ABSTRACT. Bethany A. Stahl1,2#, Emilie Peco3,4#, Sejal Davla3,5, Kazuma Murakami1,2, Don J. van Meyel3,4*, and Alex C. Keene1,2*

Sleep is critical for many aspects of brain function, and chronic sleep disturbances are associated negative health outcomes including heart disease, diabetes and metabolic syndrome. Growing evidence suggests that glia contribute to diverse aspects of sleep regulation, including neuronal and metabolic homeostasis. Moreover, glial cells regulate synaptic function and neurotransmitter clearance, raising the possibility that glial cells affect sleep by modulating neurotransmitter signaling. The fruit fly, Drosophila melanogaster, provides a powerful system for interrogating genetic mechanisms underlying sleep regulation and function. At the molecular and cellular levels, sleep is highly conserved from mammals to flies, and powerful genetic tools allow for targeted manipulation of genes in discrete cellular populations. We identify that disruption of the transporter Excitatory amino acid transporter 2 (Eaat2) in glia, but not neurons, increases sleep. In mammals, EAATs non-preferentially clear excitatory neurotransmitters from the extracellular region at synapses; however in flies, Eaat2 demonstrates selective high-affinity of aspartate and taurine. Eaat2 is exclusively expressed in ensheathing glia, and spatio-temporal manipulation reveals Eaat2 functions during adulthood in these cells to regulate sleep. The increased sleep observed in Eaat2 deficient flies is accompanied by reduced metabolic rate, revealing a novel role for ensheathing glia in metabolic regulation. Together, these findings reveal a novel wake-promoting role for Eaat2 and ensheathing glia in sleep regulation.

1. Jupiter Life Science Initiative, Florida Atlantic University, Jupiter, FL 33458, USA 2. Department of Biological Sciences, Florida Atlantic University, Jupiter, FL 33458, USA 3. Centre for Research in Neuroscience, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada 4. Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada 5. McGill Integrated Program in Neuroscience McGill University, Montreal, Quebec, Canada # Denotes equal contributions * Correspondence to don.vanmeyel@mcgill.ca or keenea@fau.edu 

ABSTRACT. Previous studies showed that circadian abnormalities often lead to human psychiatric disorders. The underlying mechanisms, however, are far from certain. We found that zebrafish per1b mutants display hyperactive-, impulsive-, and attention deficit-like behaviors and low levels of dopamine, reminiscent of human Attention Deficit Hyperactivity Disorder (ADHD) patients. Per1b directly regulates dopamine-related genes monoamine oxidase and dopamine β hydroxylase, and acts through genes important for the development or maintenance of dopaminergic neurons to regulate their number and organization in the ventral diencephalic posterior tuberculum. Using a series of behavioral assays, we show that zebrafish per2 mutant fish display lower locomotor activities, more time staying in the center region of the tank and less time for social interactions, in comparison with wild types, indicating a clear depression phenotype of the per2 mutant zebrafish. Quantitative RT-PCR shows that glucocorticoid receptor (gr) is significantly down-regulated in both the per2 mutant larvae and adult male fish brain. Luciferase reporter assays show that Per2 can enhance Rorαa-mediated expression of gr, and ChIP assays show that Per2 binds to the three RORE elements in the gr promoter, suggesting that Per2 positively regulates gr in zebrafish. Further, cortisol, and expression of pomc (proopiomelanocortin) and crh (corticotropin releasing hormone) are significantly up-regulated in the per2 mutant male fish, reminiscent of human depression patients with disruptive activities of the hypothalamic-pituitary-adrenal (HPA) axis. Taken together, our analyses of zebrafish circadian mutants shed light on new roles of the circadian clock in psychiatric disorders.

Center for Circadian Clocks, Soochow University, Suzhou 215123, Jiangsu, China Funding Support: the National Basic Research Program of China (973 Program) (2012CB947600) and the National Natural Science Foundation of China (NSFC) (31030062, 81070455, 81570171).

ABSTRACT. Abnormal circadian rhythms and circadian genes are strongly associated with several psychiatric disorders. Neuronal PAS Domain Protein 2 (NPAS2) is a core component of the molecular clock that acts as a transcription factor and is highly expressed in reward- and stress-related brain regions, such as the striatum. However, the mechanism by which NPAS2 is involved in mood-related behaviors is unclear. We measured anxiety-like behaviors in mice with a null mutation in Npas2 and found these mice exhibit less anxiety-like behavior than wild-type littermates (in elevated plus maze, light/dark box and open field assays). We assessed the effects of acute and chronic stress on striatal Npas2 expression, and found that both stressors increased Npas2. Further characterization revealed Npas2 expression is restricted to Drd1-expressing neurons. Moreover, knockdown of Npas2 in the ventral striatum resulted in a similar reduction of anxiety-like behaviors as seen in the Npas2 mutant mouse. Using ChIP-Seq, we observed that NPAS2 has distinct temporal patterns of DNA binding in ventral striatum and identified novel binding sites for NPAS2 at genes known to be important for neurotransmission (e.g. dopamine receptor Drd3, and several Gabra genes). We found that Npas2 mutant mice exhibit reduced sensitivity to the GABAa positive allosteric modulator, diazepam, and that knockdown of Npas2 reduced Gabra1 expression and response to diazepam in the ventral striatum. These results implicate Npas2 in the response to stress and the development of anxiety and provide functional evidence for the regulation of GABAergic neurotransmission by NPAS2 in the ventral striatum.

Speaker: Angela Ozburn Talk title: NPAS2 Regulation of Anxiety-Like Behavior Speaker institutional affiliations: 1. Portland Veterans Affairs Medical Center, Research and Development Service, Portland, OR, United States. 2. Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR, United States.

Funding: NARSAD Young Investigator Award, Veterans Affairs Career Development Award 2: IK2 BX002488, and NIH grants: DA07290, AA020452 and AA010760.

ABSTRACT. Most physiology and behaviors in mammal exhibit daily oscillations generated by an internal time-keeping system composed of interacting circadian clock proteins. Although circadian dysfunctions in affective disorders have been well recognized, the underlying molecular mechanisms still remain to be further clarified. Accumulating evidence suggests that molecular components of the circadian system are heavily involved in mood control. We have recently demonstrated that the circadian nuclear receptor NR1D1 (also known as REV-ERBα) constituting a stabilizing loop of the mammalian clock, has a key role in the cyclic regulation of central dopaminergic system as well as mood-related behaviors. Abrogation of Nr1d1 gene or pharmacological inhibition of NR1D1 activity induced bipolar mania-like phenotypes in mice such as hyperlocomotion, reduced depression/anxiety-like behaviors and increased aggression. In accordance with animal models, our findings in human subjects also suggest dysregulated NR1D1 function in bipolar disorder patients and imply its diagnostic applications. In conclusion, circadian molecular clock is directly and functionally linked with the midbrain dopaminergic system, thereby having an impact on mood regulation and dysregulation.

1Department of Biomedical Sciences, 2Department of Legal Medicine, Korea University College of Medicine, Seoul, South Korea.

ABSTRACT. Sarah M. Neuner, BS^1,2, Sarah Heuer, BS¹, Timothy J. Hohman, PhD³, Ryan Richholt, BS⁴, Matthew J. Huentelman, PhD⁴, Kristen M.S. O’Connell, PhD², Catherine C. Kaczorowski, PhD¹

1. The Jackson Laboratory, Bar Harbor, Maine, United States
2. The Neuroscience Institute, University of Tennessee Health Science Center, Memphis, Tennessee, United States
3. Vanderbilt Memory and Alzheimer’s Center, Department of Neurology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
4. Neurogenomics Division, Translational Genomics Research Institute, Phoenix, Arizona, United States

In Alzheimer’s disease (AD), the age of symptom onset is highly variable, with some patients exhibiting cognitive symptoms several decades later than predicted based on family history and genetic status. This variability cannot be explained by simple clinical or environmental factors, suggesting that additional genetic factors modify disease onset. The identification of modifier genes that confer resilience in high-risk patient populations would reveal new mechanisms and thus therapeutic strategies to delay disease onset. Disease-relevant protective variants are difficult to identify in human populations, primarily because asymptomatic individuals rarely enter the clinic. Mouse models represent an ideal complement to human studies, as they present many advantages, such as defined genotypes, early access to brain tissue, and precise environmental control. However, the traditional mouse models of AD have failed to translate into successful treatments that improve cognition in humans. Here, we employ a novel AD mouse genetic reference panel, designed to overcome some of the barriers presented by current AD models. Analyses of whole-genome RNA expression from the hippocampus using a variety of bioinformatics approaches revealed key hub genes as specific targets that may be leveraged to promote resilience. We also identified a strong negative association between microglia transcriptional signatures and cognitive resilience to AD. Overall, work here introduces a humanized mouse population as an innovative and reproducible resource for the study of AD and identifies potential mechanisms and candidate genes that may be targeted in at-risk individuals to promote resilience. Ongoing and future work will investigate their utility as therapeutic targets.

