sprague Dawley rats were fed an ethanol or control liquid diet for 15 days and withdrawn for 24 hours. Hippocampal RNA was isolated and subjected to RNA-Seq. Weighted gene co-expression network analysis (WGCNA) was used to identify modules of co-expressed genes and demonstrated that the genes fit into 53 co-expression modules. Genes in module 1 were significantly higher during withdrawal and were enriched in the “TNF signaling pathway” and “Epstein Barr virus Infection” by gene ontology analysis, indicating disruptions in neuroimmune signaling during withdrawal. To validate the RNA-Seq findings, hippocampal RNA and chromatin were isolated for qPCR and chromatin immunoprecipitation (ChIP) analyses, respectively. Expression of Tnfrsf1a, Stat3, Relb, Plat, Serpine1, and Timp1 were increased during withdrawal, similar to the RNA-Seq findings. Decreased acetylation of histone H3 lysine 9/14 was also observed at the promoters of these genes. Notably, treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) during withdrawal normalized expression and histone acetylation at the promoters of the Tnfrsf1a, Stat3, and Relb genes. Our results demonstrate that transcriptional changes in neuroimmune genes occur in the hippocampus during withdrawal from chronic ethanol drinking and that some of these changes can be reversed by SAHA treatment, suggesting epigenetic regulation of neuroimmune genes and possible involvement in pathophysiology of alcoholism.

1University of Illinois at Chicago, Center for Alcohol Research in Epigenetics and Department of Psychiatry & 2Jesse Brown VA Medical Center, Chicago, IL 60612 USA, NIAAA P50 AA022538 (to AWL and SCP) and U01 AA020912 (to AWL).

ABSTRACT. Sa-Ik Hong1, Seungwoo Kang1, and Doo-Sup Choi1, 2, 3

Top-down control facilitates cortico-limbic circuits for the decision-making process. Dysregulation of this process is implicated in addiction including alcohol use disorder (AUD). Activation of striatopallidal neurons is known to suppress hierarchical response in reward-seeking behavior. Adenosine A2A receptor (A2AR) is expressed in the indirect inhibitory circuit. Although A2AR is co-expressed with dopamine D2 receptor (D2R) in the medium spiny neurons (MSN) in the striatum, the precise role of A2AR in the dorsomedial striatum (DMS) in reward-seeking behaviors through top-down inhibitory pathway has not been investigated. In our study, to investigate a DMS-specific role of A2AR in top-down ethanol-seeking behavior, first, we trained mice to voluntarily seek ethanol in the nose-poke operant chambers. Then, to examine the effect of A2AR, we measured neuronal activity in the DMS and a major output targeting neurons in the external part of globus pallidus (GPe). Using in vivo pharmacologic and optogenetic techniques, we manipulated neural activities in the A2AR expressing DMS neurons to GPe. We found that mice pre-exposed to ethanol exhibited increased top-down reward-seeking behavior. Pharmacological activation of A2AR and optogenetic stimulation to A2AR-containing indirect pathway suppressed top-down response. In contrast, A2AR-inhibition and optogenetic suppression of DMS-GPe circuit reversed top-down inhibitory response. Taken together, activation of striatopallidal A2AR and indirect pathway dampens top-down ethanol-seeking behavior, indicating that activation of A2AR signaling in the striatal indirect pathway could be a potential therapeutic target for AUD.

1Department of Molecular Pharmacology and Experimental Therapeutics, 2Neurobiology of Disease Program, 3Department of Psychiatry and Psychology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA. Funding Support: NIH AA018779, Samuel C. Johnson for Genomics of Addiction Program at Mayo Clinic, Ulm Foundation, Godby Foundation, David Lehr Research Award from American Society for Pharmacology and Experimental Therapeutics

ABSTRACT. Mohammed A. AlThobiti1 and Rayan M. AlSaadi2

1 PhD. Molecular Genetics, King Fahd Security College, Riyadh, Kingdom of Saudi Arabia 2 PharmD, Collage of Pharmacy, Taif University, Taif, Kingdom of Saudi Arabia

OBJECTIVE: To explore the practice and knowledge of hospital pharmacists in Saudi Arabia regarding pharmacogenetics.

METHODS: This was a cross-sectional study conducted in 2018 that included hospital pharmacists' from the five regions in Saudi Arabia. A validated self-administered survey collected demographics and information on the pharmacist's knowledge regarding pharmacogenetics.

RESULTS: Four hundred and forty-six questionnaires were returned with a full of response rate. Only 6.5% of the participants they assessed themselves as having knowledge of pharmacogenetics. Most participants (82.6%) believed that genetic variation affects the body's response to the medication. More than half of the respondents (60.9%) said they believe that pharmacogenetics affects the provision of patient counseling. Most participants (76.4%) reported that they had never studied pharmacogenetics. All participants (100%) stated that they had never trained in pharmacogenetics. The majority of participating pharmacists (80.4%) believe that they should have knowledge of pharmacogenetics. About two-thirds (63%) of the participants believe that pharmacogenetics reduces the economic losses resulting from medication substitution.

CONCLUSION: Pharmacists at hospital pharmacies had lacking knowledge of pharmacogenetics. Lack of pharmacogenetics training and education has been identified as the major barrier to knowledge. We recommend that pharmacy colleges be required to introduce pharmacogenetics into the curriculum and to increase the number of pharmacogenetics courses for hospital pharmacists'.

ABSTRACT. A Ferrer1, MT Zimmermann2, K Namekata3, T Harada3, MA Cousin1, JL Kemppainen4, KJ Mack5, M Shinawi6, D Babovic-Vuksanovic4, EW Klee1.

Affordable next generation sequencing has increased drastically the number of variations linked to neurologic diseases; however, functional studies remain the gold standard to establish the causal genotype-phenotype link. Here, we describe the functional validation of a homozygous mutation in the gene for the Dedicator Of CytoKinesis 3 (DOCK3) found in a patient with a neurological phenotype.

