ABSTRACT. NN Gong1, L Chakravarti Dilley1, M Szuperak1, CE Williams1, MS Kayser1-3.

All animals exhibit changes to sleep throughout development, suggesting a crucial role for sleep in early life. Sleep disturbances are also a common co-morbidity in neurodevelopmental disease (NDD), perhaps increasing severity of deficits. However, the genetic and molecular factors that drive the juvenile sleep state are unknown. Indeed, accumulating evidence suggests that genetic determinants of sleep in early developmental periods are distinct from sleep genes in maturity. We used Drosophila to define molecular mechanisms controlling sleep maturation, and to investigate mechanisms linking sleep and NDD. An RNAi-based screen converged on the transcription factor pdm3 as a novel genetic regulator of sleep ontogeny. Loss of pdm3 prevents young flies from achieving the high sleep amounts typically observed. Temporal mapping experiments indicate that pdm3 alters dopaminergic innervation and intrinsic architecture of a sleep promoting brain region. Pdm3 therefore coordinates an early developmental program that prepares the brain to later execute high levels of juvenile adult sleep. Next, to study the link between early life sleep disturbances and NDD, we conducted a reverse genetic screen in flies targeting human NDD risk genes. We identified the chromatin remodeler ISWI as required for normal sleep. In addition to sleep abnormalities, ISWI knockdown in flies leads to memory and social deficits. Together, these approaches yield new insights into developmental sleep control under normal and pathologic conditions, and provide a platform for investigating behavioral sequelae of developmental sleep abnormalities.

1Department of Psychiatry, 2Department of Neuroscience, 3Chronobiology and Sleep Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

ABSTRACT. Richa Saxena, Center for Genomic Medicine, Harvard Medical School, Boston, MA

Sleep is an essential daily behavior important for many functions, such as energy metabolism, immune function, DNA repair, memory, learning and metabolic waste clearance from the brain, but the molecular basis of sleep regulation in humans is still poorly understood. Although social and environmental factors contribute substantially to sleep duration, timing and sleep quality, these traits are heritable. Recent studies leveraging population-scale databases with subjective and objective sleep measures have identified genetic factors that contribute to natural variation in sleep behaviors and sleep disorders. These discoveries are beginning to illuminate diverse biological and physiologic mechanisms, implicating the circadian clock, development of brain regions, muscle function and synaptic neurotransmission, as well as inflammatory processes. Shared genetic links are found with anthropometric, cognitive, metabolic, and psychiatric traits. While sleep changes dramatically throughout the lifespan, the genetic contributors to sleep patterns in children and during adolescence have remained understudied. Recent GWAS in children suggest that sleep duration in childhood may be driven by different genetic factors than adult sleep duration. In contrast, the genetic architecture of morningness-eveningness preference may overlap between children and adults. Our work is beginning to provide insights into the genetic basis for inter-individual variation in sleep that should advance biological understanding and open avenues for new drug discovery for sleep disorders and downstream adverse health outcomes.

ABSTRACT. Željko Agić1, Eva Mihelec 1, Valentina Dukić1, Ana Filošević Vujnović1 and Rozi Andretić Waldowski1

Circadian genes regulate many aspects of addiction to drugs in Drosophila and mammals. Cells use redox regulation to regulate their functioning and neuronal plasticity. However, role of redox changes in the addictive drug-induced neuronal plasticity is just beginning to be studied. To determine if flies with mutations in circadian genes have disturbed redox balance, as a potential explanation for their unique behavioral phenotype, we measured activity of antioxidant enzymes catalase (CAT) and Superoxid Dismutase (SOD) and the amount of hydrogen peroxide (H2O2) in period, timeless, Clock and cycle Drosphila male mutant flies. Amount of H2O2 changes as a function of the time of day, it is higher during the day then at night, while in constant darkness H2O2 is on average higher at all time points. Normalized for the amount of tissue the activity of CAT is significantly higher in heads than bodies in all genotypes. SOD activity and the amount of H2O2 is higher in the heads of wild type flies, but in other mutants is genotype dependent. Similarly, CAT is several folds higher early in the day in all genotypes, while SOD activity and H2O2 are genotype dependent. Since each circadian mutant showed specific deficits in the activity of antioxidant enzymes and H2O2 concentration we propose a gene specific role in the regulation of the antioxidant defense. Many neuropsychiatric conditions, including addiction, are influenced by irregularities in the circadian rhythm and have disrupted redox balance, thus our findings contribute to the understanding of biological mechanisms of these diseases.

1 Department of Biotechnology, University of Rijeka, Rijeka, CROATIA Funding support: Croatian Science Foundation (HRRZ) # 2794 and University of Rijeka Research Support, # 13.11.1.4.06 141

ABSTRACT. Justin Palermo1, Shilpa Sonti2, Chiara Lasconi2, Alessandra Chesi2,3, Phillip Gehrman3, Struan F.A. Grant2,3, Alex Keene1

Sleep is nearly ubiquitous throughout the animal kingdom and has been linked to a wide range of human disorders and diseases. It is a complex behavior that has a genetic basis that is modulated by many environmental factors making it difficult to identify genetic loci associated with sleep. GenomeWide Association Studies (GWAS) in humans have been used to identify single nucleotide polymorphisms (SNPs) critical to several heritable diseases or traits. We have applied an ATACseq/promoter focused Capture C methodology in iPSCs-derived neural progenitors to carry out a ‘variant-to-gene’ mapping campaign in order to identify candidate sleep regulators from sleep associated SNPs. We are validating the role of these genes in sleep regulation through a neuron-specific RNAi screen in the fruit fly, Drosophila melanogaster. This approach has implicated the PIG-Q gene, which encodes phosphatidylinositol N-acetylglucosaminyltransferase subunit Q (), an enzyme involved in the first step of glycosylphosphatidylinositol (GPI)-anchor biosynthesis. When PIG-Q is knocked down in flies, the animals present with significantly longer sleep both in the day and the night and increased sleep bouts. Current studies seek to localize PIG-Q function within the brain and identify its impact on sleep intensity and function. These results establish a reliable method for confirming effector genes at loci identified in human GWAS studies by leveraging the extensive tools available in Drosophila.

1 Department of Biological Sciences, Florida Atlantic University, Jupiter, Florida, United States of America. 2 Division of Human Genetics, The Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA, United States 3 Perelman School of Medicine, University of Pennsylvania

ABSTRACT. G Sala1, H Shoji1, T Miyakawa1

Numerous tests of mouse behaviour are conducted over multiple time points (i.e., time series). The statistical methods commonly employed to analyse time series are linear regression techniques (e.g., repeated-measures ANOVA). However, in most cases, such approaches are not powerful enough to handle time-series data’s complexity. More specifically, these methods make unrealistic assumptions (e.g., linear relationships between variables and normality of response variables). The consequences of violating these statistical assumptions are twofold: first, the model’s estimates (e.g., p-values) may be miscalculated; second, phenotypes of interest may be overlooked. Here, we show how to resolve these issues with Generalized Additive Models for Location Scale and Shape (GAMLSS). GAMLSS is a supervised machine-learning technique that relaxes the abovementioned statistical assumptions (i.e., linearity and normality). Furthermore, the assumption of linearity can be relaxed even for the model’s random structure. As a demonstrative example, we analyse longitudinal (i.e., recorded over seven days) home-cage locomotor activity in wild-type and genetically engineered subjects from 94 mouse strains (Mouse Phenotype Database). The results show that the statistical assumptions, which determine the model’s reliability, are always met. Most notable, this modelling approach can easily discriminate between different mutation-induced phenotypes. Some mutants exhibit an alteration of their locomotor activity over the whole experimental period. By contrast, in other strains, only the subjects’ circadian cycle is disrupted (e.g., enhanced activity during specific hours in mutants). Given its superior accuracy and pattern-detection capabilities over commonly employed methods, we recommend using GAMLSS for any analysis involving time-series data of mouse behaviour.

1 Institute for Comprehensive Medical Science (ICMS), Fujita Health University. Funding: This work was supported by the Japan Society for the Promotion of Science [20K14267 granted to GS].

ABSTRACT. Elam Cutts1, Ningxiang Zeng1, Karen Gamble1, Andrew Hardaway1.

