Neurotranscriptome profiles of multiple zebrafish strains

Behavioral displays or physiological responses are often influenced by intrinsic and extrinsic mechanisms in the context of the organism\u27s evolutionary history. Understanding differences in transcriptome profiles can give insight into adaptive or pathological responses.We utilize high throughput sequencing (RNA-sequencing) to characterize the neurotranscriptome profiles in both males and females across four strains of zebrafish (Danio rerio). Strains varied by previously documented differences in stress and anxiety-like behavioral responses, and generations removed from wild-caught individuals. Here we describe detailed methodologies and quality controls in generating the rawRNA-sequencing reads that are publically available in NCBI\u27s Gene Expression Omnibus database (GSE61108)

Investigating COMT Influence on the Proactive-Reactive Stress Coping Axis in Zebrafish

Individuals of the same species often display differences in correlated suites of behaviors which are made conspicuous when challenges – stressful, fear-inducing, etc. – are presented. In many species, a specific suite of behaviors (risk-aversion, aggression, exploration, learning, and memory) characterize an alternative set of stress coping styles (proactive and reactive). Such behaviors are regulated in the brain by specific neurotransmitters along with proteins that regulate them. One neurotransmitter regulator protein, catechol-O-methyltransferase (COMT) shows higher baseline whole-brain expression in proactive relative to reactive animals. However, it is not known whether its expression is a cause or a consequence of the stress coping style. In this ongoing study, I am testing the hypothesis that alteration of comta activity will lead to changes in stress coping behavior. Specifically, I attempted to knockout comtain zebrafish embryos using CRISPR/Cas9 with the intent of comparing stress-related behaviors and comtabrain-gene expression between mutant and control individuals in each stress coping style. I predict knocking out comtawill result in proactive zebrafish displaying similar stress-related behaviors as their reactive conspecifics and that reactive individuals will display elevated stress-related behaviors compared to controls. Across 155 breeding attempts, I found that approximately 23.75% (285/1200) of treatment embryos survived to 72 hours post-fertilization. High resolution melt analysis revealed 78.25% (18/23) to have variants in the target region, of which 11/18 (84.6%) showed successful mutations by Sanger sequencing. I intend to generate an F1 line in which to compare stress-related behaviors and comtabrain-gene expression by in situ hybridization

Contextual fear learning and memory differ between stress coping styles in zebrafish

Animals frequently overcome stressors and the ability to learn and recall these salient experiences is essential to an individual’s survival. As part of an animal’s stress coping style, behavioral and physiological responses to stressors are often consistent across contexts and time. However, we are only beginning to understand how cognitive traits can be biased by different coping styles. Here we investigate learning and memory differences in zebrafish (Danio rerio) displaying proactive and reactive stress coping styles. We assessed learning rate and memory duration using an associative fear conditioning paradigm that trained zebrafish to associate a context with exposure to a natural olfactory alarm cue. Our results show that both proactive and reactive zebrafish learn and remember this fearful association. However, we note significant interaction effects between stress coping style and cognition. Zebrafish with the reactive stress coping style acquired the fear memory at a significantly faster rate than proactive fish. While both stress coping styles showed equal memory recall one day postconditioning, reactive zebrafish showed significantly stronger recall of the conditioned context relative to proactive fish four days post-conditioning. Through understanding how stress coping strategies promote biases in processing salient information, we gain insight into mechanisms that can constrain adaptive behavioral responses

Electrical and behavioral courtship displays in the mormyrid fish Brienomyrus brachyistius

Mormyrid electric fish rely on the waveform of their electric organ discharges (EODs) for communicating species, sex, and social status, while they use the sequences of pulse intervals (SPIs) for communicating rapidly changing behavioral states and motivation. Little is known of electric signaling during courtship behavior because of two major difficulties: (1) the fish are not easily bred in captivity and (2) there is no reliable means of separating electric signals from several individuals in natural communication settings. Through simulating artificial rain conditions, we have successfully induced courtship and succeeded in breeding a mormyrid electric fish (Brienomyrus brachyistius) in the laboratory. We have also developed a system of video recording and editing combined with cross correlation analysis to precisely record and view behavior and separate EODs from two individuals in non-breeding and breeding contexts. Knowing the electrical and motor patterns during courtship allows for further exploration of topics such as mate choice and neural basis of pattern generation in these fish. Here we describe nine common motor displays and 11 SPIs. Analysis of frequency of occurrences suggests that some SPI patterns are sex and season specific. We also observed electrical duetting called `rasp matching\u27 during courtship signaling among pairs; males and females exchange `rasps\u27 and `bursts\u27, respectively, in alternation. Our study employs new techniques to separate and document SPIs in the context of courtship. We show that some SPIs correlate with specific behavioral acts around the time of spawning