ABSTRACT. E Petruccelli1, KM Scaplen1, M Feyder1, N Ledru1, N Savory1, KR Kaun1

The ability to associate a rewarding stimulus with a sensory cue from the environment is critical for an animal’s ability to find food and mates. Mapping the circuits in which these associations are formed, then initiate an output response provides a scaffold for understanding how experiences can influence decisions. Investigating the molecular makeup of this scaffold is key to understanding how memories for rewarding stimuli are remembered, and can be manipulated. The genetic accessibility afforded in Drosophila provides an ideal platform to understand how molecules act within reward circuits to form appetitive memories, and how drugs of abuse such as alcohol can manipulate these circuits to induce cravings. Here we describe a how memories for the intoxicating properties of alcohol are acquired and expressed through different mushroom body circuits. Intriguingly expression of these memories requires a remarkably complex multilevel circuit whereby dopamine directly, and indirectly via the mushroom body, modulates the activity of glutamatergic and cholinergic output neurons. We then reveal molecular changes that occur within mushroom body neurons to chance activity within these circuits required for memory retention. We show the importance of Scabrous/Notch signaling in formation of alcohol memory in this circuit, and demonstrate how alcohol can disrupt this highly conserved signaling cascade in the adult brain to induce transcriptional changes that persist in a reward memory circuit. Together this work provides a snapshot of how alcohol can affect the dynamic molecular and circuit mechanisms required for behavioral decisions.

ABSTRACT. JG Tasker1,2, GW Weiss1, C Chen1, Z Jiang1

Noradrenergic afferent inputs stimulate corticotropin releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) to activate the hypothalamic-pituitary-adrenal (HPA) axis in response to systemic stress. Glucocorticoids secreted in response to HPA activation feed back to the hypothalamus to inhibit HPA activation. Patch-clamp recordings of CRH neurons in brain slices revealed a rapid glucocorticoid-induced, endocytosis-dependent desensitization of alpha1 adrenoreceptors (AR1a) in PVN CRH neurons following acute stress. The glucocorticoid regulation of AR1a sensitivity is dependent on the nuclear glucocorticoid receptor (GR), as it is lost in a conditional GR knockout. Live-cell imaging of the AR1a in a hypothalamic cell line revealed that corticosterone does not itself cause internalization of the AR1a, but facilitates the norepinephrine (NE)-induced AR1a internalization by causing the accumulation of the AR1a in the late endosome. Thus, corticosterone increased AR1a interaction with the late endosomal marker Rab 11, but had no effect on AR1a interaction with the early endosomal marker Rab 5. Corticosterone also caused a reduced interaction of AR1a with the rapid recycling endosomal marker Rab4, which suggested that it caused redirection of the AR1a from the rapid recycling pathway into the late endosome. Therefore, glucocorticoids prevent rapid AR1a recycling to the membrane following ligand-mediated internalization by routing the receptor into the late endosome. These findings demonstrate a stress desensitization of CRH neurons to noradrenergic activation that likely contributes to the rapid negative feedback regulation of the HPA axis by glucocorticoids.

1Department of Cell and Molecular Biology, 2Tulane Brain Institute, Tulane University, New Orleans, Louisiana, USA

Funding support: National Institutes of Health MH066958.

ABSTRACT. Department of Biomedical Sciences, Charles E Kubly Mental Health Research Center, Marquette University, Milwaukee, WI Glucocorticoid hormones exert powerful influences on behavior and brain function through a variety of cellular mechanisms. In addition to actions mediated by the intracellular glucocorticoid receptors, accumulating evidence suggests that many glucocorticoid effects, particularly those that appear with short latencies, involve interactions with other, membrane-associated, proteins. One such protein is organic cation transporter 3 (OCT3), a bidirectional transporter for norepinephrine, dopamine, serotonin and histamine. In contrast to the presynaptic norepinephrine (NET), dopamine (DAT) and serotonin transporters (SERT), OCT3 has higher capacity and lower affinity for substrates, is insensitive to inhibition by cocaine and antidepressants, and is inhibited directly by glucocorticoid hormones. Thus, OCT3 represents a stress hormone-sensitive monoamine transport mechanism that may mediate context-dependent effects of stress on monoaminergic neurotransmission and behavior. We have demonstrated that OCT3 is expressed widely throughout the brain, localized to plasma membranes in axonal, dendritic and astrocytic processes, and on neuronal somata, consistent with a role in regulating extracellular monoamines. Indeed, we have recently demonstrated that injection of rats with stress levels of corticosterone acutely decreases dopamine clearance in the nucleus accumbens via a DAT-independent mechanism likely involving the inhibition of OCT3-mediated transport. Treatment of rats with stress levels of corticosterone led to increases in the duration and amplitude of both naturally occurring dopamine transients and electrically evoked dopamine signals. We further provided evidence that, through this mechanism, corticosterone potentiates the actions of cocaine on dopamine signaling and reinstatement of drug-seeking behavior in rats and mice.

ABSTRACT. The hypothalamic-pituitary-adrenal axis regulates plasma glucocorticoid levels. Under basal conditions, the synthesis and release of glucocorticoids occur in a circadian rhythm overarching ultradian pulses. Peak levels of glucocorticoids are seen at the start of the active phase. Limbic areas express high levels of corticosteroids (CORT) receptors and are affected by changes in CORT levels. We demonstrated earlier that repeated CORT application results in immediate (non-genomic) and long-lasting (genomic) changes in glutamate transmission in the basolateral amygdala (BLA), an area that regulates fear behavior. To study the effect of circadian fluctuations of CORT levels on glutamatergic neurotransmission in the BLA, we investigated spontaneous glutamate transmission in BLA slices of mice sacrificed at the start of the inactive and the start of the active phase. To mimic the ultradian glucocorticoid pulses, ‘inactive phase slices’ were exposed to pulses with increasing concentrations of CORT. Active phase slices were exposed to decreasing concentrations of CORT. Results showed that the basal miniature excitatory postsynaptic current (mEPSCs) frequency was increased in inactive phase slices with pulses of increasing concentration, while the basal mEPSC frequency was reduced in active phase slices exposed to the paradigm. Furthermore, tone-cued fear conditioning experiments showed that mice at the start of the inactive phase show more freezing behavior, a measure of fear, than mice at the start of the active phase. Mice treated with metyrapone, normally causing a reduction in the basal CORT level, showed more freezing behavior than controls, suggesting that fear learning depends on circadian variations in CORT levels. 

ABSTRACT. HB Leea,1, TL Schwabb,1, AN Sigafoosb, JL Gauerkeb, RG Krug, IIa, MR Serresb, DC Jacobsb, RP Cotterb, B Dasb,2, MO Petersenb, CL Dabyb, RM Urbanb, BC Berryb, and KJ Clarka,b,3

aNeurobiology of Disease program, Mayo Clinic Graduate School of Biomedical Sciences, 200 First St. SW, Rochester, MN 55905; and bDepartment of Biochemistry and Molecular Biology, Mayo Clinic, 221 Fourth Ave. SW, Rochester, MN 55902

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response (SR). Rapid physiological changes in heart rates and blood sugar levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal (HPA) axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent HPA axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires HPI axis activation. In teleost fish, interrenal cells (I) are functionally homologous to the adrenal gland cortical layer. We derived 8 frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), two in each of exon 2 and exon 5 of nr3c1 (glucocorticoid receptor), and two in exon 2 of nr3c2 (mineralocorticoid receptor). Using larval zebrafish and mild environmental stressors, acute changes in salinity or light illumination, we demonstrate that rapid locomotor response to acute stressors requires a functioning HPI axis via the action of mc2r (adrenocorticotropic hormone receptor) and the canonical glucocorticoid receptor encoded by nr3c1 gene, but not mineralocorticoid receptor (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology may prove useful to screen for genetic and pharmacological modifiers of the HPA axis.

ABSTRACT. William Jons1, Colin Colby1, Sue McElroy2, Mark Frye3, Joanna Biernacka1,3, Stacey Winham1

1Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA, 2Lindner Center of HOPE/University of Cincinnati, Cincinnati, Ohio, USA, 3Department of Psychiatry and Psychology, Mayo Clinic, Rochester, Minnesota, USA 

Bipolar disorder (BD) affects 3% of both men and women in the US, but has sex-specific differences in its symptoms. We previously discovered that cycle acceleration, rapid cycling, and an increased severity of mood episodes are more prevalent among women. Using data from the Mayo Clinic Bipolar Disorder Biobank, we now investigate whether X chromosome genetic variants might be associated with sex-specific differences in symptoms or comorbid behaviors of BD (cycle acceleration, increased severity of mood episodes, rapid cycling, attempted suicide, substance use disorder, or binge eating behavior). Logistic regression was used to test the association of each X chromosome variant with each phenotype using three approaches: (1) an approach that ignores X chromosome inactivation (XCI), (2) an approach that allows for XCI at all locations, and (3) our new approach that uses biological data regarding which regions are subject to XCI to inform the statistical model selected. None of the approaches identified significant associations of the phenotypes with X chromosome variants. Results approaching the significance threshold with our approach (p<3.3x10-6) included the association of substance use disorder with rs787088 (p= 8.77x10-6), and association of binge eating behavior with rs5915827 (p= 4.41x10-06). Both of these variants are intergenic, the first being located near DDX3X, NYX, USP9X and a gene encoding the calcium signaling kinase (CASK) protein, and the second being located between PRKX and NLGN4X, which has been implicated in autism and possibly schizophrenia. Further work will be needed to validate our findings in a larger, independent sample.