The proband is a 3-year-old female with macrocephaly, global developmental delay, hypotonia and autism spectrum disorder. High arched palate and dental anomalies were also noted. Brain and spinal MRI showed ventriculomegaly and hippocampal head atrophy, and diffuse atrophy of the thoracic spinal cord. Other tests were unremarkable. Using whole exome sequence (WES) in blood samples from the trio, we uncovered a homozygous mutation in DOCK3 (c.5020A>T; p.Met1674Leu). Her healthy parents were both heterozygous.

DOCK3 induces axonal growth in the brain, where it is mainly expressed. This variant is not described in mutation databases (ClinVar and HGMD), and represented at low percentage (0.12%) in gnomAD. In silico tools predict the variant as benign, although DOCK3 is statistically intolerant to missense variation. Protein 3D modeling indicated that the residue change occurred in the hydrophobic core of the protein’s catalytic domain, and in vitro transfection experiments concluded that the catalytic activity was impaired as a consequence. This information made us conclude that this variant is responsible for the proband’s neurologic symptoms.

This study highlights the relevance of using functional experiments to complement and validate the connection between WES findings and the phenotype from the patient tested.

1Center for Individualized Medicine. Mayo Clinic, Rochester MN. USA 2Genomics Science & Precision Medicine Center. Medical College of Wisconsin, Milwaukee WI. USA 3Visual Research Project. Tokyo Metropolitan Institute of Medical Science, Tokyo. Japan 4Clinical Genomics. Mayo Clinic, Rochester MN. USA 5Pediatric Neurology. Mayo Clinic, Rochester MN. USA 6Division of Genetics and Genomic Medicine. Washington University School of Medicine, St. Louis MO. USA

ABSTRACT. LY El Khoury1, HB Lee1, RG Krug1,2, AN Sigafoos1, KJ Clark1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, MN, USA 2Mayo Clinic School of Medicine, Mayo Clinic, MN, USA

The hypothalamic-pituitary-adrenal (HPA) axis mediates vertebrate-specific stress responses. Alterations in HPA axis activity are a causative and critical prognostic factor in many psychiatric disorders including depression. Stress during the early critical period is deemed to have a long-lasting effect throughout an organism’s lifespan. However, early life stressors can either lead to increased susceptibility or resilience to future stressors. To better understand gene-environment interactions during these critical developmental periods, we are developing assays to mimic chronic stress in the rapidly developing zebrafish. Currently, we have subjected larval zebrafish (5 days post fertilization) to prolonged, unpredictable stress (10-hr random on/off shaking overnight). Unpredictable shaking increased levels of glucocorticoid receptor (GR) activation and decreased innate immune response to a wound site. We will be further testing parameters of the unpredictable shaking assay, including the impact of various HPA axis mutants, and the impact of this early stressor on adult response to forced beach test (FBT), which measures coping in a similar manner as the forced swim test in rodents. Our assay suites will characterize the effect of early life stress on the physiology and behavioral of larval fish and the coping styles of adults, thereby providing a platform for genetic screening on potential susceptibility or resilience factors.

ABSTRACT. Price E. Dickson1, Tyler A. Roy1, Troy D. Wilcox1, Guy Mittleman2, Elissa J. Chesler1

1 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA 2 Department of Psychological Science, Ball State University, Muncie, IN 47306, USA

Substance abuse is a critical public health issue with genetic and environmental causes. Sensation seeking is a multifaceted, heritable trait which predicts the development of substance use and abuse in humans, and similar phenomena have been observed in rodents. Genetic correlations among substance use and sensation seeking indicate shared biological mechanisms. Environmental enrichment attenuates both traits suggesting that effects occur through these shared mechanisms. The molecular and neurobiological mechanisms underlying these relationships remain elusive. We used a systems genetics approach in BXD recombinant inbred (RI) mice to identify (1) genetic mechanisms driving intravenous cocaine self-administration and (2) shared genetic mechanisms underlying operant sensation seeking and alcohol preference. To assess the feasability of using the BXD RI panel to discover the mechanisms through which environmental factors influence the shared mechanisms underlying substance use and sensation seeking, we quantified the effects of environmental enrichment on operant sensation seeking, preference for a novel environment, and locomotion in a novel environment in the C57BL/6J and DBA/2J inbred strains, the founder strains of the BXD RI panel. We identified strain-dependent effects of housing condition on each of these distinct indexes of sensation seeking. Collectively, these data provide novel and, in some cases, shared biological mechanisms driving substance use and sensation seeking in the BXD RI mouse panel and provide evidence of genotype-dependent effects of environmental enrichment on sensation seeking traits in the BXD founder strains.

This work was supported by NIDA K99 DA043573 to PED and NIDA R01 DA037927 to EJC.

ABSTRACT. Acute food deprivation results in increased voluntary locomotion in a variety of vertebrate and invertebrate species, and increases the subjective, behavioral, and motivational effects of multiple drugs of abuse. The current experiments leveraged the locomotor stimulating effects of food deprivation and low-dose ethanol in an attempt to disentangle genes that regulate food- and drug-related behaviors in D melanogaster. Male and female flies from 37 Recombinant Inbred (RI) lines were ad lib fed or acutely food deprived prior to testing for basal and ethanol-stimulated locomotion. Food deprivation resulted in a marked increase in basal locomotion and a modest increase in ethanol-stimulated locomotion overall, with RI line accounting for 30-40% of the total phenotypic variation in these measures. Genome-wide association analyses identified 36 variants associated with basal locomotion in food deprived flies at LOD > 6, 13 of which were also associated with ethanol-stimulated locomotion, and two of which, VMAT and DAT, have been implicated previously in the expression of drug-related behaviors. Analysis of results from fed flies identified 3 variants associated with ethanol-stimulated locomotion, including one, fili, that also associated with basal and ethanol-stimulated locomotion in food deprived flies, as well as with body weight. These results suggest that food deprivation and low-dose ethanol induce locomotion via pathways that are largely genetically distinct in D melanogaster, but which might share some underlying genetic modifiers. Ongoing studies are examining the function of several of the identified genes in basal and ethanol-stimulated locomotion, as well as in other food- and drug-related behavioral assays. 1Drake University, Department of Psychology and Neuroscience, Des Moines IA

