Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior

Histone Methylation Mechanisms in the Adult Brain after Adolescent Alcohol Exposure

The developmental period of adolescence is characterized by profound changes in brain structure, function, and gene expression patterns that are at least partially epigenetically programmed. These biochemical alterations are responsible for the behavioral characteristics of adolescence, including increased impulsivity and the propensity to consume binge amounts of alcohol. Binge alcohol consumption during adolescence is associated with increased risk for alcohol use disorder (AUD) and comorbid psychiatric disorders such as depression and anxiety in adulthood. Rats exposed to adolescent intermittent ethanol (AIE) display increased alcohol consumption and increased anxiety-like behaviors in adulthood that are accompanied by changes to epigenetic and synaptic systems in the amygdala, a brain region that is crucial for the both expression of anxiety and the actions of ethanol. In this thesis, I focus on elucidating novel treatment targets for AUD and comorbid anxiety related to adolescent alcohol consumption in two separate but interrelated projects. In the first project, I examined the expression of microRNAs (miRNAs) in the amygdala of adult rats following AIE and found that microRNA-137 (miR-137) is increased in the AIE adult amygdala compared to saline-exposed (AIS) controls. The miR-137 target gene and epigenetic modifier lysine-specific demethylase 1 (Lsd1) and the neuron-specific splice variant Lsd1+8a show reduced mRNA levels in the AIE adult amygdala, and this is associated with an increase in repressive histone 3 lysine 9 dimethylation (H3K9me2) but unchanged H3K4me2. LSD1 levels locally at the exon IV promoter of brain-derived neurotrophic factor (Bdnf) are decreased in the AIE adult amygdala, correlating with decreased Bdnf IV mRNA expression. Infusion of an antisense antagomir of miR-137 directly into the central nucleus of the amygdala (CeA) rescues the increased alcohol drinking and anxiety-like behavior seen in AIE adult animals. miR-137 antagomir infusion in the CeA also attenuates Lsd1 and Lsd1+8a mRNA expression, levels of LSD1 at the Bdnf exon IV promoter, and Bdnf IV mRNA levels in the amygdala. These data suggest that upregulation of miR-137 is crucial to the long-lasting behavioral and molecular effects of adolescent alcohol exposure. In the second project, I found that synaptic density was reduced in the AIE adult amygdala compared to AIS rats, and that several synapse-associated genes including activity-regulated cytoskeleton-associated protein (Arc) show reduced mRNA levels in the AIE adult amygdala. AIE adult rats also show reduced occupancy of both lysine-specific demethylase 6B (KDM6B) and CREB binding protein (CBP), leading to increased H3K27me3 and decreased H3K27 acetylation (H3K27ac) at the Arc synaptic activity response element (SARE) site in the amygdala. The SARE site encodes an enhancer RNA (eRNA) upstream of Arc, and AIE leads to decreased Arc eRNA expression and increased binding of negative elongation factor (NELF) repressor complex to the Arc promoter in the amygdala which agrees with previous studies suggesting that Arc eRNA binds to NELF in order to de-repress Arc mRNA transcription. Acute ethanol challenge in adulthood normalizes the anxiety-like behavior seen in AIE adult rats, as well as the chromatin alterations at the Arc SARE site and promoter in the amygdala and altered Arc eRNA and mRNA expression. Knockdown of Kdm6b expression in the CeA provokes anxiety-like behavior in AIS rats and decreases Arc eRNA and mRNA expression, possibly via increased H3K27me3 and decreased H3K27ac observed at the SARE site and Arc promoter in the amygdala following Kdm6b small-interfering RNA (siRNA) infusion. Lastly, knockdown of the minus strand of Arc eRNA [Arc eRNA (-)] in the CeA leads to marked anxiety-like behavior in alcohol-naïve control rats as well as increased NELF repressor complex occupancy at the Arc promoter and decreased Arc mRNA expression in the amygdala. Therefore, Arc eRNA controls Arc mRNA expression in the amygdala and disruption of this system by adolescent alcohol exposure underlies the risk for anxiety-like behaviors seen in adulthood. In summary, adolescent alcohol exposure leads to an upregulation of miR-137 in the adult amygdala which alters LSD1-related chromatin remodeling pathways at Bdnf, as well as a decreased in Arc eRNA expression in the same brain region that is encoded by epigenetic alterations at the SARE site and downregulates Arc mRNA expression. These findings identify novel treatment targets and neurobiological pathways in the amygdala that contribute to the pathogenesis of AUD and comorbid anxiety. Finally, the work presented here furthers the knowledge of the epigenetic consequences of adolescent binge-like alcohol exposure in adulthood

