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      Sustained Effects of Developmental Exposure to Ethanol on Zebrafish Anxiety-Like Behaviour

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          Abstract

          In zebrafish developmentally exposed to ambient ethanol (20mM-50mM) 1–9 days post fertilization (dpf), the cortisol response to stress has been shown to be significantly attenuated in larvae, juveniles and 6 month old adults. These data are somewhat at variance with similar studies in mammals, which often show heightened stress responses. To test whether these cortisol data correlate with behavioural changes in treated animals, anxiety-like behaviour of zebrafish larvae (9dpf and 10dpf) and juveniles (23dpf) was tested in locomotor assays designed to this end. In open field tests treated animals were more exploratory, spending significantly less time at the periphery of the arena. Behavioural effects of developmental exposure to ethanol were sustained in 6-month-old adults, as judged by assessment of thigmotaxis, novel tank diving and scototaxis. Like larvae and juveniles, developmentally treated adults were generally more exploratory, and spent less time at the periphery of the arena in thigmotaxis tests, less time at the bottom of the tank in the novel tank diving tests, and less time in the dark area in scototaxis tests. The conclusion that ethanol-exposed animals showed less anxiety-like behaviour was validated by comparison with the effects of diazepam treatment, which in thigmotaxis and novel tank diving tests had similar effects to ethanol pretreatment. There is thus a possible link between the hypophyseal-pituitary-interrenal axis and the behavioural actions of developmental ethanol exposure. The mechanisms require further elucidation.

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          Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.

          The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.
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            Nonlinear mixed effects models for repeated measures data.

            We propose a general, nonlinear mixed effects model for repeated measures data and define estimators for its parameters. The proposed estimators are a natural combination of least squares estimators for nonlinear fixed effects models and maximum likelihood (or restricted maximum likelihood) estimators for linear mixed effects models. We implement Newton-Raphson estimation using previously developed computational methods for nonlinear fixed effects models and for linear mixed effects models. Two examples are presented and the connections between this work and recent work on generalized linear mixed effects models are discussed.
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              Using the rat forced swim test to assess antidepressant-like activity in rodents.

              The forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior. This protocol details using the FST in rats, which takes place over 48 h and is followed by the video analysis of the behavior. The swim test involves the scoring of active (swimming and climbing) or passive (immobility) behavior when rodents are forced to swim in a cylinder from which there is no escape. There are two versions that are used, namely the traditional and modified FSTs, which differ in their experimental setup. For both versions, a pretest of 15 min (although a number of laboratories have used a 10-min pretest with success) is included, as this accentuates the different behaviors in the 5-min swim test following drug treatment. Reduction in passive behavior is interpreted as an antidepressant-like effect of the manipulation, provided it does not increase general locomotor activity, which could provide a false positive result in the FST.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 February 2016
                2016
                : 11
                : 2
                : e0148425
                Affiliations
                [1 ]School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
                [2 ]School of Health Sciences and Social Work, University of Portsmouth, Portsmouth, United Kingdom
                University Zürich, SWITZERLAND
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: GPV CHB. Performed the experiments: MB. Analyzed the data: MB MOP. Wrote the paper: GPV MOP MB CHB.

                Article
                PONE-D-15-42946
                10.1371/journal.pone.0148425
                4749633
                26862749
                d62e4304-e11f-4aad-8751-94ffaad3fac8
                © 2016 Baiamonte et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 29 September 2015
                : 18 January 2016
                Page count
                Figures: 4, Tables: 0, Pages: 14
                Funding
                This work was supported by https://www.nc3rs.org.uk, G1000053, National Centre for the replacement, refinement and reduction of animals in research, UK, to CHB and MOP; http://www.bbsrc.ac.uk, Biotechnology and Biological Science Research Council, UK, to MB; and https://royalsociety.org, Royal Society, UK, to CHB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and Analysis Methods
                Model Organisms
                Animal Models
                Zebrafish
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Osteichthyes
                Zebrafish
                Biology and Life Sciences
                Developmental Biology
                Metamorphosis
                Larvae
                Medicine and Health Sciences
                Pharmacology
                Drugs
                Diazepam
                Biology and Life Sciences
                Biochemistry
                Hormones
                Lipid Hormones
                Hydrocortisone
                Biology and Life Sciences
                Biochemistry
                Hormones
                Steroid Hormones
                Hydrocortisone
                People and Places
                Population Groupings
                Age Groups
                Adults
                Medicine and Health Sciences
                Mental Health and Psychiatry
                Psychological Stress
                Biology and Life Sciences
                Psychology
                Psychological Stress
                Social Sciences
                Psychology
                Psychological Stress
                Biology and Life Sciences
                Behavior
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Mammals
                Rodents
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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