The dopaminergic system has been extensively studied for its influence on brain circuit function and behavior. In addition to this, the rhythmic expression of molecular core clock genes such as PER1, PER2, and BMAL1 in the suprachiasmatic nucleus has been thoroughly characterized. While rhythmicity has been shown in behaviors such as feeding and exercise, it remains to be determined if dopamine (DA) signaling itself is rhythmic, and if rhythmicity exists in primary outputs of dopamine neurons, such as the dorsal striatum. Selective loss of function and/or degeneration of DAergic neurons is a hallmark feature of Parkinson’s disease (PD), and transgenic PD mouse models have shown circadian abnormalities associated with dysfunction of the primary circadian pacemaker in the suprachiasmatic nucleus. Thus, examination of the molecular clock’s role in striatal DAergic expression has the potential to give insight to the underlying mechanisms of the disease. Utilizing the DA indicator dLight1.2 and fiber photometry, we measured DA transients in the dorsal striatum in freely behaving mice for periods of 24 hours under a 12:12 LD cycle and a 12:12 DD cycle. To ensure the DAergic identity of optical signals, we performed systemic injections of the DA transporter effluxer amphetamine (AMPH), and the D1 antagonist SCH23390 simultaneously with open-field assessments. DA signals significantly increased and decreased following AMPH and SCH23390 injection, respectively. Future studies will include the use of these same techniques in full body or nigrostriatal specific BMAL knockout animals to determine if circadian rhythms of striatal DA require core molecular clock genes.

1Department of Psychiatry and Behavioral Neurobiology, Center for Psychiatry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America Funding Support: K01DK115902 to AH

ABSTRACT. Recogntion: Outstanding Travel Awardee

ABSTRACT. Dana R. Hodorovich & Kurt C. Marsden

CHARGE syndrome is a congenital disorder affecting 1 in every 10,000 births. It is characterized by a spectrum of physical manifestations including eye, heart, craniofacial, and ear defects. CHARGE patients also frequently present with a range of behavioral difficulties such as autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (AD/HD), obsessive compulsive disorder (OCD), and sensory deficits, greatly diminishing their overall quality of life. Most CHARGE cases arise from de novo, loss-of-function mutations in the transcriptional regulator, chromodomain-helicase-DNA-binding-protein-7 (CHD7). CHD7 is an ATP-dependent chromatin remodeling protein that has been found to regulate key neurodevelopmental proteins, promote neuronal differentiation and neural crest cell development, and is required for neural processes such as cerebellar organization. While it is clear that CHD7 is required for normal neural development, how it affects neural circuit formation and function is not well understood. To investigate the pathophysiology of behavioral symptoms associated with CHARGE syndrome, we established a mutant chd7 zebrafish line using CRISPR/Cas9. With a panel of unbiased and high-throughput behavioral assays, we have defined multiple sensorimotor behavioral phenotypes. Our data show chd7 mutants have specific auditory and visually-driven behavioral deficits that are independent of defects in sensory structures, implicating chd7 in the regulation of underlying brain circuits. To identify brain regions impacted by chd7 loss of function, we are analyzing brain-wide activity using an unbiased immunofluorescence approach with phosphorylated ERK as an indicator of recent neuronal activity. Taken together, these studies will define mechanisms of chd7-dependent neurobehavioral phenotypes and empower future work to identify potential therapeutic targets.

Department of Biological Sciences, NC State University, Raleigh NC

ABSTRACT. Jacob A. Beierle1,2,3, Emily J. Yao1, Julia L. Scotellaro1, Olga Averin4, David E. Moody4, Gary Peltz5, Martin Ferris6, Camron D. Bryant1

1Laboratory of Addiction Genetics, Department of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine; 2T32 Biomolecular Pharmacology Training Program, Boston University School of Medicine; 3Transformative Training Program in Addiction Science, Boston University School of Medicine; 4Department of Pharmacology and Toxicity, University of Utah, 5Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University School of Medicine, 6Department of Genetics, University of North Carolina

Understanding the pharmacokinetic profile of an opioid drug is vital to therapeutic success, and mutations in human PK genes can alter therapeutic efficacy of opioids. Oxycodone (OXY) is a semisynthetic opioid metabolized into noroxycodone (NOR) and oxymorphone (OMOR) by CYP450 enzymes. We observed that 30 min post-oxycodone administration (1.25 mg/kg, i.p.), BALB/cJ mice show higher whole brain concentrations of OXY (t(14)=-2.55, p=0.023), NOR (t(14)=-1.917, p=0.076), and OMOR (t(14)=-2.06, p=0.058) compared to BALB/cByJ. This mirrors previous findings where BALB/cJ mice showed increased state-dependent conditioned place preference (SD-CPP) compared to BALB/cByJ. To potentially link behavioral differences with PK differences, we conducted quantitative trait locus (QTL) mapping of whole brain OXY, NOR, and OMOR concentrations in a reduced complexity cross (RCC). Because BALB/cJ and BALB/cByJ substrains differ by only ~10,000 SNPs/indels, large genetic loci mapped in F2 studies are offset by a dramatic reduction in the density of potentially causal variants. QTL mapping in 133 BALB/cJ x BALB/cByJ F2 mice (68F, 65M) revealed a single genome wide significant locus on chromosome 15 significantly associated with OMOR but, importantly, not NOR or OXY whole brain concentration. Oxymorphone is a bioactive metabolite at the mu opioid receptor, with 8x the potency of oxycodone. This region encompasses a cluster of 12 Cyp2d genes, the family of CYP450 genes responsible for metabolism of OXY to OMOR in humans. Two of these genes contain polymorphisms. Future studies will overlay these findings with OXY behavioral QTLs, cis-eQTLs, and proteomics to refine our list of candidate genes for validation.

ABSTRACT. L. Carvalho, H. Chen, and A.W. Lasek

The insula has emerged as an important region involved in aversion-resistant ethanol drinking (ARD). Within this region, GABAergic parvalbumin expressing interneurons are surrounded by specialized extracellular matrix known as perineuronal nets (PNNs), which regulate synaptic plasticity. In this study, we examined sex differences in ARD and a potential role for PNNs in this behavior. We also tested whether PNN removal in the insula by enzymatic digestion could affect ARD in males and females. In experiment 1, 4-hour ARD was assessed using two-bottle choice between water and 15% ethanol adulterated with quinine (0, 50, 100, 250 or 500 µM). In experiment 2, sex-differences in PNNs in ethanol naïve mice was examined by fluorescent staining of insular brain sections. In experiment 3, we injected chondroitinase ABC (ChABC) or PBS into the anterior insula to disrupt PNNs and then tested for ARD. In experiment 1, 250 and 500 µM quinine in ethanol reduced ethanol preference in males but not females, indicating sex differences in ARD. In experiment 2, females presented a higher PNNs intensity compared to males. In experiment 3, females treated with ChABC did not have altered ethanol preference, while males treated with ChABC exhibited an overall decrease in ethanol preference when quinine was included. These results demonstrate that females are inherently less sensitive to the presence of quinine in ethanol and that digestion of PNNs in the insula of females does not influence aversion sensitivity, at least at the quinine doses tested. These results suggest that sex-specific mechanisms regulate compulsive drinking

Center for Alcohol Research in Epigenetics, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL 60612 USA

Supported by NIAAA R01 AA027231, U01 AA020912, and P50 AA022538 (AWL).

ABSTRACT. A.M. Barkley-Levenson1 and A.A. Palmer1,2

Recent well-powered human genome-wide association studies (GWAS) have identified numerous novel genetic variants associated with alcohol consumption and problematic use. Mutant mouse lines provide a first step to determine whether individual gene manipulations are sufficient to alter alcohol consumption and to begin to explore the underlying mechanisms of action. Here, we investigated two genes (Fut2 and Dpp6) that have been associated with problematic alcohol use in humans. Fut2 knockout mice (heterozygous and homozygous) and wild type littermates were tested on a 4-day ethanol Drinking in the Dark (DID) procedure. Homozygous knockout mice showed a trend toward increased drinking compared to wild type on the final day of the DID test. In a separate experiment, heterozygous Fut2 knockout mice and wild type littermates were tested for intermittent access escalation of drinking. Mutant mice showed less escalation of drinking during week 1 of the test. However, no genotype differences were seen in later weeks, with all mice ultimately reaching the same level of intake. Finally, heterozygous Dpp6 knockout mice and wild type littermates were tested for ethanol DID. Mutant mice did not show any significant differences in intake from wild type mice. These findings highlight the complexity of translating genetic findings between humans and model organisms, even for phenotypes that may appear to be similar across species (e.g. ethanol consumption). We are continuing to evaluate these genes and others for their effects on a variety of phenotypes in mice to determine how they may increase the risk of problematic alcohol use.