Funding Support: NIH R25 GM075148, and Marriott Foundation, Bethesda, Maryland, USA

ABSTRACT. Dushyant Mehra1*, Thomas Burghardt1,2*, Joshua D.Trzasko1, Han B. Lee2,, Tanya L. Schwab2, Wil A. Gendron2,, Brandon W. Simone2, Stephen E. Ekker2,, Tamas Ordog2, and Karl J. Clark2,

*Denotes Equal Contribution 1Department of Biomedical Engineering and Physiology, Mayo Clinic Rochester MN 2Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN

Understanding the role that epigenetics plays in stress response is important for studying how stressors affect the body. DNA based epigenetic events often involve a change in chromatin conformation prior to an alteration in gene expression and capturing these dynamics can potentially provide a mechanism for understanding how different stressors affect gene regulation. Current conformation capture methods such as 3C, 4C, and Hi-C that are used map chromatin architecture only capture chromatin architecture in a snapshot and are difficult to use to characterize different chromatin conformational states over time. This makes it difficult for these methods to capture dynamic chromatin changes that can occur during transcription. Conformation capture based methods are further constrained by the location of the restriction enzyme sites and the depth of sequencing which currently limits inter loci distance to approximately 500 bp. Current dynamic imaging based methods are diffraction limited to approximately 500 bp as well due to conventional microscopy resolution limits. By combining a Crispr dCas9 protein fused to a photoactivatable GFP protein, a multiplex gRNA delivery system, and photoactivatable localization microscopy (PALM) for single molecule imaging, we are developing methods to quantitatively track dynamics and distances between individual loci at resolutions below conventional microscopy limits. This method can be used be used to determine how chromatin architecture changes in response to stress events and should be applicable to transparent model organisms such as C. Elegans and Zebrafish.

ABSTRACT. Media coverage of animal behavior genetics research often misrepresents the complex etiology of behavioral disorders, typically portraying these disorders as arising from the effects of single genes rather than multiple genetic and environmental causes. This paper explores one mechanism through which these misrepresentations develop: the separation of knowledge about environmental contributors to behavior from knowledge about genetic contributors during the publication process. Using ethnographic research from animal behavior genetics laboratories, I describe how researchers informally acquire knowledge about how environmental factors (noise, smells, other stressors) alter mouse behavior, and how the designation of this knowledge as “methodological” or “tacit” limits its circulation outside of the laboratory. The limited circulation of environmental knowledge, as compared to the much wider circulation of genetic findings, contributes to a distorted public perception of disease etiology. As a counterfactual, I present media coverage of published instances of environmental knowledge (Crabbe, Wahlsten and Dudek’s well-known 1999 paper on mouse behavior and interactions with the laboratory environment), demonstrating that alternative media narratives about behavioral disorders are possible. This research has important implications for the way that animal behavior geneticists communicate their findings publically, suggesting that more effort to circulate environmental knowledge is needed to counterbalance genetic knowledge.

ABSTRACT. Approximately 8.5% of American adults are afflicted by either moderate or severe alcohol use disorder (AUD), defined as excessive alcohol use within the last twelve months that impedes the safety of oneself and others, while being unable to reduce one’s drinking. The Collaborative Study on the Genetics of Alcoholism used linkage analysis to suggest, in European Americans, a significant association between alcohol dependence and a 140kb haplotype block in GABRA2. Synonymous SNP rs279858 tags this haplotype block, and has a minor allele frequency of 0.45. In addition to AUD, rs279858 has been associated with a number of neuropsychiatric phenotypes including comorbid illicit drug use and childhood conduct disorder. Neuro-endophenotypes such as increased activation of the insular cortex and nucleus accumbens in reward anticipation and differential activation of the ventral tegmental area and medial frontal cortex in response to alcohol cues are also associated with this haplotype block. The chr4p12 locus codes for γ1, α2, α4, and β1 GABA receptor subunits; iPSC lines carrying the minor allele at rs279858 have reduced expression not only of GABRA2, but the other three genes within this cluster. Virtual chromatin conformation capture (4C) data supports this hypothesis of cis regulation of GABA gene expression. Identification and characterization of allele-specific variants involved in mediating long-range intrachromosomal interactions in this locus is a step forward in the process of elucidating genetic risk variants for AUD and subsequently developing more-specific therapies for those at increased genetic risk.

ABSTRACT. Alcohol-induced violence causes an immense social and economic burden worldwide. Despite the pervasiveness of this phenomenon, it is an understudied behavior and it's neurogenetic underpinnings are unknown. In this study, we describe a novel fly alcohol behavior; male Drosophila melanogaster become more aggressive after being exposed to a single low dose of alcohol. After this same low dose of alcohol female flies become more receptive to courtship, but do not exhibit alcohol-induced aggression. We predicted that alcohol was cross-potentiating the endogenous cVa (11-cis-vaccenyl acetate) sex-pheromone pathway, which resulted in potentiation of aggression and receptivity to courtship. The cVa sensitive olfactory receptor neurons (ORNs) express both FruM and Or67d. Here we show that both FruM and Or67d are necessary for alcohol-induced aggression. When flies are exposed to high levels of alcohol they have lower levels of FruM and have specifically downregulated FruM production in the Or67d expressing ORNs that project to the DA1 glomerulus. Flies previously treated with this higher dose of alcohol show reduced aggression. Taken together, these results suggest that ethanol regulates FruM to produce changes in aggression in a dose dependent manner.

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. TP Wijesekera1 and NS Atkinson1

Chronic alcohol consumption is shown to affect conserved gene networks in the human brain; one such network being the innate immune system. The innate immune system in Drosophila melanogaster consists of the Toll and IMD pathways, which culminate in NfKB proteins. The NfKB proteins connected to the Toll pathway are Dorsal and Dif (Dorsal like immunity factor). The functioning of the Toll pathway, including Dorsal and Dif, in ethanol response was previously demonstrated in flies. Dif is expressed in alternative splice isoforms. Based on the presence or absence of a nuclear localization signal in the genomic sequence, it is hypothesized that Dif A is nuclear and Dif B is non-nuclear. This study demonstrates that the functioning of Dif on ethanol response is splice variant specific. The function of the two isoforms was tested using fly lines that are Dif null, while engineered to express a specific (A or B) isoform. Flies deficient in Dif B show an increase in sensitivity to ethanol induced sedation as indicated by a significant decrease in KD50 (time for 50% sedation). Absence of the Dif A isoform does not change sensitivity to ethanol. Additionally, we demonstrate the significance of the Dif splice isoforms in other adult behaviors such as male courtship, circadian rhythm and sleep. Cellular localization of Dif B was seen to be non-nuclear. We document the expression of Dif B in the mushroom bodies and the antennal lobes of the adult brain, further strengthening the significance of the isoform in ethanol response.

1Department of Neuroscience and Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, Austin, TX, USA.

ABSTRACT. MA Cousin1, 2, †, EL Veale3, †, MT Zimmermann4, 5, †, M Bialer6, MR Bekheirnia7, C Boelman8, G Douglas9, D Doummar10, C Gilissen11, J Juusola9, B Keren12, T Kleefstra11, TM Kruisselbrink2, 13, MS Leduc7, K Machol7, S Mahida14, C Mignot12, J McLaughlin6, V Narayanan15, C Nava12, R Pfundt11, K Ramsey15, F Scaglia7, C Smith-Hicks14, AS Trentesaux16, R Willaert9, N Zadeh17, 18, D Babovic-Vuksanovic2, 13, RA Urrutia5, 19, A Mathie3, EW Klee2, 11, 13

The paternally imprinted KCNK9 gene encodes a two-pore-domain potassium ion transporter expressed primarily in the brain. A variant (G236R) in KCNK9 has been reported to cause Birk-Barel mental retardation dysmorphism syndrome (BBMRDS) in several families, but no additional variants have been reported to date. Here, we describe a cohort of 15 affected individuals from 11 families, one with the G236R variant and 10 affected by 9 additional novel KCNK9 alterations. The phenotypes in all patients are generally consistent with BBMRDS. Protein modeling showed that variants affect protein structure, dynamics, and K+ ion distribution. Compared to WT, a subset of variants caused an increased probability of K+ near the channel pore and were associated with more K+ transport events. This is in stark contrast to the decrease seen with G236R, suggesting distinct channel dysregulation by these novel variants. Patient variant characterization using electrophysiological techniques was consistent with the modeling results. Four of five variants assessed by these methods caused a gain of conductance compared to WT. All five variants were outwardly rectifying with reversal potentials close to the WT equilibrium potential. This is in contrast to the severely reduced current amplitude and pronounced inward rectification seen with G236R. We describe a cohort of patients with novel KCNK9 variants causing BBMRDS and with functional impact of these variants that is distinct from the classical G236R. Based on this observation, treatment with channel agonists, with reported positive outcomes in patients with the G236R variant, may not benefit patients with gain-of-function variants.

1Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA, 2Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA, 3Medway School of Pharmacy, University of Kent and University of Greenwich, Central Avenue, Chatham Maritime, Kent, ME4 4TB, United Kingdom, 4Clinical and Translational Sciences Institute, Medical College of Wisconsin, Milwaukee, WI 53226, USA, 5Bioinformatics Research and Development Laboratory, Genomics Sciences and Precision Medicine Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA, 6Division of Medical Genetics, Northwell Health, Manhasset, NY 11030, USA, 7Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, 8Division of Neurology, BC Children's Hospital, Vancouver, British Columbia, Canada, 9GeneDx, 207 Perry Parkway, Gaithersburg, MD 20877, USA, 10APHP, Department of Neuropediatrics, National Reference Center for Neurogenetic Disorders, Hôpital Armand-Trousseau, GHUEP, Paris, France, 11Department of Human Genetics, Donders Centre for Neuroscience, Radboud University Medical Center, Nijmegen, the Netherlands, 12APHP, Département de Génétique et Centre de Référence Déficiences Intellectuelles de Causes Rares, Hôpital de la Pitié-Salpêtrière, Assistance Publique - Hôpitaux de Paris, Paris 75651, France, 13Department of Clinical Genomics, Mayo Clinic, Rochester, MN, USA, 14Department of Neurogenetics, Kennedy Krieger Institute, Baltimore, MD, USA, 15Center for Rare Childhood Disorders, Translational Genomics Research Institute, Phoenix, AZ, USA, 16Service de Néonatologie, CHU de Caen, France, 17Genetics Center, Orange, CA, USA, 18Division of Medical Genetics, CHOC Children's Hospital, Orange, CA, USA, 19Department of Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA, †Authors contributed equally. Funding Support: Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA.

ABSTRACT. Ranad Shaheen, PhD,1 Mohammed Adeeb Sebai, MD,1 Nisha Patel, PhD,1 Nour Ewida, BSc,1 Wesam Kurdi, MD,2 Ikhlass Altweijri, MD,3 Sameera Sogaty, MD,4 Elham A lmardaw i, MD,2 Mohammed Zain Seidahmed, MD,5 Abdulrahman Alnemri, MD,6 Sateesh Madirevula, PhD, 1Niema Ibrahim, BSc, 1Firdous Abdulwahab, BSc,1Mais Hashem, BSc,1Tarfa Al-Sheddi, BSc,1Rana Alomar, MSc,1Eman Alobeid, BSc,1Bahauddin Sallout, MD,7Badi AlB aqawi, MD,7Wajeih AlAali, MD,7,8Nouf Ajaji, MD,7Harry Lesmana, MD,9Robert J. Hopkin, MD,9Lucie Dupuis, MD,10Roberto Mendoza-Londono, MD,10Hadeel Al Rukban, MD,10Grace Yoon, MD,10,11Eissa Faqeih, MD,12andFowzan S. Alkuraya, MD1,13,14

Objective: Congenital hydrocephalus is an important birth defect, the genetics of which remains incompletely under-stood. To date, only 4 genes are known to cause Mendelian diseases in which congenital hydrocephalus is the main orsole clinical feature, 2 X-linked (L1CAM and AP1S2) and 2 autosomal recessive (CCDC88C and MPDZ). In this study, we aimed to determine the genetic etiology of familial congenital hydrocephalus with the assumption that these cases repre-sent Mendelian forms of the disease. Methods: Exome sequencing combined, where applicable, with positional mapping. Results: We identified a likely causal mutation in the majority of these families (21 of 27, 78%), spanning 16 genes,none of which is X-linked. Ciliopathies and dystroglycanopathies were the most common etiologies of congenital hydrocephalus in our cohort (19% and 26%, respectively). In 1 family with 4 affected members, we identified a homo-zygous truncating variant in EML1, which we propose as a novel cause of congenital hydrocephalus in addition to its suggested role in cortical malformation. Similarly, we show that recessive mutations in WDR81, previously linked to cerebellar ataxia, mental retardation, and disequilibrium syndrome 2, cause severe congenital hydrocephalus. Fur-thermore, we confirm the previously reported candidacy of MPDZ by presenting a phenotypic spectrum of congenital hydrocephalus associated with 5 recessive alleles. Interpretation: Our study highlights the importance of recessive mutations in familial congenital hydrocephalus andexpands the locus heterogeneity of this condition.

1. Departments of Genetics and 2. Department of Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Center, Riyadh, SaudiArabia; 3. Department of Surgery, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 4. Department of Medical Genetics, King FahadGeneral Hospital, Jeddah, Saudi Arabia; 5. Department of Pediatrics, Security Forces Hospital, Riyadh, Saudi Arabia; 6. Department of Pediatrics, Collegeof Medicine, King Saud University, Riyadh, Saudi Arabia; 7. Maternal Fetal Medicine Department, Women’s Specialized Hospital, King Fahad MedicalCity, Riyadh, Saudi Arabia; 8. Bnoon Medical Center, Riyadh, Saudi Arabia; 9. Division of Human Genetics, Cincinnati Children’s Hospital Medical Center,Cincinnati, OH; 10. Division of Clinical and Metabolic Genetics and 11. Neurology, Department of Paediatrics, Hospital for Sick Children, University ofToronto, Toronto, Ontario, Canada; 12. Department of Pediatric Subspecialties, Children’s Hospital, King Fahad Medical City, Riyadh, Saudi Arabia; 13. Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; and 14. Saudi Human Genome Program, KingAbdulaziz City for Science and Technology, Riyadh, Saudi Arabia

ABSTRACT. KE Preston1, RL Corwin2, JO Bader1, SL Crimmins1.

The goal of this study was to use the limited access (LA) model of binge eating to evaluate the impact of relatively enriched housing conditions on binge-type behavior in rats. In the LA model, bingeing is established when a group with intermittent access to a palatable food consumes significantly more of the food during the access period than a group with daily access. Historically, the LA model is used in nonenriched housing conditions. However, from an animal care perspective and with respect to the etiology of binge eating, it is important to determine how enrichment affects binge eating. Rats were divided into four groups: Control-Enriched, Control-Nonenriched, Intermittent-Enriched, and Intermittent-Nonenriched. Control groups received daily 30-minute access to vegetable shortening, while intermittent groups received 30-minute access to shortening on Monday, Wednesday, and Friday only. Enriched groups were housed with bedding, nesting material, toys, and a solid floor, while nonenriched groups were housed on wire flooring with no additions to cages. Conditions were reversed for intermittent groups halfway through the study. Results indicate that a relatively enriched environment delays binge onset, but does not significantly alter the amount of fat consumed during binge sessions or reverse established binge behavior. Intermittent access to vegetable shortening induces greater consumption of shortening than does daily access regardless of enrichment condition; however, rapid establishment of enduring binge-type eating appears to require austerity in housing conditions. Also, it is not likely that the level of enrichment provided in this study can prevent or reverse binge-type eating in rats.

1Dept of Clinical Investigation, William Beaumont Army Medical Center, El Paso, TX, USA 2Nutritional Sciences Dept, The Pennsylvania State University, University Park, PA, USA. Funding Support: Department of Defense. The views expressed in this presentation are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or U.S. Government.

ABSTRACT. RP Cotter, HB Lee, AN Sigafoos, BL Clarke, MI Lapid, KJ Clark

Selective serotonin reuptake inhibitors (SSRIs) are commonly used antidepressant medications that regulate mood and alleviate depression by increasing amounts of circulating serotonin in the synaptic cleft. The serotonin transporter, encoded by SLC6A4 gene in humans, recycles serotonin back to the axonal boutons of pre-synaptic neurons. However, pleiotropic roles that serotonin plays in different brain tissues are largely unknown. Moreover, we are only now becoming aware of potential long-term adverse effects of SSRIs involving other tissues than the nervous system since the first prescribed SSRI, fluoxetine, was released in 1987. Recent studies proposed a link between long-term exposure to SSRIs and decreased bone mineral density and increased fracture risk. In addition, the short form of the polymorphic region in the serotonin transporter gene (SLC6A4) is implicated in decreased bone density. Importantly, the relationship between the functions of SLC6A4, SSRIs, and bone have not been systematically investigated. We hypothesize that SLC6A4 regulates bone density by modulating osteoblast formation and/or differentiation. Using zebrafish, we are generating revertible slc6a4a mutant alleles by targeted integration of a gene-break cassette to interrupt gene function and test the role of slc6a4a on bone mineralization. We will also test the effect of commonly prescribed SSRIs on bone mineralization in conjunction with genetic manipulations. This project may demonstrate a link between slc6a4a levels and the propensity of individual SSRIs to impact bone metabolism that could inform patients and clinicians as they weigh the benefits and risks of SSRIs.