Adolescent alcohol exposure: Burden of epigenetic reprogramming, synaptic remodeling, and adult psychopathology

Adolescence represents a crucial phase of synaptic maturation characterized by molecular changes in the developing brain that shape normal behavioral patterns. Epigenetic mechanisms play an important role in these neuromaturation processes. Perturbations of normal epigenetic programming during adolescence by ethanol can delay these molecular events, leading to synaptic remodeling and abnormal adult behaviors. Repeated exposure to binge levels of alcohol increases the risk for alcohol use disorder (AUD) and comorbid psychopathology including anxiety in adulthood. Recent studies in the field clearly suggest that adolescent alcohol exposure causes widespread and persistent changes in epigenetic, neurotrophic, and neuroimmune pathways in the brain. These changes are manifested by altered synaptic remodeling and neurogenesis in key brain regions leading to adult psychopathology such as anxiety and alcoholism. This review details the molecular mechanisms underlying adolescent alcohol exposure-induced changes in synaptic plasticity and the development of alcohol addiction-related phenotypes in adulthood

High-Throughput Screening of Stem Cell Therapy for Globoid Cell Leukodystrophy Using Automated Neurophenotyping of Twitcher Mice

Globoid cell leukodystrophy (Krabbe\u27s disease) is an autosomal recessive neurodegenerative disorder that results from the deficiency of galactosylceramidase, a lysosomal enzyme involved in active myelination. Due to the progressive, lethal nature of this disease and the limited treatment options available, multiple laboratories are currently exploring novel therapies using the mouse model of globoid cell leukodystrophy. In order to establish a protocol for motor function assessment of the twitcher mouse, this study tested the capability of an automated system to detect phenotypic differences across mouse genotypes and/or treatment groups. The sensitivity of this system as a screening tool for the assessment of therapeutic interventions was determined by the administration of murine bone marrow-derived stem cells into twitcher mice via intraperitoneal injection. Animal behavior was analyzed using the Noldus EthoVision XT7 software. Novel biomarkers, including abnormal locomotion (e.g., velocity, moving duration, distance traveled, turn angle) and observed behaviors (e.g., rearing activity, number of defecation boli), were established for the twitcher mouse. These parameters were monitored across all mouse groups, and the automated system detected improved locomotion in the treated twitcher mice based on the correction of angular velocity, turn angle, moving duration, and exploratory behavior, such as thigmotaxis. Further supporting these findings, the treated mice showed improved lifespan, gait, wire hang ability, twitching severity and frequency, and sciatic nerve histopathology. Taken together, these data demonstrate the utility of computer-based neurophenotyping for motor function assessment of twitcher mice and support its utility for detecting the efficacy of stem cell-based therapy for neurodegenerative disorders

Zebrafish models of autism spectrum disorder

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by motor, social and cognitive deficits that develop early during childhood. The pathogenesis of ASD is not well characterized and involves a multifaceted interaction between genetic, neurobiological and environmental factors. Animal (experimental) models possess evolutionarily conserved behaviors and molecular pathways that are highly relevant for studying ASD. The zebrafish (Danio rerio) is a relatively new animal model with promise for understanding the pathogenesis of complex brain disorders and discovering novel treatments. As a highly social and genetically tractable organism, zebrafish have recently been applied to model a variety of deficits relevant to ASD. Here, we discuss the developing utility of zebrafish models of ASD, as well as current behavioral, toxicological and genetic models of ASD, and future directions of research in this field. © 2017 Elsevier Inc

Zebrafish Models of Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by motor, social and cognitive deficits that develop early during childhood. The pathogenesis of ASD is not well characterized and involves a multifaceted interaction between genetic, neurobiological and environmental factors. Animal (experimental) models possess evolutionarily conserved behaviors and molecular pathways that are highly relevant for studying ASD. The zebrafish (Danio rerio) is a relatively new animal model with promise for understanding the pathogenesis of complex brain disorders and discovering novel treatments. As a highly social and genetically tractable organism, zebrafish have recently been applied to model a variety of deficits relevant to ASD. Here, we discuss the developing utility of zebrafish models of ASD, as well as current behavioral, toxicological and genetic models of ASD, and future directions of research in this field