ABSTRACT. EL Catudio Garrett1,2, T Pallister1, S Dufner1, and SJ Certel1,2

The extracellular matrix (ECM) provides critical biochemical and physical signals to initiate and support a diverse array of cellular functions. Within the nervous system, both neurons and glia secrete molecules that contribute to the ECM. The CCN (CYR61, CTGF, NOV) family of proteins functions in a primarily regulatory role rather than structural. Specific members are highly expressed in the cerebral cortex, hippocampus, and cerebellum, contributing to neuron development, differentiation, and synaptic plasticity. However, the contributions of CCN family members and matricellular proteins to neuron communication and synaptic transmission remain understudied. Here we examine the role of the sole Drosophila CCN (dCCN) family member in the developing and mature nervous system. dCCN is expressed in motorneurons and interneurons throughout embryonic, larval, and adult stages. In the adult, dCCN expression is found in peripheral neurons located in the legs, wing, and proboscis that respond to sensory information and in the ventral nerve cord in neurons that innervate the reproductive system and motorneurons. In the central brain, our results demonstrate dCCN is expressed in octopamine (OA) neurons, and neurons that express the male form of Fruitless, a key regulator of male-specific behavior. Therefore, we examined the requirement for dCCN function in OA neurons using male aggression and courtship as readouts of neuron output. Our observations indicate that a reduction of dCCN in OA or all neurons results in decreased male aggression. Taken together, this work will contribute to understanding the role of matricellular proteins and the ECM in regulating neuron function and communication.

1Division of Biological Sciences, The University of Montana, Missoula, MT, United States 2Cellular, Molecular and Microbial Biology Graduate Program, The University of Montana, Missoula, MT, United States, Funded by NIH RO1 GM115510, USA

ABSTRACT. L.R. Goldberg1, M.G. Kutlu1, D. Zeid1, S., Gadiwalla1, T.J. Gould1

Cognitive deficits, such as disrupted learning, are a major symptom of nicotine withdrawal. These deficits are heritable, yet the genetic basis is unknown. Mice are valuable for identifying novel genes that contribute to variation in traits associated with various stages of addiction, including withdrawal. Our lab has developed a mouse model of nicotine withdrawal deficits in hippocampus-dependent learning, using chronic nicotine exposure via osmotic minipumps and fear conditioning. Here, we are using the recombinant inbred BXD genetic reference panel to identify novel genetic variants related to nicotine withdrawal deficits in learning. Male and female mice (n=4-8 per sex per strain, 30 strains) received either chronic saline or nicotine (6.3 mg/kg per day for 12 days), and then were tested for hippocampus-dependent learning deficits using fear conditioning. Preliminary QTL analyses using Genenetwork (1000 permutations) identified a significant QTL on chromosome 4 (96.9 Mb, LRS =35.7, p<0.05). A key advantage to utilizing the BXD lines is the wealth of available data. Upon completion of behavioral phenotyping, we will begin to prioritize candidate genes using behavioral and gene expression correlations in Genenetwork. Finally, RNA-sequencing in the BXD lines exhibiting extreme phenotypic variation will be used to identify hippocampal transcriptome changes associated with nicotine withdrawal.

1Department of Biobehavioral Health, Penn State University, University Park, PA, USA Funding Support: U01DA04163202

ABSTRACT. W.E. Crusio1, V. Lemaire-Mayo1, W. Fyke1,2, M. Premoli1,3, E. Subashi1, A. Delprato1,4, S. Pietropaolo1.

Fragile X Syndrome (FXS), the most common heritable cause of mental retardation, is due to a triplet repeat in the X-linked FMR1 gene that results in a loss of expression of the protein, FMRP. There exists a mouse model for the disorder, in which the homologous mouse gene, Fmr1, has been knocked out, also resulting in a loss of expression of the FMRP protein. This model thus has a good construct validity and therefore likely also has good predictive validity. One drawback of the mouse model is that the behavioral deficits that it displays are much milder than those observed in humans. We therefore wanted to see whether adding an environmental insult, prenatal stress, would result in more severe deficits. We subjected pregnant dams (genetic background C57BL/6J) that had been housed with a male for 2 weeks to unpredictable chronic mild stress and tested the offspring (WT and KO males, WT and heterozygous females) at the age of 7 weeks. Stress altered activity in the open field and social interaction of WT mice only. Stress also induced the appearance of cognitive deficits in the Y maze in KO mice, usually absent at this young age. These effects were sex dependent (mostly observed in males) and demonstrate interesting gene-environment interactions in the development of FXS in this mouse model.

1CNRS and University of Bordeaux, Institut de Neurosciences Cognitives et Integratives d'Aquitaine- UMR5287, Pessac, France. 2SUNY Downstate Medical Center - Neural & Behavioral Science Program, Department of Pathology, Brooklyn- NY, USA. 3University of Brescia, Department of Molecular & Translational Medicine, Brescia, Italy. 4BioScience Project, Wakefield, MA, USA.

ABSTRACT. The prevalence, age of onset, and clinical symptoms of many neuropsychiatric disorders - including alcoholism - differ substantially between males and females. We hypothesize, that the sexual differentiation of the human brain during development, is a major factor contributing to the difference in susceptibility to neurological disorders between males and females. The current concept of central nervous system sexual differentiation during development includes, not only the action of gonadal hormones, but also genes encoded on the sex chromosomes. We are investigating whether gametologous regions of the X and Y chromosome are involved in the formation of sex differences in the human brain, especially during very early central nervous system development (less than 12 weeks after gestation), before the production of sex hormones by the primordial sex organs. To do this, we differentiate human embryonic and neuronal stem cells to mature neurons/glia and investigate differences in gene expression patterns between male and female cells. In addition, marker genes for differentiation, as well as parameters for proliferation, motility and morphology are being observed. We are also using CRISPR/Cas9 gene editing for a functional investigation of novel long non-coding RNAs encoded on the Y chromosome, which have been previously identified by our group and are implicated in the early development of the nervous system in human male (manuscript under revision). Our study will shed light on differences between male and female brain development, and thus sets the basis for advances in one of the most neglected issues in medical science, sex differences.