ABSTRACT. Kyle Schaefbauer, Hirotaka Ata, Mark Wishman, Karl J Clark and Stephen C. Ekker

The gene break transposon (GBT) cassette is a unique insertional mutagen that reports endogenous gene expression including protein and RNA, causes a 99+% knockdown of the tagged gene, and is revertible by Cre recombinase. We have generated a library of 1000+ GBT alleles, searchable by gene name and expression pattern (www.zfishbook.org). A number of lines show expression in diverse neural circuits including known and novel patterns. In addition, a subset of GBT alleles are in nuclearly encoded mitochondrial genes, loci that play critical roles in neurodegenerative diseases through unknown or understudied mechanisms. The GBT system presents a unique opportunity to study genes that are associated with Parkinson’s Disease (PD) and other neurodegenerative disorders. Notably, of the over 10 mitochondrial GBT loci we have identified to date, GBT 599 is integrated in the mitochondrial calcium uniporter (mcu) gene. Finally, the recently developed VALET gene editing-based approach enables the targeted integration of this gene break insertional cassette. We will report our efforts to generate engineered GBT alleles in pink1 and park2. Together, these new genetic lines represent new revertible zebrafish models suitable for mechanistic analyses and high-throughput drug screening applications of PD and related disorders.

International Protein Trap Consortium Mayo Clinic, Rochester, MN 55906

ABSTRACT. EM Litkowski1, RA Radcliffe2, WJ Shi3, LM Saba1,2, K Kechris1

To increase our understanding of genetic effects on acute ethanol sensitivity as it relates to alcohol consumption, we examined the differential brain co-expression of genes between saline and ethanol treated LXS recombinant inbred mice. Differential co-expression can be defined as a change in the relationship between two genes after a perturbation, such as exposure to ethanol. We estimated posterior probabilities for differential co-expression by employing a mixture model that bins gene pairs based on their relationship before and after the perturbation, e.g., no correlation between the two genes in the saline group and a positive correlation between genes in the ethanol group. Preliminary results unveiled approximately 450 unique genes in co-expressed gene pairs after ethanol exposure that had no relationship in the saline group. Furthermore, approximately 900 unique genes were in co-expressed gene pairs in the saline group that had no relationship after ethanol exposure. Gene enrichment analysis applied to these gene classes highlights several pathways known to have roles in ethanol exposure and alcohol use disorders, such as dopaminergic-synapse, alcoholism, thyrotropin-releasing hormone receptor signaling, and endogenous cannabinoid signaling. Performing differential co-expression analysis allowed us to distinguish between treatment groups, furthering our knowledge of the genetics of acute ethanol sensitivity and how that might relate to alcohol consumption.

1Department of Biostatistics and Informatics, Colorado School of Public Health, 2 Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, 3Computational Bioscience Program, School of Medicine, University of Colorado, Anschutz Medical Campus, 13001 E. 17th Place, Aurora, CO 80045, Funding Support: NIH grant R01-AA016957, NIH grant R01-AA021131, NIH grant T15LM009451

ABSTRACT. Ashley N. Sigafoos1,2, Nicole J. Boczek1, Han B. Lee2, Tanya L. Schwab 1,2 , Patrick R. Blackburn1, Margot A. Cousin1, Eric W. Klee1, and Karl J. Clark1,2

1Center for Individualized Medicine and 2Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA

Precision medicine is conceptualized as a medical care and research model to provide individualized and tailored care for each patient based on one’s genetic make-up. By far, the most common phenotypic category of undiagnosed diseases is neurologic disorders, accounting for 53% of all accepted patients to the NIH. Arguably, neuronal phenotypes are the most difficult to study since the tissue is not easily accessible. Our Translational Genomics Core (TGC) within the Center for Individualized Medicine at Mayo Clinic have been developing functional and behavioral assay suites using models including zebrafish. The overarching goal and hypothesis of TGC is that we enhance the diagnosis and understanding of neuronal phenotypes of rare disorders via functional characterization and validation of the roles that Variants of Unknown Significance (VUS) play. Leveraging the high degree of conservation of gene functions to humans and rapid development of the fish model, we are characterizing the functions of VUSs of human GABBR2, SLC2A10, SOX5, and DNM1L. We revealed that zebrafish gabbr2 knockouts show profoundly decreased locomotor response while overexpression of human GABBR2 in transgenic fish lines leads to moderately decreased locomotion to light change stimulation. Additional functional work is required to understand how patient VUSs influence the observed phenotypes. We envision our pioneering functional and behavioral work will both aid in patient diagnosis and provide options for patient care and wellbeing.

ABSTRACT. M M Johansson1, P Pottmeier1, P Suciu1, T Ahmad1, A Zaghlool2, J Halvardson2, E Darj3, 4, L Feuk2, C Peuckert1, 5 and Elena Jazin1*

1Department of Organismal Biology, EBC, Uppsala University, Sweden 2Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden 3Department of Women's and Children's Health, International Maternal and Child Health (IMCH), Uppsala University, Sweden 4Department of Public Health and General Practice, Norwegian University of Science and Technology, Trondheim, Norway 5 Department of Molecular Biology, Stockholm University, Stockholm, Sweden

Global microarray gene expression analyses demonstrated differences in female and male embryos during neurodevelopment. In particular, before sexual maturation of the gonads, the differences seem to concentrate on the expression of genes encoded on the X and Y chromosomes. To investigate the genome-wide differences in expression during this early developmental window, we combined high resolution RNA sequencing with qPCR to analyse brain samples from human embryos during the first trimester of development. Our results show that the largest biased group consisted of genes encoded on the sex chromosomes and the majority of all differentially expressed genes were male-biased. 10 out of 13 expressed gametolog pairs showed unbalanced gene dosage and as a consequence, a male biased expression. Among X-chromosome genes, three genes had higher expression in female embryos but a balanced gene dosage due to the Y gametolog expression. In addition, we found 6 novel non-annotated long non-coding RNAs on the Y-chromosome with conserved expression patterns in new-born chimpanzees. The tissue specific and time restricted expression of these long non-coding RNAs strongly suggests important functions during central nervous system development in human males.

*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. A Park, C Stojanik, T Tran, N Atkinson Department of Neuroscience, University of Texas at Austin

Despite the pervasiveness of alcohol-induced violence, little is understood about what genes contributes to this behavior. Previous work has identified serotonin and monoamine oxidases as being important for regulating alcohol-induced aggression. However, there are extremely few studies investigating novel genes that contribute to alcohol-induced aggression, likely due to the lack of model organisms with robust genetic toolsets. Using Drosophila melanogaster we investigate the role of NPF (Neuropeptide F) and TH (Tyrosine Hydroxylase) in mediating alcohol-induced aggression. NPF is a neuropeptide that has previously been implicated in sexually dimorphic alcohol behaviors. Tyrosine Hydroxylase, which is an enzyme necessary for dopamine synthesis has also been previously implicated in alcohol behaviors. Both of these genes have mammalian orthologues with nearly identical pathways and functions. Investigating the role of these genes in Drosophila will enable us to better understand the unknown basis of how alcohol-induced aggression is regulated in humans.

ABSTRACT. R. Maiya1,2, A. Beckham1,2, G.N. Tiwari1, S.P. Farris1, R.D. Mayfield1, and R.O. Messing1,2.

Repeated alcohol exposure leads to changes in gene expression that are thought to underlie the transition from moderate to excessive drinking. Gene expression profiling studies have identified a multitude of alcohol-responsive gene networks, but the mechanisms by which these networks are mobilized to a neuroadaptive response are not well understood. One mechanism could involve alcohol regulation of transcriptional co-regulators that bind and modulate the activity of several transcription factors. Our results indicate that the transcriptional regulator LMO4 is one such candidate regulator. Mice harboring a gene trap insertion at the Lmo4 (Lmo4gt/+) locus consumed significantly more and showed enhanced preference for alcohol in a 24-h intermittent access procedure. shRNA-mediated knockdown of LMO4 in the NAc significantly enhanced whereas knockdown in the BLA decreased alcohol consumption and preference and reduced conditioned place preference to alcohol. To ascertain the molecular mechanisms that underlie the contrasting effects of LMO4 knockdown in the BLA and NAc, we performed unbiased transcriptome profiling of these two brain regions in WT and Lmo4gt/+ mice using RNASeq. We identified approximately 1000 genes that were differentially expressed in both brain regions of which only 48 were common between the two brain regions. We validated several of these differentially expressed genes by quantitative real time PCR. Weighted gene co-expression network analysis implicated genes related to the extracellular matrix in the BLA and genes related to phosphatidylinositol-3,4,5-triphosphate binding in the NAc as primary transcriptional targets of LMO4. Future experiments will determine the effects of perturbation of these networks on alcohol consumption.