1Department of Psychiatry, University of California San Diego, La Jolla, CA, USA; 2Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA. Funding support: NIH-NIAAA grants K99 AA027835 and R01 AA026281

ABSTRACT. Brian P. Wang 1, Dan Landayan 1, Jennifer Zhou 2, Fred W. Wolf 1,2

The brain uses both the carrot and the stick (reward and punishment) to coax survival-promoting behaviors at the right time. Thirst coaxes Drosophila to search for water when dehydrated. However, it is unclear how the brain encodes thirst, especially downstream of interoceptive and hygrosensory inputs. We used an unbiased screen and intersectional genetics to identify and characterize two pairs of thirst interneurons that are both necessary and sufficient for thirsty water seeking. Janu-GABA neurons are local inhibitory interneurons in the subesophageal zone (SEZ) that specifically promote water seeking and not other actions in thirst-motivated behavior. Janu-AstA neurons project from the SEZ to Neuropeptide F (NPF; Drosophila NPY) neurons in the superior medial protocerebrum (SMP). Janu-AstA neurons also specifically promote water seeking without affecting water intake, like Janu-GABA. However, Janu-AstA also suppress feeding behavior. Janu-AstA regulates thirst and feeding by inhibiting NPF release. Activation of the Janu water-seeking neurons imparts a positive valence, suggesting that they encode the rewarding carrot of impending water repletion rather than the punishing stick of thirst. The JanuAstA and Janu-GABA together constitute novel parts of the water-seeking circuit to coordinate between antagonistic internal states through interesting motivational properties.

1 Quantitative and Systems Biology Graduate Program, UC Merced, CA 953434 2 Department of Molecular and Cell Biology, UC Merced, CA 953435

ABSTRACT. Mice selectively bred for high methamphetamine drinking (MAHDR), compared to those bred for low drinking (MALDR), exhibit greater sensitivity to methamphetamine reward and insensitivity to methamphetamine aversive and hypothermic effects. The trace amine-associated receptor 1 gene (Taar1) is a quantitative trait gene for methamphetamine intake that also impacts methamphetamine thermal response. The MAHDR line is homozygous for the mutant Taar1m1J allele, whereas MALDR mice are is Taar1 +/+ or Taar1 +/m1J. To determine if differential sensitivity to methamphetamine-induced hypothermia extends to drugs of similar and different classes, we examined sensitivity to the hypothermic effect of cocaine, the amphetamine-like substance 3,4-methylenedioxymethamphetamine (MDMA), and the opioid morphine in these lines. The lines did not differ in thermal response to cocaine, only MALDR mice developed hypothermia to MDMA, and MAHDR mice developed greater hypothermia to morphine. We speculated that the mu-opioid receptor gene (Oprm1) impacts morphine response, and genotyped the mice tested for morphine-induced hypothermia. We found genetic linkage between Taar1 and Oprm1; MAHDR mice more often inherit the Oprm1D2 allele and MALDR mice more often inherit the Oprm1B6 allele. Additional testing demonstrated that the line-specific thermal effects of the full OPRM1 agonist morphine are not mimicked by the partial OPRM1 agonist buprenorphine. Data from a family of recombinant inbred mouse strains support the influence of Oprm1, but not Taar1, genotype on thermal response to morphine. These results nominate Oprm1 as a genetic risk factor for morphine-induced hypothermia, and provide additional evidence for a connection between drug preference and drug thermal response.

1Department of Behavioral Neuroscience and Methamphetamine Abuse Research Center, Oregon Health & Science University, 2Veterans Affairs Portland Health Care System, Portland, OR USA. Funding Support: NIDA R01DA046081, NIDA U01DA041579, NIDA T32DA07262, NIAAA R24AA020245, the Department of Veterans Affairs I01BX002106, and the VA Research Career Scientist program

ABSTRACT. Saeedeh Hosseinian1, Aleksandra Mech1, Adele Leggieri1, William Havelange1, Munise Merteroglu2, Ian Sealy2, Elisabeth Busch-Nentwich2, Caroline Brennan1

Impulsivity is defined as acting on a momentary basis without consideration of outcomes. Two forms of impulsivity are impulsive action involving deficiency in response inhibition and impulsive choice referring to impairment in delayed gratification. Both forms of impulsivity are associated with many psychiatric disorders such as addiction, ADHD and bipolar disorder. While impulse control disorders show moderate to high degrees of heritability, the genetics of impulsivity is not widely studied. We aimed to identify genes and pathways underlying impulsivity using a forward genetic screen of ENU-mutagenised zebrafish. We screened 64 families estimated to cover 5072 dominant and 1327 recessive alleles. We assessed impulsive action using a zebrafish version of 5-choice serial reaction time task. In this task, after the animal learns the association between a stimulus and a reward, a pre-stimulus interval is applied during which a premature response is recorded as a measure of impulsive action. We identified seven candidate families of which one has been further analysed revealing a heritable deficit in impulse control. The exome sequencing of the founder of the line identified 29 candidate mutations of which three are associated with ADHD including one associated with four other psychiatric disorders as well in GWASs. Future work should identify genes and pathways underlying this phenotype. We demonstrated for the first time that a forward genetic screen of zebrafish for impulsivity could identify lines with potential translational relevance to human.

1School of Biological and Chemical Sciences, Queen Mary University of London, London, UK 2Department of Medicine, University of Cambridge, Cambridge, UK Funding Support: NIH R01 grant number U01 DA044400-01 to CHB and EMB

ABSTRACT. A Leggieri1, J García-González1, JV Torres-Perez1, W Havelange1, S Hosseinian1, AM Mech1, and CH Brennan1

ANKK1 is suggested to be involved in vulnerability to addictions. The mechanism by which ANKK1 may impact addiction vulnerability is poorly understood but has been suggested to involve effects on development and/or functioning of dopaminergic pathways. To test this hypothesis, we generated a CRISPR-Cas9 loss of function ankk1 zebrafish line. We assessed ankk1 mutants and wild-type (wt) siblings for behavioral phenotypes at 5 days post fertilization (dpf) Ankk1-/- show decreased locomotor activity and recovered slowly in forced light/dark test. To test impact of ankk1 loss of function on dopamine regulated behavior associated with addiction vulnerability, we examined the effects of amisulpride on habituation to acoustic startle, observing that ankk1 mutants were less sensitive to effect of amisulpride then wt siblings. As chronic alteration in dopamine signaling is predicted to affect brain dopamine receptor expression, we examined the expression of components of the dopamine pathway by qPCR, and of dopamine D2 receptor by immunohistochemistry. At 5dpf, we found a significant up-regulation of drd2b mRNA expression levels. In adult zebrafish brain, drd2 protein was detected in cerebral cortex, cerebellum, hippocampus, and caudate homologue regions, resembling the pattern in humans. In contrast, in ankk1 mutants drd2 expression was reduced in cortical regions being predominantly found in the hindbrain. Our findings support a role for ANKK1 in the development of the dopaminergic pathway.

1 School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Rd, London, E1 4NS, United Kingdom Funding Support: NIH R01 grant number U01 DA044400-01 to CHB

ABSTRACT. S. Shams and L. Westberg

Oxytocin is a neuropeptide that regulates a wide range of mammalian social and non-social behaviors, such as bonding, social recognition, and anxiety. Zebrafish (Danio rerio) is a highly social vertebrate with phylogenetic conservation in oxytocin and relevant neurotransmitter systems, and relies heavily on visual social information (not olfactory). Since the involvement of the oxytocin system in zebrafish social behavior is not well-established we used adult CRISPR-Cas9-mutants lacking either of the two zebrafish oxytocin receptor genes, oxtr and oxtrl, and studied them in a social behavioral task. We observed socialization between four fish (same-genotype, n ≥ 20 for each group) in a large open-field (40x body length) and measured shoaling, schooling, group excursions, general activity, and specific motor patterns of mutant and sibling control fish. Following automated social stimulation, we also measured the levels of neurotransmitters and amino acids (dopamine, serotonin, norepinephrine, glutamate, GABA, and glycine) in multiple brain regions using HPLC for these mutant fish and their wild-type control fish. Our data show that in the large ethologically-relevant open-field, zebrafish lacking oxytocin receptors display subtle differences in locomotion and anxiety-related behaviors, and significant deficits in social behavior. We also found differences in neurotransmitter levels in the measured brain regions, indicating a role of oxytocin in regulation of neurotransmitters associated with social and emotional behavior. These results suggest that the two oxytocin receptors may play important roles in zebrafish social behavior. Further, these findings advance our understanding of neural mechanisms underlying oxytocin-regulated social interaction in zebrafish.