Uppsala University, Department of Organism Biology, Unit for Evolution and Development, Uppsala, Sweden. Funding support: Swedish Research Foundation, Grant name: Sex determination factors in the brain encoded in the Y chromosome (Project number K2012-61X-22089-01-3)

ABSTRACT. Townsley, K.G. 1, 2, 3, Tran, A.T.D. 1, 2, Firsick, E.J. 1, 2, Hack, W. 1, 2, Batish, T. 1, 2, Kanadibhotla, S. 1, 2, Crabbe, J.C. 1, 2, Ozburn, A.R. 1,2

Recent studies provide strong evidence regarding a potential role for phosphodiesterase (PDE) inhibitors in the regulation of alcohol drinking in mice, rats, and humans. PDE4 inhibitors increase cAMP signaling and striatal activity in the brain. Rolipram and apremilast target multiple PDE4 isoforms with varying affinities. PDE4A and PDE4B are highly expressed in the nucleus accumbens (NAc), an important point of convergence in stress/reward-pathways. We investigated the role of PDE4 in high-intensity, binge-like alcohol drinking in High Drinking in the Dark mice (HDID-1).

HDID-1 female mice experienced 8 weeks of a 4-days/week Drinking in the Dark (DID; ethanol or water) paradigm. NAc tissue was collected and processed for multiplex qRT-PCR (n=6 mice/treatment/time point) to determine whether ethanol altered the expression of PDE4. Next, we tested whether rolipram (0, 5, 7.5, or 10mg/kg) or apremilast (0, 20, or 40 mg/kg) reduced ethanol drinking during DID (n=11-12/sex/dose). To determine whether inhibition of PDE4 in the NAc was sufficient to reduce DID, we administered apremilast intra-accumbens (0 or 2ug, via bilateral cannulae; n=15-17/group). We also tested the effects of apremilast on intake of other fluids and tastants.

Chronic binge-like drinking increased Pde4b expression in the NAc (p<0.01). Rolipram and apremilast reduced binge-like drinking (both drugs p<0.0001) and BALs (rolipram p<0.05; apremilast p<0.0001). Further, intra-accumbens administration of apremilast selectively reduced ethanol intake (p<0.05). These data provide support for the use of apremilast as a potential new therapeutic for the treatment of high-intensity binge-like drinking.

1Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon, 97239 USA; 2Portland Alcohol Research Center, Research and Development, VA Portland Health Care System, Portland, Oregon, 97239 USA; 3Department of Biology, Portland State University, Portland, Oregon, 97201 USA

Supported by NIH grants (AA13519; AA10760; AA020245), US Department of Veterans Affairs Grants (BX000313; CDA2 BX002488), NIH Common Fund and Office of Scientific Workforce Diversity (UL1GM118964; TL4GM118965; RL5GM118963), and the John R. Andrews Family.

ABSTRACT. Jessica Huebschman1, Miles Fontenot2, Feba Thomas2, Chris Cowan2,3, Laura N. Smith1,2

Cocaine, and other drugs of abuse, cause synaptic changes in the brain, altering circuitry to produce potentially long-lasting effects. Such exposure in rodents causes an increase in dendritic spine density and synaptic strength on medium spiny neurons (MSNs) in both the nucleus accumbens (NAc) and dorsal striatum – brain regions important for goal-oriented and habitual behaviors that are likely involved in the development of addiction. In this study, we identify a role for the fragile X mental retardation protein (FMRP), an RNA binding protein, in regulating these changes. FMRP controls the translation of hundreds of brain RNAs, many of which are involved in synaptic function. Loss of this protein, as seen in fragile X syndrome (FXS), results in an increase in dendritic spines, particularly immature spine types, in cortical and hippocampal brain regions, suggesting that FMRP is involved in regulating spine maturation and elimination. Here we show that lack of FMRP in Fmr1 knockout (KO) mice allows cocaine-induced increases in dendritic branching and spine density in NAc at a time point when it is not yet observed in wild-type animals, suggesting that FMRP limits this process. Interestingly, the observed increase primarily involves thin spine types, typically considered more immature and labile. We also observed a significant cocaine-induced increase in overall spine density in the dorsal lateral striatum of Fmr1 KO animals at this time point. Ongoing work in our lab is investigating the role of FMRP more specifically in regulating synaptic morphology and function.

1 Department of Neuroscience and Experimental Therapeutics, College of Medicine Texas A&M Health Science Center, 2 Department of Psychiatry, The University of Texas Southwestern Medical Center, 3 Departments of Neuroscience and Psychiatry & Behavioral Sciences, Medical University of South Carolina

ABSTRACT. B.L. Backus1, J.M. Martinez2, D.C. Curtis2, J.E. Bertrand2, P.J. Syapin2, and S.E. Bergeson2

1Texas Tech University and 2Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Only three pharmacotherapeutics for Alcohol Use Disorder (AUD) are FDA approved and none are widely used (<10%) or show a strong effect to reduce alcohol consumption in the long-term (<20% see sustained outcomes). Unfortunately, ~10% of the population suffers from AUD and over 5% of all medical morbidities share risky ethanol consumption as an underlying issue. As a consequence, intoxication, in general, and ‘alcohol addiction’ (severe AUD), in particular, are important clinical problems with a compelling need for new treatment. Although the age of ‘big data’ and high throughput genomics has increased potential targets for medications development, overwhelming rodent use is an increasing research concern. We recently showed that tetracycline analogs reduced drinking, and wished to test a species with more biological similarity to humans. Swine have successfully been used for medical purposes and share better genetic similarity to humans than rodents. At TTU’s research farm we used a Large White x Landrace hybrid cross to test the overarching hypothesis that swine would effectively model AUD. Escalating % ethanol, two-bucket choice showed preference levels of ~70% and intoxication. Pharmacokinetic elimination was similar to humans. Finally, we tested whether naltrexone and minocycline would reduce consumption; both drugs reduced drinking, and minocycline decreased preference. Voluntary consumption to biologically relevant BACs, human-relevant alcohol elimination, and drug reduction of ‘risky’ drinking support the hypothesis that swine may be a useful translational model for AUD.