1Waggoner Center for Alcohol and Addiction Research, 2Department of Neuroscience, The University of Texas at Austin, Austin, TX, 78712

ABSTRACT. Authors: Katheryn M Wininger1, Yun Fang Jia2, Lee Peyton2, Seungwoo Kang2, Daniel Lindberg1, Doo-Sup Choi1,2,3

1Neurobiology of Disease Program, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN 55905, USA. 2Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School of Biomedical Sciences, Rochester, MN 55905, USA.3Department of Psychiatry and Psychology, Mayo Clinic, Rochester, MN 55905, USA.

Glutamate transporter 1 (GLT1) is responsible for roughly 90% of glutamate clearance from the synaptic environment. Consequently, dysregulation of GLT1 results in hyper or hypo-glutamatergic states, which have been implicated in many psychiatric disorders such as anxiety, depression, addiction, and post-traumatic stress disorders. Although GLT1 is expressed by neurons and plays an important role in regulating neural physiology and glutamatergic signaling, numerous studies have suggested that GLT1 is predominantly expressed by astrocytes. Therefore, to investigate the astrocytic function of GLT1, we generated a line of astrocyte-specific GLT1 knockout mice by crossing floxed GLT1 mice with astrocyte-specific protein GFAP (glial fibrillary acidic protein) driven Cre recombinase expressing transgenic mice. As anticipated, these animals displayed marked deficiencies in GLT1 mRNA and protein expression in brain regions associated with psychiatric disorders, including the medial prefrontal cortex, nucleus accumbens, caudate putamen, and hippocampus. Importantly, mice lacking astrocytic GLT1 exhibited significantly reduced depressive and anxiety-like behavior, as well as decreased freezing behavior to both context and cue in fear conditioning. Interestingly, astrocytic GLT1 deficient mice displayed reduced behavioral sensitivity to acute ethanol exposure without changes in ethanol preference. No differences were observed in in cognition, memory, or overall movement, although GLT1 deficient mice reared significantly more than their wild type counterparts. Combined, this data suggests that astrocytic GLT1-deficient animals may be less sensitive to the effects of aversive stimuli or stress. However, because stress and corticosterone are known to regulate GLT1 expression, further investigation is necessary to clarify the region or circuit-specific roles of GLT1 and to more fully understand how GLT1 may contribute to psychiatric disorders.

ABSTRACT. G Risi1,2, E Ausmus3, G Salinas1,2, AG Vidal-Gadea3*

About one-quarter of the world population is estimated to be infected with parasitic nematodes. There is widespread resistance to currently used drugs in the veterinary field, and concern is growing that resistance may arise in humans parasitic nematodes. C. elegans is a free living nematode that has been especially suggested as a good model for anthelmintic drug, and target discovery. This project takes advantage of C. elegans’ burrowing behavior to model the need of parasitic worms to cross biological barriers during infections. We are searching for new molecular targets to prevent nematodes from infecting livestock and other hosts. Worms will be placed in multi-well plates and fed bacteria using RNA interference to target a specific gene in each well. We are targeting genes with no homology in humans, but with homology in the parasitic nematode Haemonchus contortus. This will allow us to identify genes likely to be involved in parasitic nematode biology, but with no similar targets in humans (or livestock). Burrowing will be assessed in RNAi silenced animals. This assay will allow us to identify potential drug targets that can be used to impede nematocidal infections.

1Pasteur Institute of Montevideo, Montevideo, Uruguay, 2School of Chemistry, University of the Republic, Montevideo, Uruguay, 3School of Biological Sciences, Illinois State University, Normal, IL *avidal@ilstu.edu Funding support: National Agency for Investigation and Innovation (ANII) of Uruguay internship to GR. (MOV_CA_2017_1_137068); NIH 1R15AR068583-01A1 grant to AGVG.

ABSTRACT. T-H Kuo, Y-S. Su Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu, Taiwan (ROC), Funding support: Ministry of Science and Technology, Taiwan (ROC)

Animals perform a wide range of social behaviors for survival and reproduction. Understanding these behavior may provide a framework of social interactions in human and eventually help us to elucidate abnormalities in social behaviors under various psychiatric disorders. Allogrooming, or social grooming, is defined as grooming behavior between members of the same species and can be widely observed throughout animal kingdoms. This behavior plays an important role in social bonding and is generally believed to be an altruistic behavior because groomers face several immediate costs to benefit recipients. Although allogrooming has been documented in mice, the functions and mechanisms have never been well characterized due to intensive male-male aggression, which limits the observations of other social behaviors during resident-intruder assay. By comparing dominant and subordinate males under resident-intruder assay, we found that subordinate males mainly perform allogrooming instead of aggression while there is no change in other social behaviors. Covering exotic materials, like mineral oil, on a intruder significantly enhanced the allogrooming behavior from a groomer (resident), supporting the function of allogrooming in hygienic purpose. We also observed increase of immobile time of intruder during allogrooming interaction, suggesting that grooming recipients are directly benefited from this social interaction. Our study showed that allogrooming in mice is altruistic and can be easily observed in subordinate animals. This study established a new platform to investigate the biological mechanisms of altruistic behaviors for the future study.

ABSTRACT. Authors: Lauren Bailey, Samantha Cermak, Jared Bagley, J. David Jentsch

Affiliation: Department of Psychology (Behavioral Neuroscience), Binghamton University, Binghamton NY 13902

Drugs of abuse, including alcohol and stimulants like cocaine, produce subjective effects that are subject to individual variability, and genetic variation accounts for at least a portion of those differences. Notably, research in both animal models and human subjects point towards reward sensitivity and impulsivity as being trait characteristics that predict relatively greater positive subjective responses to stimulant drugs. Unfortunately, past efforts have yet to yield convincing insights into underlying genetic influences on these traits due to the characteristics of the mouse panels used. The Collaborative Cross (CC) recombinant inbred mouse strains, their inbred founders, and the Diversity Outbred (DO) mice that are derived from them are a powerful genetic reference panel that has potential as a tool for revealing genetic contributions to cocaine abuse and related traits. Here we describe use of the eight CC/DO founder strains to examine the heritability of reward sensitivity and impulsivity traits, as well as genetic correlations between these measures and existing addiction-related phenotypes. Methods. Founder strains were all tested for activity in an open field and reward sensitivity (intake of chocolate BOOST®). Mice were then divided into two counterbalanced groups and underwent reversal learning (impulsive action) or delay discounting (impulsive choice). Results. The founder mice demonstrate significant heritability for premature responding within the reversal task, locomotor activity, and reward sensitivity. At this preliminary stage, significant strain differences for delay discounting are unclear. This research was conducted within the broader, inter-laboratory effort of the Center for Systems Neurogenetics of Addiction (CSNA) to characterize CC and DO mice for multiple, cocaine abuse related traits. These data will facilitate the discovery of genetic correlations between predictive traits, which will then guide discovery of genes and genetic variants that contribute to addictive behaviors.

Funding: These studies were supported in part by PHS Grant P50-DA041602. 

ABSTRACT. 1Shabani S, 1Houlton S, 1Ghimire B, 1Casiquin C, 2,3Phillips TJ

Avidity for methamphetamine (MA) is genetically associated with the balance in sensitivity to the rewarding and aversive effects of MA. Studies using bidirectional selective breeding of mice for high and low voluntary MA drinking (MADR) identified a quantitative trait locus on chromosome 10 associated with MA intake. Further study identified the trace amine-associated receptor 1 (Taar1) gene as a relevant gene in that region. The gene product, TAAR1, is a receptor that regulates monoamine neurotransmission. The MADR mice were selectively bred from a founding population of C57BL/6J x DBA/2J F2 (B6D2F2) mice, using a two-bottle choice drinking procedure. The D2 strain and MA high drinking (MAHDR) mice are homozygous for a Taar1 allele that codes for a non-functional receptor and are insensitive to the aversive and hypothermic effects of MA. B6 and MA low drinking (MALDR) mice have at least one copy of the alternative Taar1 allele that codes for functional TAAR1, are sensitive to these MA effects and have low MA consumption. MA is a direct agonist at TAAR1, but also impacts monoamine levels via transporter actions. Whether MA induces aversion and hypothermia via TAAR1 stimulation is not known. To study this, we used the MALDR mice to determine whether the effects of a TAAR1 full agonist would be similar to those of MA. The agonist, RO-5256390, was studied for its ability to induce a conditioned taste aversion (CTA), a conditioned place aversion (CPA), and hypothermia. The ability of IP 2 or 4 mg/kg RO-5256390 to induce a CTA to sodium chloride was measured, and the ability of IP 0.05, 0.1, 0.5 or 4 mg/kg RO-5256390 to induce CPA was examined. One day following the end of each procedure, the mice were tested with the same RO-5256390 doses for hypothermic effects. Rectal temperatures were measured at time 0 and then at times 60, 120 and 180 min after injection. Both doses of the TAAR1 agonist induced robust CTA, compared to vehicle treatment, but there were no differences between the 2 and 4 mg/kg dose groups. Significant and robust hypothermic effects of both TAAR1 agonist doses were found at 60 min post injection, with no significant difference between these two groups. In the place conditioning procedure, even the lowest dose of 0.05 mg/kg RO-5256390 induced robust CPA. Hypothermic effects were apparent after all doses of the agonist and were most profound 60 min post-injection; however, doses >0.05 mg/kg had larger hypothermic effects. Thus, similar to MA, a TAAR1-specific agonist has conditioned aversive and hypothermic effects in MALDR mice, suggesting that TAAR1 stimulation plays an important role in sensitivity to the aversive effects of MA. Aversive actions of MA mediated by TAAR1 may curb MA intake in MALDR mice.

