ABSTRACT. Rainer Spanagel Institute of Psychopharmacology, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany rainer.spanagel@zi-mannheim.de The characterization of disease-related neurocircuitries is fundamental for understanding mental disorders and for drug development. Neuroscientists are currently making a major effort to map those neurocircuitries, mainly by means of tracing studies and optogenetic methodology. Here I will present an alternative approach based on the first global neurochemical connectome and a novel database on neurochemical fingerprints produced by psychoactive drugs. By using advanced data mining, supervised machine learning, and network analysis we have generated a multiscale, multilayer neurochemical connectome of the rat brain (www.chemnetdb.org). The connectome consists of 125 scale-consistent cerebral nuclei and 2,931 multiscale, multi-chemical connections. In addition we have used a further big data approach to extract and integrate data from pharmacological microdialysis experiments. We systematically extracted data from 3,383 original in vivo microdialysis experiments (110,674 rats) which resulted in a large database (Systematic Pharmacological Database or Syphad (www.syphad.org)). We have then developed an in silico learning system that integrates Syphad and ChemNetDB data for predicting neurochemical fingerprints of various drug consumption patterns. In this lecture I will present data on neurochemical fingerprints of repeated intoxicating cycles of alcohol exposure. Our simulation of neurochemistry following chronic alcohol exposure predicts switches in neurochemical activity, especially a hyper-dopaminergic state in the nucleus accumbens and a hyper-glutamatergic state in the infralimbic cortex. These predictions can indeed be validated in post-mortem brain tissue from deceased alcoholics and in the alcohol-dependent rat brain. Furthermore, we show how mGluR2 agonist can restore these neurochemical switches and can thereby reduce alcohol craving and relapse.

ABSTRACT. David G. Mets1 and Michael S. Brainard1 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. 1 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.

ABSTRACT. AV Kukekova1, JP Hekman1, JL Johnson1, W Edwards2, AV Vladimirova3, RG Gulevich3, AV Kharlamova3, LT Raetzman2, LN Trut3 Domesticated species are characterized by reduced fearfulness, increased social tolerance, and increased resistance to stress. These behaviors are closely linked to reduced reactivity of the hypothalamic-pituitary-adrenal (HPA) axis, the hormonal cascade associated with the stress response in mammals. Specifically, reductions in circulating levels of adrenocorticotrophic hormone (ACTH), released by the anterior pituitary, and glucocorticoids, released by the adrenals, have been demonstrated in several domesticated species. Here we used the tame and aggressive strains of the red fox to explore mechanisms associated with the HPA axis reactivity. RNA extracted from the anterior pituitaries of six tame and six aggressive foxes and from the right adrenal glands of 11 tame and 11 aggressive foxes was sequenced on an Illumina HiSeq2500. The gene expression and network analyses looking at the differences in anterior pituitaries of tame and aggressive foxes indicated the importance of genes related to the regulation of exocytosis, specifically mediated by cAMP, the organization of pseudopodia, and cell motility. In adrenals, differential gene expression analysis suggested differences in ectodermal cell differentiation and interleukin-8 production, while weighted gene co-expression network analysis found differences in cholesterol biosynthesis and cell migration, suggesting that the biosynthesis of cholesterol (a steroid hormone precursor), rather than the biosynthesis of adrenal steroid hormones may differ between the two strains. These finding suggest that the tame and aggressive foxes may have differences in HPA reactivity due to differences in regulation of the hormone release. 1Department of Animal Sciences, College of Agriculture, Consumer, and Environmental Sciences, University of Illinois at Urbana-Champaign, IL 61801, USA 2Department of Molecular and Integrative Physiology, School of Molecular and Cellular Biology, College of Liberal Arts and Sciences, University of Illinois at Urbana-Champaign, IL 61801, USA 3Institute of Cytology and Genetics of the Russian Academy of Sciences, Novosibirsk, 630090, Russia

ABSTRACT. Halie M. Rando1,2, Jennifer L. Johnson2, Guojie Zhang3,4,5, Lyudmila N. Trut6, Anna V. Kukekova2 1Illinois Informatics Institute, 2Department of Animal Science, College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL USA; 3China National Genebank, BGI-Shenzhen, Shenzhen, Guangdong, China; 4Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark; 5State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China; 6Institute of Cytology and Genetics of the Russian Academy of Sciences, Novosibirsk, Russia; Funding Support: NIH R01 GM120782 and National Defense Science & Engineering Graduate Fellowship (NDSEG) Program

Sociality is fundamental to human and animal behavior, and atypical social behavior is implicated in many human psychiatric and neurodevelopmental disorders. Though mapping behavioral traits in humans is very challenging, the selective breeding of red foxes (Vulpes vulpes) presents a novel model for the genetic regulation of social behavior. Fifty generations of breeding has produced hypersociable “tame” foxes and defensively “aggressive” foxes in which 9 behavioral quantitative trait loci (QTL) were identified, but low marker resolution prevented the identification of candidate genes. With the new red fox genome assembly, these regions can now be fine mapped. The genomes of 30 foxes (10 each from the tame, aggressive, and conventional farm-bred strains) were each sequenced at 2.5x and pooled by population for SNP calling. SNPs were analyzed within sliding windows for within-population selective sweeps and between-population divergence (FST). Additional foxes comprising the least-related individuals from the tame (N=20) and aggressive (N=22) strains were then sequenced at 15x each, and putative regions under selection were identified. In the pooled analysis, either Hp and/or FST identified 103 genomic regions as likely to be under selection. Thirty of these regions overlapped 5 previously identified fox QTL. Loci within QTL intervals identified by both the pooled and individual analyses are strong candidates for social behavioral regulation. The deep sequencing data also presents the first opportunity to characterize haplotypes and even polymorphisms differentiating the tame and aggressive strains to identify novel candidates for the genetic regulation of mammalian social behavior.