Supported by the TTU/TTUHSC Presidential Initiative and The Laura W. Bush Institute for Women’s Health.

ABSTRACT. AN Moore1, J Linden1, JD Jentsch1

There is substantial evidence to suggest a relationship between a lack of inhibitory control and an increased susceptibility to drug abuse and addiction. Previous research suggests that an individual’s capacity for inhibitory control is heritable, and genome-wide linkage studies in BxD mice identified Syn3, which encodes the synaptic phosphoprotein synapsin III, as a potential candidate gene. Specifically, low Syn3 expression was found to be genetically correlated with poor inhibitory control. We hypothesize that mice underexpressing Syn3 will exhibit poor reversal learning performance, signaling diminished inhibitory control. Methods. Male and female mice, aged 2-9 months, that were homozygous for a deletion of the Syn3 gene (Syn3 -/-), heterozygous (Syn3 -/+), or wild-type (Syn3 +/+) were tested in a reversal learning task. Results. Genotype was not found to significantly alter reversal learning performance in terms of trials to criterion, omissions, or feedback learning. Although genotype did not significantly alter feedback learning, sex did, with males outperforming females on their ability to use previous outcomes to optimize future choice behavior. Additionally, genotype was found to have a significant sex-dependent effect on the ability to complete a sustained, variable duration observing response, with homozygous Syn3 deleted males having the greatest difficulty completing the observing response under the longest time requirement. Taken together, these findings suggest that Syn3 may have a complex effect on impulsivity, altering waiting but not action impulsivity. Further investigation is required to clarify the full effects of underexpressing Syn3 on motivated and impulsive behaviors.

1 Department of Psychology (Behavioral Neuroscience), Binghamton University, Binghamton NY 13902.

ABSTRACT. J.R. Bagley1, L. S. Bailey1, J. D. Jentsch1,2

Alcohol consumption and associated subjective effects are individually variable, and genetic factors account for a substantial proportion of that variance. Most forward genetic research to date has originated from reduced complexity intercrosses with limited genetic and phenotypic variability. The collaborative cross (CC) recombinant inbred (RI) panel, its inbred founders and the diversity outbred (DO) populations, are a cutting-edge tool for genetic and genomic research in part because of their remarkable genetic diversity. For these reasons, we assessed both sexes of all eight inbred CC/DO founder strains for voluntary alcohol drinking (20% ethanol, 2-bottle choice with water) during the active phase of the mouse circadian cycle (in the dark). The CC/DO founder strains demonstrate substantial and statistically significant strain differences in terms of alcohol lick preference scores, as well as total alcohol intake, with the high drinking strain consuming up to 10 times the body-weight adjusted amount of alcohol relative to the low drinking strain. Furthermore, strain mean distributions suggest this is a quantitative trait in both sexes, however substantial sex effects are present in some strains with females drinking more alcohol. This work has established heritability of voluntary ethanol drinking in the CC founder strains and will serve as a foundation for further characterization of CC RI strains. This research is performed in the context of the Center for Systems Neurogenetics of Addiction (CSNA) project and will be integrated into expansive data sets that will allow for in depth analysis of genetic relationships of addiction related behaviors and gene expression.

1. State University of New York at Binghamton, Binghamton, New York 13902 USA, 2. The Jackson Laboratory, Bar Harbor, Maine 04609 USA

ABSTRACT. Current treatment of major depression is not medically satisfying, partly due to the fact that the heterogeneity of the disease was not clearly defined, and thus treatment with differential drugs was not plausible. Traditional Chinese Medicine (TCM) is a personalized medicine in which the diagnosis and treatment is based on syndrome pattern identification for a given condition. It has been found that most prevalent TCM syndrome patterns in depression clinically include Qi stagnation, the stress responsiveness-like syndrome and Qi deficiency, fatigue-like syndrome. Balb/cJ and 129/S1 both were depression-prone, but they appeared to have different phenotypes of syndrome. Here, to further validate the plausible subtypes of depression, we used the formula that specifically treats the TCM syndrome and characterized the associated gene networks. Balb/cJ and 129/S1 received same protocol of chronic mild stress. Stressed or non-stressed mice also received administration of Qi stagnation alleviating TCM drug Yueju, Qi tonifying drug Sijunzi. After stress, both strain of mice reduced the sucrose preference, indicating a depression-like condition. However, only Balb/cJ mice showed early-onset and lasting Qi deficiency phenotype, tested with grip strength of forelimbs and loaded swimming. In Balb/cJ mice, treatment with Sijunzi and Yueju both improved performance in sucrose preference, although Sijunzi but not Yueju improved Qi-deficiency; In 129/S1 mice, Yueju improved performance in both sucrose preference and Oi-stagnation, whereas neither was improved by Sijunzi. Finally, syndrome specific gene networks were dissected using RNA-seq on the hypothalamus in these mice to identify the molecular signatures of subtypes of depression. Together, this study suggests genetic predisposition of Balb/cJ and 129/S1 to different syndromes may be useful to model the subtypes of depression for better and more precise treatment of depression.

1, Center for Translational Systems Biology and Neuroscience, 2, key Laboratory of Integrative Biomedicine of Brain Diseases, Support: by the National Science Foundation of China (81673625) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).   

ABSTRACT. Anna Delprato1 and Wim Crusio2,3 1BioScience Project, Wakefield, MA, USA 2University of Bordeaux, Institut de Neurosciences Cognitives et Intégratives d'Aquitaine (UMR 5287), Pessac Cedex, France 3CNRS, Institut de Neurosciences Cognitives et Intégratives d'Aquitaine (UMR 5287), Pessac Cedex, France.