Department of Pharmacology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden Funding support: Swedish Research Council

ABSTRACT. C. Reed1, A.M. Stafford1, J. R. K. Mootz1, H. Baba1, J. Erk1, T.J. Phillips1,2

The trace amine-associated receptor 1 gene (Taar1) impacts methamphetamine (MA) intake. A mutant form of Taar1 (Taar1m1J) codes for a non-functional receptor, and Taar1m1J/m1J mice exhibit greater levels of MA intake than mice possessing the reference Taar1+ allele. To study the impact of this mutation in a genetically diverse population, male and female heterogeneous stock-collaborative cross (HS-CC) mice were tested for MA intake, interbred with DBA/2J mice to transfer the mutant allele onto the diverse background, and used for selective breeding. To study residual variation in MA intake existing in Taar1m1J/m1J mice, selective breeding for higher (MAH) vs. lower (MAL) MA intake was performed from Taar1m1J/m1J individuals; a control line of Taar1+/+ individuals (MAC) was retained. The lines were examined for MA-induced locomotor and thermal responses, as well as for fluid volume and tastant consumption. HS-CC mice consumed little MA. HS-CC x DBA/2J cross Taar1m1J/m1J mice consumed significantly more MA than Taar1+/+ mice. Response to selection was significant by the S2 generation and there were corresponding changes in fluid volume consumed. Fluid consumption was not different in non-MA drinking studies. Taar1m1J/m1J genotype (MAC vs. MAL or MAH mice) was associated with heighted MA locomotor responses and reduced hypothermic response. MAL mice exhibited greater sensitization than MAH mice, but the selected lines did not consistently differ for the thermal or tastant phenotypes. Residual variation among high risk Taar1m1J/m1J mice appears to involve mechanisms associated with neuroadaptation to MA, but not mechanisms underlying sensitivity to hypothermic effects of MA.

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 R01DA046081, and NIH NIAAA R24AA020245.

ABSTRACT. Nitisha Ponnappan1, Esha Dhawan1, Mehek Dedhia1, Alisha Chunduri1, Lu Lu2, Robert W. Williams2, Anna Delprato1,3, and Wim E. Crusio1,3

We propose that the variability of a character should be seen as a phenotype different from the mean of a character. Previously we have shown low but significant heritabilities for the variances of behaviors exhibited in an open field and for morphometric measurements of the hippocampus in inbred and hybrid mice (W.E. Crusio. Inheritance of behavioral and neuroanatomical phenotypical variance: hybrid mice are not always more stable than inbreds. Behavior Genetics, 36: 723-731, 2006). In the present experiment, we studied behavioral and neuroanatomical variation in more than 50 recombinant inbred strains between C57BL/6J and DBA/2J. Our aim was to localize QTL influencing the phenotypical stability of behavior in an open-field, inter-male aggression, spatial learning in a radial maze, and morphometric variation in the hippocampus. Our results confirmed the existence of significant genetic influences on phenotypical variability. Several QTLs and gene candidates were identified and will be discussed.

1Department of Research and Education, BioScience Project, Wakefield, MA, 01880, USA, 2Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, TN, USA, 3Institut de Neurosciences Cognitives et Intégratives d'Aquitaine, UMR 5287, CNRS and University of Bordeaux, Pessac cedex, Aquitaine, 33615, France

ABSTRACT. Kristin M Scaplen1,2,5, Mustafa Talay1,6, John D Fisher1, Raphael Cohn3,7, Altar Sorka.1,Yoshinori Aso4, Gilad Barnea1, Karla R Kaun1.

The Mushroom Body (MB) is a well-characterized associative memory structure within the Drosophila brain. Analyzing MB connectivity using multiple approaches is critical for understanding the functional implications of this structure. Using the genetic anterograde transsynaptic tracing tool, trans-Tango, we identified divergent projections across the brain and convergent downstream targets of the MB output neurons (MBONs). Our analysis revealed at least three separate targets that receive convergent input from MBONs: other MBONs, the fan shaped body (FSB), and the lateral accessory lobe (LAL). We describe, both anatomically and functionally, a multilayer circuit in which inhibitory and excitatory MBONs converge on the same genetic subset of FSB and LAL neurons. This circuit architecture enables the brain to update and integrate information with previous experience before executing appropriate behavioral responses. Our use of trans-Tango provides a genetically accessible anatomical framework for investigating the functional relevance of components within these complex and interconnected circuits.

1 Department of Neuroscience, Brown University, Providence, RI 02912 USA, 2 Department of Psychology, Bryant University, Smithfield RI 02917, USA, 3 Laboratory of Neurophysiology and Behavior, The Rockefeller University, New York, NY 10065, USA, 4 Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States, 5 Center for Health and Behavioral Sciences, Bryant University, Smithfield, RI 02917 USA, 6 Current address: Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 02138, USA, 7 Current address: Department of Biological Studies, Columbia University, New York, NY, 10027

ABSTRACT. Emily Xiao1*, Devika Manoj1* and Anna Delprato1

Genetic variants of DCX, COMT and FMR1 have been linked to both neurodevelopmental disorders related to social behavior and intellectual disability (ID). In this systems based study we examine the roles of the DCX, COMT and FMR1 genes in the context of hippocampal neurogenesis with respect to these disorders to identify hubs and signaling pathways that bridge these conditions. Our approach includes a literature review, functional analysis of RNA Seq data for genes correlating to DCX, COMT and FMR1 expression in the developing hippocampus, and an assessment of experimentally validated protein-protein interactions. Evaluation of the gene correlates identified genes linked to early Wnt signaling, social behavior, mental retardation and neurogenesis. The neurogenesis related genes include many that are also associated with axon, dendrite, and neuron processes. A topological assessment of protein-protein interactions for DCX, COMT and FMR1 indicates that the DCX and FMR1 networks are highly interconnected via proteins associated with RNA binding and cell cycle such as FXR1/2 and CYFIP2, whereas the COMT network is linked to the DCX and FMR1 networks via the neurotrophic factor S100B as well as the microtubule associated proteins MAPT and TUBA1A. Our findings suggest that factors connecting DCX, COMT and FMR1 in hippocampal neurogenesis, social behavior and ID disorders such as Fragile X Syndrome converge at Wnt signaling and synapse related processes such as dendrite and axon guidance and branching.

*Equal contributions

1Department of Research and Education, BioScience Project, Wakefield, MA, 01880, USA

ABSTRACT. Shu Ye 1, 2, 3 #, Biao Cai 1, 2, 4, #, Peng Zhou 1, 2, 4 , Hua-Wu Gao 1, 2, Meng-Ting Zhang 1, 2, Da-Bao Chen1, 3, Yun-Peng Qin 1, 3 , Xin Lei 1, 3, Xin-Quan Li 1, 3, Juan Liu 1, 3, Ya-Xun Cheng 1, 3, Yong-Chuan Yao 5, Guo-Ming Shen 1, 2, 3 *

Background: Huang-Pu-Tong-Qiao formula (HPTQ), a traditional Chinese medicine (TCM) formula used to improve cognitive impairment. However, the underlying neuroprotective mechanism of HPTQ treated for diabetic cognitive dysfunction (DCD) remain unclear. The purpose of this study was to investigate the neuroprotective mechanism of HPTQ in DCD mouse based on the molecular docking. Methods: To investigate the neuroprotective effect of HPTQ in DCD, the Morris water maze (MWM), novel object recognition (NOR) test was used to detect the learning and memory changes of mice; hematoxylin-eosin (HE) staining were used to investigate damage of hippocampal neurons; the western blot (WB) were used to examine the level of brain-derived neurotrophic factor (BDNF) of hippocampus. To investigate the neuroprotective mechanism of HPTQ in DCD, the molecular docking was used to predict the possible target proteins of different active components in HPTQ and then the WB was used to verify the expression of key target proteins in hippocampus of mice. Results: HPTQ improved the learning and memory ability, hippocampal neuron damage and the level of BDNF in hippocampus of DCD model treated with HFD/STZ for 12 weeks. Besides, the results of molecular docking showed that the main chemical components of HPTQ could be well combined with the targets of Bcl-2-associated X (Bax) and B-cell lymphoma2(Bcl-2) and caspase-3. The levels of Bax/Bcl-2 protein ratio and caspase-3 increased in DCD model while and the HPTQ inhibited the expression. Conclusions: These data indicated that HPTQ improved the cognitive performances of DCD mice via the Bax/Bcl-2 and caspase-3 Pathway.