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

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

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

ABSTRACT. MA Ehringer1, MS Powers1, LM Evans1, JC DeFries1 1Institute for Behavioral Genetics, University of Colorado Boulder; Boulder, Colorado, USA.

Anxiety disorders lead to debilitation in individuals and families, leading to high social and economic costs. Genetic factors have been shown to contribute to underlying risk for anxiety, but little is known about the specific genes and gene networks involved. Open-field activity is commonly used as a mouse model for anxiety-related behaviors, and bidirectional selection for high and low open-field activity was initiated in 1965 (DeFries et al, 1978). This process generated replicate inbred strains that differ ~40-fold in activity. We recently completed whole-genome sequencing of one male and one female from each of the four strains (H1, H2, L1, L2) using an average 10X coverage depth to pursue three separate questions. First, we examine strain distribution patterns in the four strains for all variants that differed between the BALB/cJ and C57BL/6J parental strains. Next, we assess genome-wide allele sharing both between and within the High and Low strains (viz., H1 vs. L1, H2 vs. L2, H1 vs. H2, and L1 vs. L2). Finally, genomic regions previously identified with high confidence from QTL studies will be carefully investigated for differences shared by both High compared to Low strains. This will be the first report of whole genome sequencing for these unique strains, which will provide a valuable resource for future studies that may integrate data generated from other genomic technologies to reveal underlying molecular genetic differences contributing the extreme phenotypic differences.

Funding Support: T32 AA007464; University of Colorado Institutional funds

ABSTRACT. Advances in genomics and proteomics have implicated a number of genes involved in actin polymerization in psychiatric and neurological disorders associated with irregular synaptic function. Here we examine the function of Nck1, an intracellular scaffolding protein known to play a role in actin dynamics, in the mouse brain and behavior. Our study found that mice lacking Nck1 display increased levels of anxiety-like behaviors, while other sensorimotor responses, including vision, olfactory responses and locomotion are indistinguishable from controls. The anxiety-like phenotype was rescued by treatment with diazepam, an anxiolytic that functions as a positive allosteric modulator of the GABAA receptor, implicating a deregulation of inhibitory control. Given that the amygdala is closely coupled with anxiety-like behaviours, we focused our further analysis on this brain region. We show that in the amygdala Nck1 is expressed in neurons but not microglia. Further analysis revealed that mice lacking Nck1 had fewer synapses and changes in synaptic morphologies in the spiny principal neurons of the basolateral amygdala. Taken together our study proposes that Nck1 is likely important in regulating neuronal excitability. Finally, our data suggests that Nck1 is necessary for normal synapse development in the amygdala and that the loss of Nck1 leads to the development of behaviors linked to anxiety.

1Department of Pharmacology, 2Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada. Funding Support: CIHR (MOP 341174)

ABSTRACT. A primary challenge facing Autism Spectrum Disorder genetics is the large and growing number of genes and gene variants of unknown functional significance. To determine the functional effects of ASD-associated variants we developed a novel strategy based on CRISPR-Cas9 genome engineering in the high-throughput genetic model organism Caenorhabditis elegans to generate single-copy integrated knock-in lines expressing the exact human gene variants identified in ASD. We have begun by focusing on the high-confidence ASD-associated gene PTEN. In C. elegans, the sole ortholog of PTEN, daf-18, regulates attractive navigation behaviour up a concentration gradient of NaCl (this behaviour is called NaCl chemotaxis). Using a novel automated machine vision chemotaxis paradigm we have shown that either directly replacing daf-18 with human WT PTEN using CRISPR or nervous system specific expression of human WT PTEN is able to substitute for loss of daf-18 and rescue this behavioural deficit. Surprisingly, all ASD-associated missense mutations in PTEN assessed resulted in partial or complete loss-of-function and failed to rescue this sensory deficit. Collaborative complementary in vivo functional assays in yeast, and fly as well as in vitro assays in HEK293 cells and rat neural culture directly corroborate our finding that ASD-associated PTEN variants are loss-of-function. The wealth of in vivo empirical data from this research will improve algorithms that estimate the pathogenicity of missense mutations, improve diagnostic accuracy, and further precision medicine efforts to treat ASD.

Troy A. McDiarmid1, Kathryn Post3, Riki Dingwall3, Payel Ganguly3, Matthew Edwards3, Ben Callaghan4, Manuel Belmadani4, Fabian Meili1, Warren Meyers1, Barry Young3, Sanja Rogic4, Chris Loewen3, Douglas Allan1,3, Timothy O’Connor1,3, Shernaz Bamji1,3, Paul Pavlidis4, Kurt Haas1,3, Catharine H. Rankin1,2

1. Djavad Mowafaghian Centre for Brain Health, University of British Columbia, 2211 Wesbrook mall, Vancouver, B.C., V6T 2B5; 2. Dept. of Psychology, University of British Columbia, 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. 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.