Differences in stress reactivity between zebrafish with alternative stress coping styles

Animals experience stress in a variety of contexts and the behavioural and neuroendocrine responses to stress can vary among conspecifics. The responses across stressors often covary within an individual and are consistently different between individuals, which represent distinct stress coping styles (e.g. proactive and reactive). While studies have identified differences in peak glucocorticoid levels, less is known about how cortisol levels differ between stress coping styles at other time points of the glucocorticoid stress response. Here we quantified whole-body cortisol levels and stress-related behaviours (e.g. depth preference, movement) at time points representing the rise and recovery periods of the stress response in zebrafish lines selectively bred to display the proactive and reactive coping style. We found that cortisol levels and stress behaviours are significantly different between the lines, sexes and time points. Further, individuals from the reactive line showed significantly higher cortisol levels during the rising phase of the stress response compared with those from the proactive line. We also observed a significant correlation between individual variation of cortisol levels and depth preference but only in the reactive line. Our results show that differences in cortisol levels between the alternative stress coping styles extend to the rising phase of the endocrine stress response and that cortisol levels may explain variation in depth preferences in the reactive line. Differences in the timing and duration of cortisol levels may influence immediate behavioural displays and longer lasting neuromolecular mechanisms that modulate future responses

Limited sex-biased neural gene expression patterns across strains in zebrafish (Danio rerio)

Background: Male and female vertebrates typically differ in a range of characteristics, from morphology to physiology to behavior, which are influenced by factors such as the social environment and the internal hormonal and genetic milieu. However, sex differences in gene expression profiles in the brains of vertebrates are only beginning to be understood. Fishes provide a unique complement to studies of sex differences in mammals and birds given that fish show extreme plasticity and lability of sexually dimorphic characters and behaviors during development and even adulthood. Hence, teleost models can give additional insight into sexual differentiation. The goal of this study is to identify neurotranscriptomic mechanisms for sex differences in the brain. Results: In this study we examined whole-brain sex-biased gene expression through RNA-sequencing across four strains of zebrafish. We subsequently conducted systems level analyses by examining gene network dynamics between the sexes using weighted gene coexpression network analysis. Surprisingly, only 61 genes (approximately 0.4% of genes analyzed) showed a significant sex effect across all four strains, and 48 of these differences were male-biased. Several of these genes are associated with steroid hormone biosynthesis. Despite sex differences in a display of stress-related behaviors, basal transcript levels did not predict the intensity of the behavioral display. WGCNA revealed only one module that was significantly associated with sex. Intriguingly, comparing intermodule dynamics between the sexes revealed only moderate preservation. Further we identify sex-specific gene modules. Conclusions: Despite differences in morphology, physiology, and behavior, there is limited sex-biased neural gene expression in zebrafish. Further, genes found to be sex-biased are associated with hormone biosynthesis, suggesting that sex steroid hormones may be key contributors to sexual behavioral plasticity seen in teleosts. A possible mechanism is through regulating specific brain gene networks

Characterizing the neurotranscriptomic states in alternative stress coping styles

Background: Animals experience stress in many contexts and often successfully cope. Individuals exhibiting the proactive versus reactive stress coping styles display qualitatively different behavioral and neuroendocrine responses to stressors. The predisposition to exhibiting a particular coping style is due to genetic and environmental factors. In this study we explore the neurotranscriptomic and gene network biases that are associated with differences between zebrafish (Danio rerio) lines selected for proactive and reactive coping styles and reared in a common garden environment. Results: Using RNA-sequencing we quantified the basal transcriptomes from the brains of wild-derived zebrafish lines selectively bred to exhibit the proactive or reactive stress coping style. We identified 1953 genes that differed in baseline gene expression levels. Weighted gene coexpression network analyses identified one gene module associated with line differences. Together with our previous pharmacological experiment, we identified a core set of 62 genes associated with line differences. Gene ontology analyses reveal that many of these core genes are implicated in neurometabolism (e.g. organic acid biosynthetic and fatty acid metabolic processes). Conclusions: Our results show that proactive and reactive stress coping individuals display distinct basal neurotranscriptomic states. Differences in baseline expression of select genes or regulation of specific gene modules are linked to the magnitude of the behavioral response and the display of a coping style, respectively. Our results expand the molecular mechanisms of stress coping from one focused on the neurotransmitter systems to a more complex system that involves an organism’s capability to handle neurometabolic loads and allows for comparisons with other animal taxa to uncover potential conserved mechanisms