School of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine

ABSTRACT. The CDC reports that 52.9% of people who used MA in the last year have a MA use disorder. Quantitative trait locus mapping in selectively bred high and low MA drinking (MADR) mouse lines identified a single nucleotide polymorphism (SNP) that accounts for 60% of the genetically determined differences in MA intake in the MADR lines. This nonsynonymous substitution mutation within the trace amine-associated receptor 1 (Taar1) gene alters the protein amino acid sequence and results in the expression of a nonfunctional TAAR1 receptor. MA is a full TAAR1 agonist, and homozygosity for this mutation (Taar1m1J) segregates with high MA drinking and low sensitivity with MA-induced aversion. MA-induced potentiation of NMDA-mediated excitatory postsynaptic currents (EPSCs) is dependent on TAAR1 in midbrain dopamine neurons and is observed in MALDR mice but not MAHDR mice. These results suggest that increased activation of NMDA receptors is important for aversive effects of MA. Since the lateral habenula-dorsal raphe (DR) circuitry, specifically serotonin (5-HT) neurons in the DR, regulate aversive behaviors, we also measured the effects of MA on EPSCs in DR brain slices from C57BL/6J mice, one of the two progenitor strains of the MADR lines. MA potentiates NMDA EPSCs in larger neurons (putative 5-HT neurons), but not smaller cells. Post-hoc immunohistochemistry will confirm cell type. Future studies will compare the MADR lines. Sensitivity to MA-induced aversion via TAAR1 signaling may play an important protective role against developing MA use disorders.

1Department of Behavioral Neuroscience, Oregon Health & Science University, 2Neurological Surgery, Oregon Health & Science University, 3Veterans Affairs Portland Health Care System, Portland, OR USA. Funding Support: NIDA R01DA046081, NIDA U01DA041579, NIAAA T32- AA007468, the Department of Veterans Affairs I01BX002106, and the VA Research Career Scientist program.

ABSTRACT. Naltrexone-precipitated conditioned place aversion (CPA) is considered as an effective model to investigate the motivational aspects of withdrawal from acute opioid dependence. While precipitated CPA can be viewed as an abnormal memory of withdrawal, promoting the extinction of CPA might be a strategy for attenuating the opioid-related withdrawal symptoms. Studies have suggested that novel spatial exploration (novelty) facilitates the formation of extinction memories. However, whether novelty affects the extinction of CPA is unknown, Here, we examined the effect of novelty exposure on the extinction of naltrexone-precipitated CPA. We found that morphine followed by naltrexone produced significant place aversion with just one pairing in all groups. Exposure to the novel environment 1h before extinction facilitates the extinction long-term memory of CPA. However, exploring novelty 6h before the extinction had no effects. We also tested whether the novel nature of the environment is key to this effect and found that exposure to the familiar but not novel environment had no effect on the formation of extinction long-term memory of naltrexone-precipitated CPA. These results suggest that novelty exposure enhanced the extinction of the naltrexone-precipitated CPA and might be a promising behavioral strategy to reduce opioid withdrawal symptoms.

ABSTRACT. An ENU mutagenesis screen was conducted in mice and isolated a mouse line with low acute response to cocaine responses. We mapped and cloned the causative variant responsible for the lowered response to cocaine. A mutation in Slit-Robo GTPase activating protein 3 (Srgap3) is responsible for this lowered locomotor activation phenotype. We recreated the mutation in C57BL/6J using CRISPR/Cas9 based precise engineering and observed the lowered cocaine locomotor activity and behavioral sensitization, which confirmed the causative mutation. We carried out intravenous self-administration (IVSA), the gold standard for addiction phenotype in this line. We find lowered voluntary cocaine intake and reinstatement in IVSA in the Srgap3 mutant mice. Biochemical analysis revealed that the ENU mutation leads to a highly destabilized protein. Finally, electrophysiological characterization of Srgap3 mutant neurons in the nucleus accumbens core showed preexisting hyperexcitability that resist the cocaine-induced further increase in excitability, robustly observed in control group. To the best of our knowledge, this is the first report of SrGAP3 as a regulator of cocaine acute, sensitized, or voluntary intake. SrGAP3 is a negative regulator of Rac1 signaling and interacts with WAVE/Cyfip2 regulatory complex that we previously identified using forward genetic analysis in moue substrains. Functionally, it has been implicated in regulation of dendritic spine and lammelapodia formation. Combined, this data points towards Rac1-Srgap3-Wave/Cyfip2 as a major regulator of cocaine responses.

This work was funded by NIDA U01 DA041668 to V.K.

ABSTRACT. Authors: Brittany J. Baugher, Shama N. Huq, and Benjamin D. Sachs Affiliation: Department of Psychological and Brain Sciences, Villanova University, 800 Lancaster Ave., Villanova, PA 19085

Women are more likely than men to suffer from major depression and anxiety disorders, a fact that is thought to depend in part on sex differences in stress susceptibility. Consistent with this, several stress paradigms have been reported to exert more pronounced effects on depression- and anxiety-like behaviors in female rodents compared to males. For example, several studies have reported that females are more susceptible to a subset of depression- and anxiety-like behaviors induced by six days of exposure to sub-chronic variable stress. The current study sought to evaluate the generalizability of this increased susceptibility of females to sub-chronic stressors by examining potential sex differences in response to a modified, five-day stress paradigm. In addition to measuring behavior, the current work also evaluated the effects of stress on the expression of several genes in the nucleus accumbens that have been suggested to underlie sex differences in vulnerability to sub-chronic stress. The current results indicate that males and females are both susceptible to this new five-day stress regimen. Although males and females displayed mostly similar behavioral responses to stress, the sexes were marked by several distinct molecular alterations following sub-chronic stress exposure. These data indicate that females do not exhibit a general increase in susceptibility to sub-chronic stressors. However, the observed sex differences in gene expression could reflect an example of sexual convergence in which stress induces similar behavioral phenotypes through unique molecular mechanisms

ABSTRACT. Rebecca L Schnabel, Ryan P Cotter, Han B Lee, Muriel Metko, Peri Goldberg, and Karl J Clark

Aversive stimuli elicit a stress response (SR) in organisms. Severe short-term stress or stress experienced for an extended period can overburden stress regulating systems. Excessive employment of SR systems like the HPA axis can lead to the development or aggravation of a variety of complex diseases including psychiatric disorders. A long-term goal of ours is to gain further insight into the connection between stress and disease and potentially develop new interventions or therapeutics. Currently, we are using zebrafish to discover genes that impact vertebrate rapid stress responses. By means of a forward genetic screen to test numerous transposon-mediated insertional mutant alleles, we identified several candidate genes that impact locomotor responses to acute stress in larval zebrafish. Currently, we are generating targeted integrations into two of those candidate genes, pbx1a and diaph1. These new integration alleles are Cre-reversible and include secondary markers which allow for rapid genotyping via fluorescence. To rescue pbx1a and diaph1 function in interrenal cells (zebrafish adrenal cell population), we will cross these fish to an interrenal cell specific Cre-reporter line. Both pbx1a and diaph1 appear to be involved in cortisol production in human adrenal cell culture. Therefore, we predict that pbx1a and diaph1 are needed for normal ACTH stimulation of cortisol production in interrenal cells. However, both pbx1a and diaph1 are expressed throughout much of the CNS, and their role could be more complex or take place outside of interrenal cells.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA Funding Support: National Institutes of Health (USA) R01GM134732

ABSTRACT. Grace E. Boyum, Han B. Lee, Ashley N. Sigafoos, Amanda A. Heeren, Izzabella K. Greene, Rebecca L. Schnabel, Margot A. Cousin and Karl J. Clark

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN

A Mayo Clinic patient was assessed for symptoms including intractable epilepsy and developmental delay. Whole exome sequencing showed a variant of unknown significance in ARHGAP35. This protein binds the promoter of NR3C1, the glucocorticoid receptor (GR), which is instrumental for the hypothalamus-pituitary-adrenal (HPA) axis response to organismal stress. Therefore, it is likely that ARHGAP35 plays a role in the systemic stress response and possible that it could be involved in observed symptoms in the patient. Since our lab is interested in stress biology and tests patient variants in a wide array of rare diseases, we began testing the biological role of ARHGAP35 by developing zebrafish lines with targeted mutant alleles in Danio rerio paralogs arhgap35a and arhgap35b. We first tested these mutant alleles to determine if their behavioral response to an acute stressor was similar to wildtype siblings. Using light-exposure stress assays, we confirmed that neither arhgap35a nor arhgap35b is involved in the locomotor response to an acute stressor. This matches with our hypothesis that ARHGAP35, a transcription factor, is likely impacting the genomic or transcriptional responses to stress. To look at the impact of loss of ARHGAP35 on transcriptional changes, we collected mRNA from larval zebrafish prior to and during a prolonged unpredictable stressor, using isolated genotypes from an arhgap35b in-cross. This mRNA is currently being processed through RNA-SEQ, and we hope to present some of our early results characterizing the impact of loss of arhgap35b on the transcriptome before and during an extended, unpredictable stressor.