1 Dept of Biology, Minot State University, Minot, ND USA. 2 Methamphetamine Abuse Research Center, Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR. 3 Veterans Affairs Portland Health Care System, Portland, OR USA. Supported by: Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the NIH P20GM103442 (SS), the Department of Veterans Affairs (TJP), and NIH grants P50DA018165, U01DA041579 and R24AA020245 (TJP).

ABSTRACT. BC Jones1, JP O’Callaghan2, DB Miller2, W Zhao1

Gulf war illness (GWI) is a chronic disorder that afflicts up to one-third of the veterans of the Persian Gulf conflict. Why some individuals developed GWI while others have not, is the subject of our work. Soldiers were exposed to a number of chemicals, including organophosphates (OP) that inhibit cholinesterase. OPs also promote neuroinflammation. An animal model showed that high concentrations of corticosterone acted synergistically to increase the expression of pro-inflammatory cytokine genes. The animal model was the C57BL/6J (B6) male mouse. To address individual differences, we replicated the experiment, adding the DBA/2J (D2) strain and females. The design: Treatment groups and treatments. There were four conditions: Condition 1, Control. Injected i.p. with saline and six hours later killed by cervical dislocation and frontal cortex and hippocampus harvested. Condition 2, CORT. Corticosterone (200 mg/L) added to drinking water for seven days prior to tissue harvest as in condition 1. Condition 3, DFP. Injected i.p. with 4 mg/kg diisopropylflurophosphate (DFP), an irreversible cholinesterase inhibitor and killed 6h later for tissue harvest. Condition 4, CORT+DFP. CORT in drinking water as in group 2 for seven days and on the eighth day, injected i.p. with 4 mg/kg i.p. DFP and tissues harvested six h later. The cytokines included (IL6), IL1-β, CCL2, LIF, OSM TNFα. Results. Overall, D2 mice were much less affected by CORT+DFP than the B6 mice and females of both strains less affected than males. Conclusion. These results support a likely genetic basis for individual differences in susceptibility to GWI.

1Department of Genetics, Genomics, and Informatics, University of Tennessee Health Center, Memphis, TN 2Center for Disease Control, National Institute of Environmental Health and Safety, Morgantown, WV Funding: USPHS Grant ES022614; DoD GWI 160086

ABSTRACT. Embryonic neural patterning genes regulate both patterning and formation of autonomic circuits. These autonomic circuits require appropriate neuronal and glial integration for efficient circuit function. However, the developmental interdependencies between neurons and glia of specific circuits has not been established. Here, we report the generation of an astrocyte population derived from PHOX2B patterned precursors in the mouse brainstem. These astrocytes are necessary for appropriate function of the PHOX2B-derived chemosensory centers, sleep homeostasis and proper synapse morphology. These findings indicate that PHOX2B may act by patterning precursor cells that later generate both astrocytes and neurons destined to form functional autonomic circuits.

1 The Ohio State University College of Medicine, Department of Pathology, 2Department of Physiology and Biophysics, Institute of Biomedical Science, University of Sao Paulo, 3 The Ohio State University College of Medicine, Department of Neuroscience, 4The Ohio State University College of Engineering, Department of Mechanical and Aerospace Engineering, 5The Ohio State University Mathematical Biosciences Institute, 6 The Ohio State University Campus Microscopy and Imaging Facility, 7Department of Pharmacology, Institute of Biomedical Science, University of São Paulo. *= Co-corresponding Author. United States studies were supported by NIH/NHLBI R01HL132355. Brazil research was supported by the São Paulo Research Foundation (FAPESP; grants: 2014/22406-1 to ACT; 2015/23376-1 to TSM) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant: 471263/2013-3 to ACT). CNPq fellowship (301219/2016-8 to ACT and 301904/2015-4 to TSM).

ABSTRACT. A primary challenge facing Autism Spectrum Disorder (ASD) genetics is the large and growing number of genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system we characterized 26 quantitative phenotypes spanning morphology, locomotion, sensory, and habituation learning in 97 strains of C. elegans each carrying a mutation in an ortholog of an ASD-associated gene. This research has generated a large number of novel genotype to phenotype relationships that range from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviours. Clustering based on multi-parametric phenomic profiles revealed a set of 12 genes that all result in a strikingly similar profile characterized by hypersensitivity and impaired habituation learning. Current epistasis experiments are aimed at determining whether the phenomic similarity among members of this cluster results from previously undiscovered functional interactions. One of the genes in this cluster is the sole C. elegans ortholog of neuroligins, nlg-1. Transgenic pan-neuronal expression of human NLGN3 in nlg-1 mutant C. elegans rescued their sensory and learning impairments; confirming functional conservation. The wealth of in vivo phenomic functional data generated in this work will inform more targeted studies in vertebrates and offers novel positive and negative pathway components as therapeutic targets for ameliorating the effects of ASD.

1. Djavad Mowafaghian Centre for Brain Health, University of British Columbia, 2211 Wesbrook mall, Vancouver, B.C., V6T 2B5; 2. Dept. of Psychology, UBC, 2136 West Mall, Vancouver, B.C., V6T 1Z4 3. Department of Cell and Physiological Sciences, 2350 Health Science Mall, B.C. V6T 1Z3 4. Department of Psychiatry, 2255 Wesbrook Mall, B.C. V6T 2A1

ABSTRACT. To successfully navigate their surroundings, animals must decide how much attention they dedicate to the variety of environmental stimuli they routinely encounter. This decision is often reflected on the nervous system allocation of resources to different stimuli (e.g. humans rely on vision more than touch). In nature, animals must reconcile the physical nature, and the saliency of stimuli to optimize search strategies using finite behavioral repertoires. Most work in the field has focused on orientation strategies specific to highly salient stimuli (e.g. temperature or chemical gradients). It remains unclear how animals optimize their behavioral strategies for stimuli of different physical nature. Like many animals, the nematode C. elegans detects a large variety of stimuli and responds using a small well described repertoire of orientation strategies. Here we use C. elegans to investigate how it optimizes navigation to physically distinct stimuli of different salience. We recorded the worms orienting to temperature, chemical, or magnetic field stimuli and analyzed heading and orientation strategies. Worms appear to orient differentially in the presence of a temperature gradient by increasing velocity and clustered reversals (i.e. pirouettes) as a search strategy that was not observed in magnetic orientation or chemotaxis. Conversely, pirouettes appear suppressed in chemical gradients while gradual turning (weathervaning) is increased. These results suggest that C. elegans uses distinct orientation strategies depending on the physical nature of a stimulus.

ABSTRACT. Gap junctions facilitate intercellular communication and rapid information processing in the nervous system, mediating swift and coordinated behavioral responses. In invertebrates, innexin are the structural components of gap junctions and provide direct entryways for electrical currents between neurons. While gap junction structure and function are well known, the specific contribution of innexins to neural circuit function and behavior remain poorly understood. We use RNA interference (RNAi) to target innexin expression in the marbled crayfish, Procambarus virginalis. Crayfish are well-suited for electrical coupling and behavioral studies due to their well-characterized tail-flip escape responses and their coordinated leg movements during locomotion. We hypothesize that innexins contribute substantially to both behaviors. Our analyses indicate that marbled crayfish share homology for seven innexin genes with other species, up to 84% within Cancer borealis, Homarus americanus and Homarus gammarus. To test specific expression in the ventral nerve cord (containing neurons that mediate both behaviors), we designed and tested innexin-specific primers. Agarose gel analyses showed that at least innexin-4 and innexin-2 are expressed. We were able to characterize 90% of the putative innexin-2 exons, and thus selected innexin-2 as the first candidate for investigating the behavioral consequences of innexin silencing. To induce RNAi, we created innexin-2 double-stranded RNA (~553bp). We are currently assessing innexin-2 contribution to locomotion and escape behaviors by subcutaneous injecting of dsRNA and measuring 1) innexin-2 suppression levels using real-time PCR, 2) effects of silencing on electrical communication in the escape circuit, and 3) both behaviors at 24, 48, and 72 hours post-injection.