Slitrk family members function in nervous system development and synapse dynamics. Several Slitrk gene variants have been linked to human neurological disorders such as Tourette syndrome, schizophrenia, autism, and obsessive compulsive disorder. Slitrks are transmembrane proteins that have extracellular N-terminal tandem leucine-rich repeats (LRR domains), a single transmembrane domain, and distinct C-terminal, cytoplasmic regions of varying length. In this study, we describe a Slitrk family-wide characterization using structural modeling and other in silico methods to predict the functional consequences of Slitrk gene variants on the fidelity of the proteins and their known interactions. These data will be used to forecast additional mutations that may be risk factors for neuropsychiatric disorders as well as identify conserved and divergent patterns of naturally-occurring variation. Results: For Slitrk1, 79% of the residues were modeled with greater than 90% accuracy which enables the visualization of most of the protein and the orientation of its domains with respect to one another. The prediction of a ligand binding site occurring in the 2nd LRR domain was also detected. For the other Slitrk proteins, the structural modeling was largely confined to the N-terminal region, including both LRR domains, and ranged from 58% (Slitrk3 and Slitrk5) to 66% (Slitrk4) of the residues modeled with greater than 90% accuracy. For Slitrk3 and Slitrk4, a portion of the C-terminal region was modeled with 80% confidence. Taken together, the structural modeling results for the 6 Slitrk family members, provides a foundation for rationalizing the gene variants based on the three dimensional protein structures.

ABSTRACT. C. Reed 1, H. Baba1, J. Erk1, T.J. Phillips1,2

The trace amine-associated receptor 1 gene (Taar1) impacts MA intake and other MA-related behaviors. A mutation in Taar1 (Taar1m1J) codes for a non-functional receptor, and Taar1m1J/m1J mice exhibit greater levels of MA intake than mice that possess at least 1 copy of the alternative allele (Taar1+). This mutation spontaneously arose in DBA/2J (D2J) mice and its impact has been studied on D2J and C57BL/6J strain backgrounds, which may not possess all genetic factors that could impact MA intake and other MA-related traits. Thus, the mutation was crossed onto heterogeneous stock-collaborative cross (HS-CC) mice that capture 90% of the genetic diversity in Mus musculus. Although Taar1m1J/m1J mice from this population consumed more MA than mice with at least 1 copy of Taar1+, some Taar1m1J/m1J mice had low MA intake, suggesting the presence of genetic modifiers of the Taar1m1J/m1J genotype effect. To identify these modifiers, selective breeding of high and low MA intake lines from D2JxHS-CC-Taar1m1J/m1J individuals is underway. Parent means (±SEM) of the S1 generation were 8.1±0.7 and 1.7±0.2 mg/kg, for the high and low MA intake lines, respectively. Response to selection was significant by S2; offspring means for MA intake were 5.2±0.4 vs. 4.2±0.3 mg/kg. Acute MA-induced stimulation and sensitization were significantly greater in Taar1m1J/m1J vs. Taar1+/+ mice of the originating population, but did not differ between the Taar1m1J/m1J S1 MA intake lines. If S2 mice differ for these traits, this will indicate that modifiers of the Taar1m1J/m1J effect for MA intake are also relevant to MA-induced locomotor stimulation and sensitization.

1Oregon Health & Science University, and 2Veterans Affairs Portland Health Care System Portland, OR, 97239 USA Supported by the Department of Veterans Affairs I01BX002106, NIH NIDA U01DA041579, NIH NIDA P50DA018165, and NIH NIAAA R24AA020245.

ABSTRACT. Duchenne muscular dystrophy (DMD) is a lethal degenerative disease that affects 1 in 3,500 males. DMD is caused by mutations in the dystrophin gene, which is expressed in muscle and nervous tissue. About one-third of DMD patients show developmental delays, among other neurological and muscular phenotypes. C. elegans is unique among animals used in DMD research in its ability to model not only the genetic insult, but also the behavioral, and cellular phenotypes observed in patients. To determine if Caenorhabditis elegans can also be used to model the neurological deficits of DMD, we ran dystrophic (dys-1) worms through a battery of neurological tests. Wild type animals are attracted to low concentrations of an attractant (1% diacetyl) but are repelled by high concentrations (100% diacetyl). Dystrophic worms detected and oriented normally towards low appetitive concentrations of a chemical cue. However, knockout mutants or worms with dystrophin specifically suppressed in nervous tissue also exhibited positive chemotaxis rather than being repelled by high concentrations. These findings suggest that lack of neuronal dys-1 is responsible for the ability of the animals to be repelled by noxious concentrations of diacetyl. The current experiments suggest that, in addition to modeling the muscular aspects of this disease, C. elegans may be useful to model the neurological impairments associated with DMD as well.

ABSTRACT. Duchenne muscular dystrophy (DMD) is an x-linked degenerative disease that affects one out of every 3,500 males. This disease is produced by a mutation in the dystrophin gene that results in an absence of the dystrophin protein. The result is progressive muscle weakness, leading to loss of ambulation in late adolescence and premature death. Currently, there is no cure for DMD, and no well accepted exercise regime for DMD patients. Studies have suggested minimal improvement in strength following exercise, but the effect of type and duration of exercise has not been considered until now. Previously, we demonstrated that the dys-1(eg33) C. elegans mutant is the most faithful animal model for Duchenne muscular dystrophy. It models the disease genetically, behaviorally, and anatomically, without requiring the secondary (sensitizing) mutations common in other animal models of the disease. To determine the effects of exercise on muscle integrity we subjected dys-1 animals to control, strength, or endurance exercise regiments of multiple durations. We find that while swimming did not increase longevity in dystrophic animals, it did temporarily improved their mobility. Our results represent the most complete assessment of exercise effect on dystrophic musculature to date, and demonstrate the complex interactions taking place between factors differentially affecting dystrophic musculature health, function, and longevity. Patients often face the daunting challenge of engaging in therapeutic treatments lacking experimental basis. It is our hope that our work will enable DMD patients and physicians to make more informed decisions regarding potential treatment approaches.