ABSTRACT. Nikita Tjernström1, Erika Roman1,2

Gambling in some form is present in almost every culture and the prevalence of gambling disorder (GD) is around 0.2–5.3% worldwide. GD is the first non-substance addictive disorder listed along alcohol and substance use disorders, respectively, in the Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-5). Individuals with GD display deficits in decision-making in the Iowa Gambling task. The rat Gambling task (rGT) is a rodent analog of the Iowa Gambling task that can be used to improve our understanding of the neurobiological mechanisms underlying gambling behavior of relevance to GD. The aim of the study was to explore individual differences in gambling strategies emerging in the rGT. Furthermore, associations between the gambling strategy groups and sexual behavior, voluntary alcohol intake and response to naltrexone was examined. Subgroups of rats were found that preferred the suboptimal safest choice as well as the disadvantageous choice, i.e. the riskiest gambling strategy. No associations between gambling strategies and results from copulatory behavior tests were found. Voluntary alcohol intake was assessed and the risky group had higher voluntary alcohol intake and preference than the strategic and the safe groups. Lastly, naltrexone treatment during testing in the rGT decreased the number of completed trials and premature responses, while omissions were increased, which indicates lowered motivation. In conclusion, stable individual gambling strategies in the rGT were found and were associated voluntary alcohol intake. No associations with copulatory behavior were found.

1Research Group Neuropharmacology, Addiction and Behavior, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden 2Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden

ABSTRACT. CP King1, AS Chitre2, O Polesskaya2, SB Flagel3,4, TE Robinson5, LC Solberg Woods6, AA Palmer78, PJ Meyer1

Addiction vulnerability is influenced by genetic and environmental factors and is associated with non-drug traits such as the tendency to attribute incentive salience to reward cues. We performed a genome-wide association study (GWAS) to identify genomic regions associated with these behaviors. We examined two cohorts of phenotypically and genetically diverse N/NIH Heterogeneous Stock (HS) rats at two institutions during a Pavlovian Conditioned Approach task, in which we characterized the individual responses to food-associated stimuli (“cues”). Specifically, we measured two general categories of Pavlovian conditioned responses: cue-directed “sign-tracking” behavior, and food-cup directed “goal-tracking” behavior. We then identified genetic loci associated with multiple measures reflecting these two sets of behaviors for HS rats at each institution. We identified several loci across a number of measures for the two cohorts, with each measure associated with 1-8 loci. Many robust loci associated with sign-tracking were identified on chromosome 1. Interval sizes of loci varied largely, although some of the strongest associations for sign-tracking contained few genes (e.g. Tenm4, Mir708). We demonstrate that HS rats are useful for investigating the genetic variants underlying complex behavior and may be useful for identifying candidate genes for future testing.

1Department of Psychology, University at Buffalo, NY, USA, 2Department of Psychiatry, University of California San Diego, La Jolla, USA, 3Department of Psychiatry, University of Michigan, Ann Arbor, USA, 4Michigan Neuroscience Institute, University of Michigan, Ann Arbor, USA, 5Department of Psychology, University of Michigan, Ann Arbor, USA, 6Department of Internal Medicine, Molecular Medicine, Center on Diabetes, Obesity and Metabolism, Wake Forest School of Medicine, Winston-Salem, USA, 7Department of Psychiatry, University of California San Diego, La Jolla, USA, 8Institute for Genomic Medicine, University of California San Diego, La Jolla, USA.

Funding Support: NIDA P50 DA037844

ABSTRACT. Pamela N. Romero Villela1,2, Richard Border, Ph.D.3, Matthew C. Keller, Ph.D.1,2, Luke M. Evans, Ph.D.1,4, Marissa A. Ehringer, Ph.D.1,5 Genes coding for the neuronal nicotinic acetylcholine receptor emerged as the top replicable associations with smoking behaviors from GWAS a little over a decade ago. In particular, a non synonymous single nucleotide polymorphism (SNP) in CHRNA5, rs16969968, leads to an amino acid change (D398N) which affects the pharmacological response of the receptor. Given the magnitude of its effects, we aimed to identify other genetic variants that may interact with SNP rs16969968. Using the UK Biobank, we performed a genome-wide rs16969968-by-SNP interaction analysis for cigarettes per day in a subset of 116,257 unrelated current and former smokers. We evaluated both raw (M=18.22, SD=10.16) and log-transformed (M=1.20, SD=0.25) cigarette per day scores from 116,257 unrelated current and former smokers. We failed to detect genome-wide significant interactions between rs16969968 and other SNPs across the genome with respect to either raw (M=18.22, SD=10.16) or log-transformed (M=1.20, SD=0.25) cigarette per day scores. These results are consistent with our simulations, which demonstrated that the power to detect SNPxSNP interactions at the genome-wide level remains limited, even in large samples such as the UK Biobank. We also conducted a secondary gene-by-SNP interaction analysis of daily cigarette use using MAGMA. Two genes on chromosome 20 (TMEM230, PCNA) reached genome-wide significance (p<2.64e-06) for the rs16969968-by-Gene interaction term. Currently, we are conducting ongoing analyses to determine if genes involved in the nicotinic pathway interact with rs16969869 more strongly than the rest of the genome to influence daily cigarette usage. Results from this work provide insight regarding possible epistatic mechanisms that underlie smoking behaviors, which may inform future functional studies of the underlying biology. 1 Institute for Behavioral Genetics, University of Colorado, Boulder Colorado 2 Department of Psychology, University of Colorado Boulder, Colorado 3 Departments of Neurology and Computer Science, University of California, Los Angeles 4 Department of Ecology & Evolutionary Biology, University of Colorado Boulder, Colorado 5 Department of Integrative Physiology, University of Colorado Boulder, Colorado

ABSTRACT. Leinani Hession*,1, Gautam Sabnis*,1, Others TBD1, Gary Churchill1, **, and Vivek Kumar1, **

Chronological aging is uniform, but biological aging is heterogeneous. Clinically, this heterogeneity manifests itself in health status and mortality, and distinguishes healthy from unhealthy aging. Clinical frailty indexes (FIs) serve as an important tool in gerontology to capture health status. FIs have been adapted for use in mice and are an effective predictor of mortality risk. To accelerate our understanding of biological aging, high-throughput approaches to pre-clinical studies are necessary. Currently, however, mouse frailty indexing is manual and relies on trained scorers, which imposes limits on scalability and reliability. Here, we introduce a machine learning based visual frailty index (vFI) for mice that operates on video data from an open field assay. We generate one of the largest mouse FI datasets comprising 256 males and 195 females. From video data we use neural networks to extract morphometric, gait, and other behavioral features that correlate with manual FI score and age. We use these features to train a regression model that accurately predicts frailty within 1.03 ±0.08 (3.9% ± 0.3%) of the pre-normalized FI score in terms of median absolute error (MAE). As sex-specific aspects of aging in mice are still poorly characterized, we provide an analysis on sex. In sum, we show that features of biological aging are encoded in open-field video data and can be used to construct a vFI that can complement or replace current manual FI methods. This new method will lead to increased accuracy, reproducibility, and scalability, and potentially enable large scale mechanistic and interventional studies of aging.