ABSTRACT. Binge eating (BE) is a heritable symptom of eating disorders associated with anxiety, depression, malnutrition, and obesity. Determining its genetic basis could facilitate discovery of more efficacious therapeutics. We used an intermittent, limited access paradigm to examine inbred mouse strain differences in BE of sweetened palatable food (PF), conditioned food reward, and compulsive-like eating between C57BL/6J (B6J) and DBA/2J (D2J) inbred mouse strains. We identified a robust escalation in PF consumption in D2J and a conditioned place preference for the PF-paired side. D2J also showed compulsive-like eating in the light/dark conflict test, revealing a unique acquire-retreat hoarding-like behavior of PF to the dark side of the apparatus. To gain insight into the genetic basis of BE in D2J versus B6J, we phenotyped and genotyped a cohort of 133 B6JxD2J-F2 mice at three historic, B6J/D2J-derived quantitative trait loci (QTLs), including a chr.4 QTL associated with sweet taste (156 Mb), a chr.6 QTL associated with bitter taste (133 Mb), and a chr.11 QTL (50 Mb) associated with behavioral sensitivity to drugs of abuse, including methamphetamine, cocaine, and ethanol. Both the chr 6 and chr 11 D2J alleles were associated with increased PF consumption and slope of escalation, thus accounting for a portion of phenotypic variance. In contrast, the sweet taste locus did not affect PF consumption. Hnrnph1 haploinsufficient mice (chr.11: 50 Mb) showed an increase in PF consumption, supporting Hnrnph1 as a causal gene. QTL mapping in BXD recombinant inbred lines will identify other genes contributing to the various components of BE.

1 Laboratory of Addiction Genetics, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine,

2 Boston University Undergraduate Research Opportunity Program (UROP),

3 NIGMS Training Program in Biomolecular Pharmacology, Boston University School of Medicine,

4 Boston University Transformative Training Program in Addiction Science (TTPAS), Burroughs Wellcome Fund,

5 Departments of Genetics, Genomics and Informatics and Anatomy and Neurobiology, University of Tennessee Health Science Center.

ABSTRACT. To explore the genetic origins of methamphetamine (MA) use disorder, selective breeding was used to create lines of mice for high (MAHDR) and low (MALDR) MA drinking from the F2 cross of the DBA/2J (D2) and C57BL/6J (B6) inbred strains. Sensitivity to MA-induced conditioned taste aversion (CTA) is genetically correlated with amount of MA consumed in the selected lines, indicating some common genetic regulation. A quantitative trait locus was found that accounts for >50% of the genetic variance in MA intake in the lines. The trace amine-associated receptor 1 (Taar1) falls within this region, and MA is a direct agonist at the receptor (TAAR1). Five replicate sets of lines have been produced, and all replicates of the MAHDR lines are homozygous for a mutant Taar1 allele (Taar1m1J), derived from the D2 strain, which expresses a nonfunctional receptor. The MALDR lines are heterozygous or homozygous for the alternative Taar1 allele, derived from the B6 strain, which expresses a functional receptor. Existing data are consistent with the hypothesis that Taar1m1J homozygosity drives the higher MA intake in MAHDR mice. To confirm this, CRISPR-Cas9 allele swap was performed, replacing Taar1m1J in MAHDR mice with B6 Taar1. In two-bottle choice and CTA procedures, MAHDR allele-swapped mice consumed less MA, relative to typical MAHDR mice, and exhibited levels of these phenotypes similar to those of MALDR mice. This proves that TAAR1 is responsible for differences in MA intake and CTA between the MADR lines, and substantiates interest in the role of Taar1 in MA abuse.

 1Oregon Health & Science University, 2Veterans Affairs Portland Health Care System, Portland, OR, USA. Funding Support: Department of Veterans Affairs I01BX002106, NIH NIDA P50DA018165 and U01DA041579

ABSTRACT. Mice of different strains and transgenic mouse models of AD have marked differences in life expectancy depending on the genotype and sex of the model (Rae & Brown 2015 Neurosci Biobehav Rev. 57: 238-51). A measure of aging which is independent of chronological age is necessary to compare age-related changes in mice with different lifespans. Frailty could provide a measure of healthspan facilitate comparisons of aging between mice of different genotypes and sexes. This study used a validated mouse frailty index (FI) assessment tool based on deficit accumulation (Whitehead et al., 2014 J Gerontol Biol Sci Med Sci 69: 621–32) to explore genotype and sex differences in lifespan and healthspan of 3xTg-AD mice. Results showed that male 3xTg-AD mice aged 300-600 days had a higher FI score (Mean FI=0.21) than either male wild-type (Mean FI=0.15) or female 3xTg-AD mice (Mean FI=0.10), and the elevated frailty scores were accompanied by parallel increases in mortality. Frailty increased exponentially with age in all groups, and higher rates of deficit accumulation elevated mortality risk in all groups. When mice were stratified by FI score, frailty predicted mortality, at least in females. The mouse clinical FI provides a valuable tool that can be used to evaluate healthspan in mice of different strains and in transgenic mice.

ABSTRACT. In 2015, 36.5 million U.S. adults regularly smoked cigarettes, however 68% wished they could quit (CDC). Although there are several nicotine replacement products and prescription non-nicotine medications to facilitate the quitting process, the success rate is still less than 10%. Pre-vious human genetics studies in our lab led to an interest in a microRNA (miRNA; miR-138), which is potentially involved in expression of the CHRNB4 gene (Gallego et al., 2013). CHRNB4 is highly expressed in the medial habenula (MHb) and is important for nicotine intake/preference behaviors. To assess whether miR-138 is behaviorally relevant in vivo, an adeno-associated virus (AAV) containing either miR-138 or a scrambled control miRNA was injected into the MHb of male C57BL/6J mice. The mice were then singly housed and given a two-bottle choice procedure where one bottle contained water and one bottle contained escalating concentrations of nicotine (25 - 200 ug/mL). Compared to the mice injected with the scrambled control miRNA, those receiving the miR-138 injection showed a reduction in voluntary nicotine intake/preference. We hypothesize that this reduction of nicotine consumption is caused by reduced expression of Chrnb4 in the MHb, which will be tested using epibatidine-binding experiments following AAV injections. Finally, because of the known sex differences in smoking behaviors, we have begun the same behavioral testing procedures in females. This study will provide improved understanding of the role miR-138 contributes to nicotine addiction and may help in developing potential treatments for nicotine dependence.

1Institute for Behavioral Genetics, University of Colorado, Boulder, CO, USA 2Department of Integrative Physiology, University of Colorado, Boulder, CO, USA

ABSTRACT. There is an immense amount of diversity observable in the living world today. Understanding the genetic basis of trait evolution is critical to identifying the mechanisms that generated this diversity and the evolutionary processes that led to the establishment of new traits. Astyanax mexicanus, the blind Mexican cavefish, exists in two interfertile forms, a surface-dwelling form and multiple independently evolved cave-dwelling forms. As cavefish have evolved a number of behavioral traits and A. mexicanus are amenable to genetic manipulation, this system provides a unique opportunity to functionally test candidate genes for their role in evolution of behaviors. We have leveraged CRISPR/Cas9 genome editing techniques to mutate the oculocutaneous albinism 2 (oca2) gene in surface fish. Mutations in this gene are hypothesized to underlie both the evolution of albinism and, through changes in catecholamine levels, evolution of behaviors. Thus, through examining CRISPR-generated mutant fish, we can gain unprecedented insight into the consequences of naturally occurring mutations in the evolution of morphology and behavior.

ABSTRACT. Learning reflects the influence of experience on genetically determined circuitry, but little is known about how experience and genetics interact to determine complex learned phenotypes. Here, we used vocal learning in songbirds to study how experience and genetics contribute to inter-individual differences in learned song. Previous work has established that such differences in song within a species depend on learning, but in principle some of these differences could also depend on genetic variation. We focused on song tempo, a learned and quantifiable feature that is controlled by central neural circuitry. To identify genetic contributions to tempo we computer-tutored juvenile Bengalese finches (Lonchura striata domestica) from different genetic backgrounds with synthetic songs in which tempo was systematically varied. Computer-tutored birds exhibited unexpectedly strong heritability for song tempo, and comparatively weak influence of experience. We then tested whether heritability was fixed and independent of experience by providing a second group of birds with enriched instruction via live social tutoring. Live-tutoring resulted in not only a significant increase in the influence of experience on tempo, but also a dramatic decrease in the influence of genetics, indicating that enriched instruction could overcome genetic biases evident under computer tutoring. Our results reveal strong heritable genetic contributions to inter-individual variation in song tempo, but that the degree of heritability depends profoundly on the quality of instruction. They suggest that for more complex learned phenotypes, where it can be difficult to identify and control relevant experiential variables, heritability may similarly be contingent on the specifics of experience.

Department of Physiology, University of California, San Francisco, CA 94158; Center for Integrative Neuroscience, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158.