LDEF Space Plasma-High Voltage Drainage Experiment post-flight results

The Space Plasma-High Voltage Drainage Experiment (SP-HVDE) was comprised of two identical experimental trays. With one tray located on the leading (ram facing, B10) edge and the other located on the trailing (wake facing, D4) edge of the Long Duration Exposure Facility (LDEF), it was possible to directly compare the effects of ram and wake spacecraft environments on charged dielectric materials. Six arrays of Kapton dielectric samples of 2 mil, 3 mil, and 5 mil thicknesses maintained at +/- 300, +/- 500, and +/- 1000 voltage bias formed the experimental matrix of each tray. In addition, each tray carried two solar cell strings, one biased at +300 volts and the other at -300 volts, to study current leakage from High Voltage Solar Arrays (HVSA). The SP-HVDE provides the first direct, long-term, in-flight measurements of average leakage current through dielectric materials under electric stress. The experiment also yields information on the long term stability of the bulk dielectric properties of such materials. Data and findings of the SP-HVDE are an extension of those from shorter term flight experiments such as the PIX-1 (Plasma Interaction Experiment) and PIX-2 and are therefore valuable in the design and evaluation of long-lived space systems with high voltage systems exposed to the low earth orbital environment. A summary of the SP-HVDE post flight analysis final report delivered to the LDEF Project Office under contract to the National Aeronautics and Space Administration is presented

Repeatability and reliability of exploratory behavior in proactive and reactive zebrafsh, Danio rerio

Behavioral responses to novel situations often vary and can belong to a suite of correlated behaviors. Characteristic behaviors of different personality types (e.g. stress coping styles) are generally consistent across contexts and time. Here, we compare the repeatability and reliability of exploratory behaviors between zebrafsh strains selectively bred to display contrasting behavioral responses to stressors that represent the proactive-reactive axis. Specifically, we measure exploratory behavior of individual fish in an open field test over five weeks. We quantified the stationary time, average swimming speed and time spent by a fish in the center area. We found a number of strain differences for each behavioral measure. Stationary time was the most repeatable and reliable measure for assessing proactive-reactive behavioral differences. Reactive zebrafish generally showed the highest reliability and repeatability of exploratory behavior compared to proactive zebrafish and a separate wild caught strain. Given the increased interest in the evolutionary consequences and proximate mechanisms of consistent individual differences, it will be important to continue to investigate how different selective pressures may influence expression of stress coping styles and their effects on the consistency of an animal’s behavior

Identifying context-specific gene profiles of social, reproductive and mate preference behavior in a fish species with female mate choice

Sensory and social inputs interact with underlying gene suites to coordinate social behavior. Here we use a naturally complex system in sexual selection studies, the swordtail, to explore how genes associated with mate preference, receptivity, and social affiliation interact in the female brain under specific social conditions. We focused on 11genes associated with mate preference in this species (neuroserpin, neuroligin-3, NMDA receptor, tPA, stathmin-2, β-1 adrenergic receptor) or with female sociosexual behaviors in other taxa (vasotocin, isotocin, brain aromatase,α-1 adrenergic receptor, tyrosinehydroxylase). We exposed females to four social conditions, including pairings of differing mate choice complexity (large males, large/ small males, small males), and a social lcontrol (two females). Female mate preference differed significantly by context. Multiple discriminant analysis (MDA) of behaviors revealed a primary axis (explaining 50.2% between-group variance) highlighting differences between groups eliciting high preference behaviors (LL,LS) vs. other contexts, and a secondary axis capturing general measures distinguishing a non-favored group (SS) from other groups. Gene expression MDA revealed a major axis (68.4% between-group variance) that distinguished amongst differential male pairings and was driven by suites of “preference and receptivity genes”; whereas a second axis, distinguishing high affiliation groups (large males, females) from low (small males), was characterized by traditional affiliative-associated genes (isotocin, vasotocin). We found context-specific correlations between behavior and gene MDA, suggesting gene suites covary with behaviors in a socially relevant context. Distinct associations between “affiliative” and “preference” axes suggest mate preference maybe mediated by distinct clusters from those of social affiliation. Our results highlight the need to incorporate natural complexity of mating systems into behavioral genomics