*Equal Contribution 1The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 **Corresponding Authors: Gary.Churchill@Jax.org, Vivek.Kumar@Jax.org

ABSTRACT. Over 250 mouse inbred strains have now been sequenced, including over 200 RI strains (BXD and CC). This allows the potential for a massive diallel cross (DAX) of over 60,000 isogenic F1s. Each one of these is fully “pre-sequenced” because we have sequence for both fully inbred parents. F1 genomes can be replicated any number of times, in any environment, and studied at any age. This not only improves traditional QTL mapping, but also allows us to identify dominance effects (since the DAX includes heterozygotes), parent-of-origin effects (by using reciprocal crosses), and epistatic interactions. Causal models, predictions, and ROC curves can be made in one subset of the hypothetical 60,000 F1s, and tested or refined in another subset. With the large number of available genometypes, it is possible to identify higher-order epistatic interactions among variants. If one includes genetically engineered models into a DAX, then the complex effects of genetic background and GXE can be jointly identified, and modifier alleles can be mapped. Because all animals are isogenic, a consistent polyphenome can be built up systematically, and because all DAX are derived from known parents, this polyphenome (a phenome extended across ages, treatments, sexes, etc) is integratable with decades of previous data. Data become better with age–what we call smart quadratic data.

New technologies and new tools make a DAX an important next step towards the realization of experimental precision medicine - being able to predict gene-by-gene-by-sex-by-environment (age, treatment, etc.) interactions, and to test these predictions.

Department of Genetics, Genomics, and Informatics, The University of Tennessee Health Science Center, Memphis, TN, USA

ABSTRACT. Dana R. Hodorovich & Kurt C. Marsden

CHARGE syndrome is a congenital disorder affecting 1 in every 10,000 births. It is characterized by a spectrum of physical manifestations including eye, heart, craniofacial, and ear defects. CHARGE patients also frequently present with a range of behavioral difficulties such as autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (AD/HD), obsessive compulsive disorder (OCD), and sensory deficits, greatly diminishing their overall quality of life. Most CHARGE cases arise from de novo, loss-of-function mutations in the transcriptional regulator, chromodomain-helicase-DNA-binding-protein-7 (CHD7). CHD7 is an ATP-dependent chromatin remodeling protein that has been found to regulate key neurodevelopmental proteins, promote neuronal differentiation and neural crest cell development, and is required for neural processes such as cerebellar organization. While it is clear that CHD7 is required for normal neural development, how it affects neural circuit formation and function is not well understood. To investigate the pathophysiology of behavioral symptoms associated with CHARGE syndrome, we established a mutant chd7 zebrafish line using CRISPR/Cas9. With a panel of unbiased and high-throughput behavioral assays, we have defined multiple sensorimotor behavioral phenotypes. Our data show chd7 mutants have specific auditory and visually-driven behavioral deficits that are independent of defects in sensory structures, implicating chd7 in the regulation of underlying brain circuits. To identify brain regions impacted by chd7 loss of function, we are analyzing brain-wide activity using an unbiased immunofluorescence approach with phosphorylated ERK as an indicator of recent neuronal activity. Taken together, these studies will define mechanisms of chd7-dependent neurobehavioral phenotypes and empower future work to identify potential therapeutic targets.

ABSTRACT. Background: Bullying is a significant early life stressor linked to internalizing and externalizing behaviors, suicidal tendencies and poor academic performance. However, sex-specific differences within these correlates have not been studied in a large, diverse sample. Methods: Using ABCD 2.01 data repository (11,875 9-10-year-old participants), we evaluated associations between parent-reported bullying experienced by the child, and the child’s behavioral problems assessed using the Child Behavioral Checklist (CBCL); suicidal ideation/intent, with the Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS); cognition, using the NIH Toolbox® Cognition Battery; and academic performance. Statistical analyses included the generalized additive models, likelihood ratio and chi-square tests, with adjustments for socioeconomic and demographic status.

Results: 11,016 children from 21 ABCD sites were included in the analyses. 1,683 of these participants (15.3%) experienced bullying problems, 1.2 times more prevalent in boys than in girls. Bullying was associated with elevated CBCL scores. Boys had greater behavioral problems than girls, and associations between bullying and several CBCL domain scores were stronger in boys than in girls (P<0.001). Additionally, bullied children were 2.4 times more likely to display suicidal tendencies than non-bullied children. Worsening CBCL scores were associated with greater suicidal tendencies among bullied children compared to non-bullied peers (P<0.05). Cognitive scores varied by sex and were lower among bullied children (ΔR2<0.01, P<0.05). Boys and bullied children had poorer grades (P<0.001), worse in those with suicidal symptoms (P<0.001). Conclusions: These findings highlight the sex-specific negative consequences of bullying. Future longitudinal studies will identify factors that might promote resilience to bullying.

Acknowledgments: Data used in the preparation of this article were obtained from the Adolescent Brain Cognitive DevelopmentSM (ABCD) Study (https://abcdstudy.org), held in the NIMH Data Archive (NDA). This is a multisite, longitudinal study designed to recruit more than 10,000 children age 9-10 and follow them over 10 years into early adulthood. The ABCD Study® is supported by the National Institutes of Health and additional federal partners under award numbers U01DA041048, U01DA050989, U01DA051016, U01DA041022, U01DA051018, U01DA051037, U01DA050987, U01DA041174, U01DA041106, U01DA041117, U01DA041028, U01DA041134, U01DA050988, U01DA051039, U01DA041156, U01DA041025, U01DA041120, U01DA051038, U01DA041148, U01DA041093, U01DA041089, U24DA041123, U24DA041147.

A full list of supporters is available at https://abcdstudy.org/federal-partners.html. A listing of participating sites and a complete listing of the study investigators can be found at https://abcdstudy.org/consortium_members/. ABCD consortium investigators designed and implemented the study and/or provided data but did not necessarily participate in analysis or writing of this report. This manuscript reflects the views of the authors and may not reflect the opinions or views of the NIH or ABCD consortium investigators

ABSTRACT. M Dai1, AR Dunn1, J-G Zhang1, VM Philip1, D Bridges2, KMS O’Connell1, CC Kaczorowski1

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

ABSTRACT. WC Booher1,2, LA Vanderlinden3,4, LA Hall1, CA Lowry6-10, LM Saba3, and MA Ehringer1,2

High and Low Activity strains of mice were bidirectionally selected for differences in open-field activity (OFA) and subsequently inbred to use as a genetic model for studying anxiety-like behaviors (DeFries et al., 1978). In addition to the extreme differences in OFA, the High and Low Activity mice demonstrate corresponding differences in other anxiety-related behaviors as assessed by light-dark box, elevated plus-maze, and novel object exploration (Booher et al., 2021). Hippocampal RNA-sequencing of the High and Low Activity mice identified 3,901 differentially expressed protein-coding genes, with both sex-dependent and sex-independent effects. Functional enrichment analysis (PANTHER) highlighted 15 GO terms, which allowed us to create a narrow list of 264 top candidate genes. Of the top candidate genes, 46 encoded four Complexes (I, II, IV, and V) and two electron carriers (cytochrome c and ubiquinone) of the mitochondrial oxidative phosphorylation (OXPHOS) process. The most striking results were in the female high anxiety, Low Activity mice, where 39/46 genes relating to OXPHOS were upregulated. In addition, comparison of our top candidate genes with two previously curated High and Low Activity gene lists and a PubMed literature review suggests Ndufa13, Ndufb7, Ndufc2, Ndufs2, Coq7, Arrb1, Crtc1, Dcc, Dlg2, Lrp8, Ntkr3 and Trim32 (the first five are involved in OXPHOS) as genes of interest for anxiety-like behaviors. In addition to the current results, mitochondrial dysfunction has recently appeared as both a cause and effect of anxiety-related disorders and thus should be further explored as a possible novel pharmaceutical treatment for anxiety disorders.

1.Department of Integrative Physiology, University of Colorado, Boulder, CO, USA 2.Institute for Behavioral Genetics, University of Colorado, Boulder, CO, USA 3.Department of Pharmaceutical Sciences of the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 4.Department of Epidemiology in the Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 5.Department of Biostatistics and Informatics at the Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 6.Department of Integrative Physiology, Center for Neuroscience, and Center for Microbial Exploration, University of Colorado Boulder, Boulder, CO 80309, USA 7.Departments of Psychiatry, Neurology, and Physical Medicine & Rehabilitation, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA 8.Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Rocky Mountain Regional Veterans Affairs Medical Center (RMRVAMC), Aurora, Colorado, 80045, USA 9.Military and Veteran Microbiome: Consortium for Research and Education (MVM-CoRE), Denver, CO 80220, USA 10.Senior Fellow, inVIVO Planetary Health, of the Worldwide Universities Network (WUN), West New York, NJ 07093, USA

Funding 1.National Institute of Mental Health training grant (T32 MH016880) 2.National Institute of Mental Health (grant number 1R21MH116263) 3.The National Center for Complementary and Integrative Health (grant numbers R01AT010005 and R41AT011390) 4.The Colorado Office of Economic Development and International Trade (OEDIT) Advanced Industries Accelerator Program (grant number CTGG1-2020-3064) 5.The Department of the Navy, Office of Naval Research Multidisciplinary University Research Initiative (MURI) Award (grant number N00014-15-1-2809).