ABSTRACT. Wolfram syndrome (WFS) is a rare autosomal recessive disorder characterized by diabetes mellitus and insipidus, progressive optic atrophy, and sensorineural deafness. An increased risk of psychiatric disorders has also been reported in WFS patients. There are two subtypes of WFS. Type 1 (WFS1) is caused by mutations in the WFS1 gene and type 2 (WFS2) is characterized by mutations in the CISD2 gene.

Existing mouse models for WFS exhibit similar phenotypes to those observed in WFS patients including diabetic nephropathy, metabolic disruptions and optic atrophy. We identified a mouse mutant, Chirper, with a spontaneous mutation in the Cisd2 gene. Chirper mice emit frequent sonic vocalizations that are audible to the human ear, exhibit rapid respiration and have decreased body size and weight compared to unaffected littermates. Unlike WFS patients, Chirper mice do not appear to be diabetic.

Although behavioral phenotypes have been characterized in Wfs1 knockout mice, similar studies have been lacking for Cisd2. We tested Chirper mice in a battery of behavioral assays that model phenotypes related to neurological and psychiatric disorders including anxiety, sensorimotor gating, stress response, social interaction, learning and memory. We observed that homozygous and heterozygous mutant mice exhibit increased stress response. Homozygous mutants also show deficits in spatial learning and memory compared to wildtype littermates. Future studies will assess auditory brainstem response to observe the cochlear function of Chirper mice.

Our data indicate that the Chirper mouse strain could be a useful model to investigate the neurological and psychiatric symptoms observed in WFS.

1Department of Genetics, 2Neurobiology Curriculum, 3Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC.

ABSTRACT. Heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1) is a quantitative trait gene underlying reduced methamphetamine (MA) sensitivity. We subsequently showed that Hnrnph1 haploinsufficiency reduces MA-induced reward and reinforcement and MA-induced dopamine release in the nucleus accumbens. To explore the mechanism, we used immunoblotting and immunohistochemistry (IHC) to assay the level of tyrosine hydroxylase (TH), a precursor for dopamine synthesis and a marker of dopaminergic neurons, in the striatum and medial prefrontal cortex where the dopaminergic fibers terminate as well as the ventral midbrain where dopaminergic neuronal cell bodies originate. We hypothesized that a decrease in the number or innervation of dopaminergic neurons could underlie the neurobehavioral results. We found a small increase in TH staining in the dorsal striatum and nucleus accumbens. Immunoblot confirmed an increase in TH protein in the dorsal striatum and ventral midbrain. However, IHC of the ventral midbrain did not provide any evidence for an increase in the number of TH-positive neurons or in the amount of TH staining. Stereology of TH-positive puncta within the forebrain did not show any evidence for a difference in the number of dopaminergic fibers. These data suggest an alternate, drug-induced cell biological mechanism by which hnRNP H1 deletion affects MA neurobehavioral responding. We are investigating synaptic levels of the dopamine transporter (DAT), DAT function, and vesicular monoamine transporter expression. We are also using CLIP-seq and co-immunoprecipitation combined with mass spectrometry to identify changes in hnRNP H1 RNA targets and protein complexes following MA treatment. These studies will provide insight for future mechanistic validation.

1 Laboratory of Addiction Genetics, Departments of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine 2 NIGMS Biomolecular Pharmacology Training Program (GRANT), Boston University School of Medicine (GRANT) 3 Boston University Transformative Training Program in Addiction Science (TTPAS) Burroughs Wellcome Fund, (GRANT) 4 Department of Psychological and Brain Sciences, University of California, Santa Barbara 5 Ph.D. Program in Bioinformatics, Boston University 6 Computational Biomedicine, Boston University School of Medicine 7 Department of Anatomy and Neurobiology, Boston University School of Medicine

ABSTRACT. Our expanding knowledge about the influence of alternative polyadenylation (APA) on health and disease has created a new avenue for genetics research. APA is a tightly regulated mechanism by which a single gene encodes RNA isoforms with different polyadenylation sites. The present study utilized publicly available RNA sequencing (RNA-Seq) data from the read sequence archive in a secondary analysis to investigate APA changes in the nucleus accumbens of alcohol consuming male outbred Wistar rats in two separate experiments. For the first experiment, animals were either given continuous access to ethanol or only received water. In the second experiment, all animals were given continuous access with the exception of a period of forced consumption via gavage of either ethanol or water in between continuous access periods. Differences in APA between the groups in each experiment were determined by analyzing the RNA-Seq data using the bioinformatics tool DaPars (Dynamic analysis of Alternative PolyAdenylation from RNA-seq). Several genes displayed differences in polyadenylation site usage between groups. Some of these genes, such as Gfap, Psma4, and Ndufv3, have previously been associated with alcohol consumption. For these genes, the results here may provide an alternative understanding for the effect of ethanol on their expression. Other genes lacked any previous connection with alcohol, indicating possibly novel genetic factors were identified and, due to the nature of the analysis used for their identification, ethanol’s effect on their expression and structure. Our results indicate that APA may improve our understanding of ethanol’s effects on brain beyond expression differences.

1Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, CO, USA

ABSTRACT. ß-arrestin proteins (ß-arrestin-1 and 2) are ubiquitously expressed signaling molecules heavily implicated in the desensitization of G protein-coupled receptor signaling. While genetic knockout of a single ß-arrestin isoform has previously revealed distinct roles of these isoforms in drug-related behaviors, the majority of previous research has focused on the role of ß-arrestin-2. To better understand the role of ß-arrestin-1 in neurological behavior, we evaluated baseline and drug-related behavioral differences in ß-arrestin-1 wild-type (ßarr1 +/+), heterozygous (ßarr1 +/-), and knockout (ßarr1 -/-) male and female C57BL/6 mice. Compared with wild-type and heterozygous mice, ß-arrestin-1 knockout mice demonstrated higher baseline locomotor activity. Knockout mice also displayed higher Modified Racine Scale (MRS) scores for seizure behavior for delta-opioid agonist SNC80-induced seizures, suggesting that seizure threshold is reduced upon a global knockout of β-arrestin-1. For natural reward intake, no differences in sucrose preference were observed between genotypes or sexes. However, female ß-arrestin-1 knockout mice consumed more 10% alcohol than heterozygous females in a limited access, two-bottle choice model. In a binge-like 20% alcohol model, female ß-arrestin-1 knockout mice consumed significantly more alcohol than both heterozygous and wild-type females. A significant sex-effect was observed with females consuming more alcohol than males in both drinking models. Increased sensitivity to latency to loss of righting reflex was also observed in ß-arrestin-1 knockout mice, although no differences in duration of loss of righting reflex were observed. Overall, our findings suggest that expression of ß-arrestin-1 may be protective against seizure, hyperlocomotion in both sexes, and alcohol consumption in female mice.

1Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, 2Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, Indiana 47907, 3Interdisciplinary Life Science PhD Program (PULSe), Purdue University, West Lafayette, Indiana 47907

ABSTRACT. Modulatory transmitter systems are major contributors to nervous system plasticity and behavioral flexibility. To understand the mechanisms of neuromodulation, we must characterize modulator origins, targets, molecular pathways, and physiological responses. This is challenging using existing model organisms due to lack of identified neurons, access to cellular and circuit dynamics, or molecular tools. We seek to overcome these limitations by establishing transgenic labeling of neuronal structures, including modulatory transmitters and their receptors, in decapod crustaceans with characterized modulatory systems and neuronal physiology. We are using marbled crayfish - the first decapod crustaceans to have both genome and transcriptome sequenced. We have shown that offspring of this parthenogenetic species show little genetic variability, indicating a high probability for genetic manipulations to be carried to future generations1. As a first step toward transgenesis, we utilized our genome and transcriptome to perform BLAST analyses against related species to identify candidate genes of interest. We first developed a positive transgenesis marker by fusing GFP with the ubiquitously-activated promoter for actin-1 (Pactin-1), as actin-1 is broadly expressed early in development. Co-injection of Pactin-1::GFP will facilitate determination of transgenesis in subsequent injections of GFP-fusion products of neuronal genes. The marbled crayfish actin-1 transcript consists of 1132 bases with no introns; it is 92% homologous to cytoplasmic-type-actin-1 from Homarus americanus and 97.6% homologous to actin-1 from Penaeus monodon. We are now microinjecting Pactin-1::GFP plasmids into stage-1 oocytes to create the first transgenic line of crayfish.

1 Gutekunst, J., Andriantsoa, R., Falckenhayn, C., Hanna, K., Stein, W., Rasamy, J., and Lyko, F. Clonal genome evolution and rapid invasive spread of the marbled crayfish. Nature Ecology & Evolution. (2018). doi:10.1038/s41559-018-0467-9

ABSTRACT. Chang-Chih Huang^1,2, San-Yuan Huang^1,3*

¹ Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan² Department of Psychiatry, Buddhist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan, R.O.C.³ Department of Psychiatry, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan

Dopaminergic dysfunction is an important role in the pathogenesis of alcohol use disorder (AUD) and major depression (MD). Gene variants of the dopamine transporter (DAT) (also known as SLC6A3 gene) may influence expression of DAT. However, imaging studies on brain DAT availability in patient with AUD are limited, and the association of DAT availability with SLC6A3 gene variants in patients with AUD has not been analyzed. Hence, this study examined the relationship between brain DAT availability, SLC6A3 gene variants, cognitive function, and depressive symptoms in different subgroups of AUD. Single-photon emission tomography imaging with 99mTc-TRODAT-1 as a ligand was used to measure striatal DAT availability in 103 patients with AUD (55 pure AUD and 48 AUD/ MD) and 42 age and sex-matched healthy volunteers. Each subject was genotyped for the DAT polymorphism, and the Wisconsin Card Sorting Test (WCST), Hamilton Depression Rating Scale (HDRS) were used to assess neurocognitive function and severity of depression prior to brain imaging. Patients with AUD showed a significant reduction of DAT availability in three brain regions (p < 0.001), and this reduction was more pronounced in the pure AUD patients compared to healthy controls. The A allele of rs6350 have a greater risk to develop AUD (P < 0.05), and AUD patient with A allele shows a significant reduction trend of DAT availability in striatum region. The study results indicate that the DAT availability and SLC6A3 gene may have a role in susceptibility to AUD, and the SLC6A3 variants may influence the DAT availability.

ABSTRACT. CN Miller1,2, MJ Caruso3, and HM Kamens2

The initial response to nicotine is an important predictor of subsequent abuse. Multiple factors may alter this response including genetic background and age of first use. Here we investigated the influence of age, genetic background, and their interaction on nicotine sensitivity. We then examined whether these factors influence the relationship between initial behavioral responses and voluntary nicotine consumption in adulthood in male C57BL/6J and DBA/2J mice. The initial response to nicotine was measured during early adolescence (PND 31), middle adolescence (PND 41), late adolescence (PND 51), or adulthood (PND 71). We measured nicotine-induced changes in locomotor activity and body temperature to assess behavioral and physiological sensitivity to an acute injection of nicotine. Thirty-five days after behavioral testing, all animals were assessed for voluntary oral nicotine consumption. Results demonstrated that adult C57BL/6J mice were more sensitive to nicotine-induced hypothermia compared to early adolescence. In DBA/2J mice age and treatment interacted such that early adolescents were insensitive to nicotine’s hypothermic effects, but this response developed in later age. Locomotor sedation also differed by strain. Locomotor depression increased with age in C57BL/6J animals, but remained constant across time in DBA/2J mice. Finally, our data suggest that an acute nicotine exposure has long lasting effects on 100 ug/uL nicotine consumption in DBA/2J, but not C57BL/6J, mice. By understanding how age and genetic background influence initial behavioral responses to nicotine, we have a greater understanding of factors that promote nicotine abuse later in life.

1Department of Science, 2Department of Biobehavioral Health Penn State University Old Main, University Park, PA 16802, USA 3Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Acknowledgements: The Broadhurst Career Development Professorship for the study of Health Promotion and Disease Prevention (HMK).