ABSTRACT. Mice selectively bred for high methamphetamine drinking (MAHDR), compared to those bred for low drinking (MALDR), exhibit greater sensitivity to methamphetamine reward and insensitivity to methamphetamine aversive and hypothermic effects. The trace amine-associated receptor 1 gene (Taar1) is a quantitative trait gene for methamphetamine intake that also impacts methamphetamine thermal response. The MAHDR line is homozygous for the mutant Taar1m1J allele, whereas MALDR mice are is Taar1 +/+ or Taar1 +/m1J. To determine if differential sensitivity to methamphetamine-induced hypothermia extends to drugs of similar and different classes, we examined sensitivity to the hypothermic effect of cocaine, the amphetamine-like substance 3,4-methylenedioxymethamphetamine (MDMA), and the opioid morphine in these lines. The lines did not differ in thermal response to cocaine, only MALDR mice developed hypothermia to MDMA, and MAHDR mice developed greater hypothermia to morphine. We speculated that the mu-opioid receptor gene (Oprm1) impacts morphine response, and genotyped the mice tested for morphine-induced hypothermia. We found genetic linkage between Taar1 and Oprm1; MAHDR mice more often inherit the Oprm1D2 allele and MALDR mice more often inherit the Oprm1B6 allele. Additional testing demonstrated that the line-specific thermal effects of the full OPRM1 agonist morphine are not mimicked by the partial OPRM1 agonist buprenorphine. Data from a family of recombinant inbred mouse strains support the influence of Oprm1, but not Taar1, genotype on thermal response to morphine. These results nominate Oprm1 as a genetic risk factor for morphine-induced hypothermia, and provide additional evidence for a connection between drug preference and drug thermal response.

1Department of Behavioral Neuroscience and Methamphetamine Abuse Research Center, Oregon Health & Science University, 2Veterans Affairs Portland Health Care System, Portland, OR USA. Funding Support: NIDA R01DA046081, NIDA U01DA041579, NIDA T32DA07262, NIAAA R24AA020245, the Department of Veterans Affairs I01BX002106, and the VA Research Career Scientist program

ABSTRACT. JM Sadino1, CJ Kelly1, XG Bradeen1, ZR Donaldson1,2

The loss of a spouse is often cited as the most traumatic event in a person’s life. However, for most people, the severity and maladaptive effects of grief subside over time via a currently understudied neuromolecular adaptive process. Like humans, socially monogamous prairie voles (Microtus ochrogaster) form opposite-sex pair bonds, and upon partner separation, show behavioral and neuroendocrine stress-phenotypes that diminish over time. Eventually, they can form a new bond, a key indicator that they have adapted to the loss of their partner. Thus, prairie voles provide an ethologically-relevant model for examining potential neuromolecular changes that emerge following partner separation that regulate adapting to loss. Here, we test the hypothesis that extended partner separation diminishes pair bond associated behaviors and causes pair bond transcriptional signatures to erode. Following two weeks of pairing, nucleus accumbens tissue was collected from animals that either remained paired or were separated after 48hrs or 4wks. In a separate cohort, we assessed partner preference and selective aggression at these timepoints. Surprisingly, pair bond behaviors persist throughout separation, although partner preference metrics become more variable with prolonged separation. In addition, we found that the pair bond transcriptional signature is stable over time in animals that remain paired, but erodes following prolonged partner separation. Eroded genes are associated with mitochondrial regulation and gliogenesis, potentially indicating cellular stress. This result parallels recent findings examining the male prairie vole pair bonding transcriptional signature. Our results provide the foundational framework for studying the molecular basis of pair bond loss adaptation.

1Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, 80309, USA, 2Department of Psychology and Neuroscience, University of Colorado Boulder, 80309, USA

Funding Support: NIH DP2OD026143, Whitehall foundation, Dana Foundation

ABSTRACT. GJ Preston 1, T Kozicz 1

The etiology of psychiatric disorders is complex and poorly understood. Mitochondrial metabolic dysfunction has been strongly implicated in vulnerability to several psychopathologies, including posttraumatic stress disorder (PTSD). We recently identified both brain mitochondrial energy metabolism and the abundance of circulating acetylcarnitine as highly predictive of PTSD-like behavior in a cohort of male mice following exposure to a recently-developed paradigm for the induction of PTSD-like behavior. We therefore hypothesized that supplementation with the antidepressant L-acetylcarnitine (LAC) would mitigate PTSD-like behavior in male mice following exposure to trauma. We exposed 96 C57BL/6J male mice to a PTSD-induction paradigm comprised of two decontextualized sessions of inescapable electric foot shock. Immediately following induction, 48 animals were supplemented with ~100mg/kg/day LAC (0.06% in drinking water ad libitum). All animals were then assayed for light/dark transfer, marble burying, acoustic startle response, and light phase activity, in order to identify PTSD-like and resilient animals. Animals were sacrificed and metabolomic analysis for mitochondrial organic acids, acylcarnitines, and amino acids was performed in circulating plasma. PTSD-like animals supplemented with LAC displayed significantly reduced severity of PTSD-like behavior relative to those supplemented with vehicle alone. Supplementation with LAC also significantly mitigated the corticosterone response to restraint stress in PTSD-like animals. We also observed evidence of significant multisystem mitochondrial stress in both PTSD-like and resilient animals following exposure to the PTSD-induction paradigm, which was significantly mitigated by LAC-supplementation. These data provide valuable evidence on the mechanisms of an emerging antidepressant therapy as a potential intervention in PTSD. 1 Department of Clinical Genomics

ABSTRACT. Antonios M. Diab1, Michael Wigerius1, Dylan P. Quinn1, Jiansong Qi1, Ibrahim Shahin1, Julia Paffile1, Barbara Karten2, Stefan R. Krueger3, and James P. Fawcett1,4

Memory formation and maintenance is a dynamic process involving the modulation of the actin cytoskeleton within the synapse. Understanding the signaling pathways that contribute to this modulation is important for our understanding of synapse formation and function, and in learning and memory. Here, we assess the role of an actin regulator, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), in the hippocampus and on behaviour. We report that male mice lacking NCK1 have impairments in short-term memory in a social recognition task, in working memory in a Y-maze, and in spatial learning in the Morris water maze. We find that NCK1 is expressed in post-mitotic neurons but is dispensable for neuronal proliferation and migration in the developing hippocampus. Morphologically, we find that NCK1 is not necessary for overall neuronal dendrite development. However, NCK1-deficient neurons have lower dendritic spine and synapse densities in vitro and in vivo. EM studies reveal that mice lacking NCK1 show an increase in PSD volume and thickness in the CA1 region. Functionally, using FRAP and actin incorporation assays, we find that NCK1 influences the rate of actin turnover in dendritic spines. Together, our study implicates NCK1 as an important molecule in the stabilization of actin dynamics and functions in the formation of dendritic spines, and in hippocampal dependent memory.

1Department of Pharmacology, 2Department of Biochemistry and Molecular Biology, 3Department of Physiology and Biophysics, 4Department of Surgery, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada Funding Support: Canadian Institutes of Health Research (MOP 84366, 79413 and PJT 159738)

ABSTRACT. The habenula is among the evolutionarily most conserved parts of the brain and has been known for its role in the control of behavior to cope with aversive stimuli. Our recent studies in zebrafish have revealed the novel roles of the two parallel neural pathways from the dorsal habenula to its target, the interpeduncular nucleus, in the control of behavioral choice whether to behave dominantly or submissively in the social conflict. They are modifiable depending on the internal state of the fish such as hunger and play another important role in orientation of attention whether to direct it internally to oneself or externally to others. These studies, therefore, are revealing a novel role for the habenula as the integrated switchboard for concertedly controlling behavior either as a winner with self-centered (idiothetic) attention or a loser with others-oriented (allothetic) attention.