Hawai'i and other island communities around the globe are experiencing high exposures to micro- and nanoplastics (MNPs). Widespread use of various types of plastics and the action of wave physics coupled with abundant silica creates a constant production of MNPs. The community health effects of exposure to MNPs are not yet fully understood. The aquatic vertebrate model zebrafish, Danio rerio, was utilized to evaluate the developmental and behavioral effects of nanoplastic (NP) exposure as well as visual confirmation of plastic accumulation after embryonic and larval exposure. Eye and yolk size, heart rate, angle of development, and early larval locomotion behavior were quantified after early NP exposure lasting 3 or 5 days. In this work, a digital laboratory environment is used as the context for research trainees spanning high school to graduate and medical school in Hawai'i to explore health outcomes of developmental NP exposures. Presence of MNPs in the early developmental environment led to changes in growth and behavior of exposed fish. Data analysis showed significant effects of exposures on changes in the rate of development and yolk consumption on day 2 and reduced body length and increased locomotion on day 6. Nanoplastic exposure also affected overall body length, but this effect was not significant. These data support the potential dangers of MNP exposure and demonstrate their persistent detrimental effects on development and behavior in young zebrafish. Accordingly, our work contributes toward shifting focus toward understanding longer impacts on health, growth, and behaviors and potentially the reduction of harm in developing vertebrates.

Methyl CpG binding protein 2 (MECP2) is an essential global modulator of transcription and mutations in MECP2 are the most common cause of Rett syndrome, an X-linked neurodevelopmental disorder. Patients diagnosed with Rett syndrome have increased risk for epilepsy as well as problems with anxiety and social communication. Using the zebrafish mecp2Q63X line, this study aimed to increase our understanding of the role of Mecp2 function in regulation of pharmacologically-induced hyperlocomotion, developmental social preference, and adult socialization, anxiety-related behaviour, and baseline cortisol levels. To determine responses of mecp2−/− zebrafish to a stimulating convulsant, general locomotor activity was measured at 5 days post-fertilization (dpf) in sibling mecp2+/+, mecp2+/−, and mecp2−/− fish after treatment with a GABAA receptor antagonist pentylenetetrazol (PTZ) at varying concentrations. Responses to social stimulus were investigated in juvenile (21 dpf) and adult mecp2−/− and mecp2+/+ fish. Anxiety responses to a novel tank and whole-body cortisol levels were also measured in adult mecp2−/− and control mecp2+/+ zebrafish. The behavioural tests showed that mecp2−/− zebrafish displayed hypolocomotion at the larval stage, along with increased freezing time and thigmotaxis, and higher whole-body cortisol levels in adulthood. However, the hyper-locomotion response to PTZ at 5 dpf and social preference for visual social stimulus at 21 dpf and in adulthood were not affected by the lack of functional Mecp2. Functional Mecp2 modulated larval locomotion and behavioural anxiety at different ages and adult cortisol levels, but mecp2 null-mutation did not alter adult locomotion and socialization, and developmental sociability and PTZ-induced hyperlocomotion in zebrafish. Given the variability reported in patients and in rodent Mecp2 knockout models, studies using zebrafish can explore vital elements of MECP2’s role across development and improve our understanding of neural mechanisms underlying neurodevelopmental disorders.

The vertebrate stress response (SR) is mediated by the hypothalamic-pituitary-adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. Genetic abrogation of glucocorticoid receptor (nr3c1) decreased basal locomotor activity in light and darkness. Some key HPI axis receptors (mc2r [ACTH receptor], nr3c1), but not nr3c2 (mineralocorticoid receptor), were required to adapt to light more efficiently but became dispensable when longer illumination was provided. Such light adaptation was more efficient in dimmer light. Our findings show that the HPI axis contributes to the SR, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPI axis activity.

Microscopes are essential for research and education in science. Unlike computers and online learning tools, however, microscopes are not currently a fixed element in K-12 classrooms, due to steep cost, needless complexity, and often requiring a prohibitive level of staff training to effectively deploy. In a collaboration with Area 10 Labs, Integrated Science Education Outreach (InSciEd Out) developed a state-of-the-art alternative microscope, the InSciEdRS View, to reduce the financial barrier, prohibitive per-student cost, unnecessary complexity, and extensive staff training. Utilizing a 1080p camera and a lunchbox-style case, this Wi-Fi- and USB-connectable microscope comes with all necessary components for visualization of microscopic specimens (10 x -50 x magnification). While built to handle the rigors of classroom use, its imaging capability and battery-operation can make it flexible for a laboratory or fieldwork as well. We further highlight here K-12 curricula that we have developed using larval zebrafish to enable teachers, science outreach leaders, and parents to support active hands-on science observations. The InSciEdRS View microscope and the InSciEd Out curricula are readily scalable, translatable, and accessible for traditional and neurodiverse students and integrating these in various settings can be an efficient way to achieve better outcomes in science education.

The Ala Wai Canal is an artificial waterway in the tourist district of Waikiki in Honolulu, HI. Originally built to collect runoff from industrial, residential, and green spaces dedicated to recreation, the Ala Wai Canal has since experienced potent levels of toxicity due to this runoff entering the watershed and making it hazardous for both marine life and humans at current concentration, including Danio rerio (zebrafish). A community of learners at educations levels from high school to postbaccalaureate from Oahu, HI was connected through the Consortium for Increasing Research and Collaborative Learning Experiences (CIRCLE) distance research program. This team conducted research with an Investigator and team from Mayo Clinic in Rochester, MN, with the Ala Wai Canal as its primary subject. Through CIRCLE, research trainees sent two 32 oz bottles of Ala Wai- acquired water to a partnered laboratory at the Mayo Clinic in which zebrafish embryos were observed at differing concentrations of the sampled water against a variety of developmental and behavioral assays. Research trainees also created atlases of developmental outcomes in zebrafish following exposure to environmental toxins and tables of potential pesticide contaminants to enable the identification of the substances linked to structural defects and enhanced stress during Ala Wai water exposure experiments.

The vertebrate stress response (SR) is mediated by the hypothalamic-pituitary-adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. The glucocorticoid receptor (nr3c1) was necessary to maintain basal locomotor activity in light and darkness. The HPA axis was required to adapt to light more efficiently but became dispensable when longer illumination was provided. Light adaptation was more efficient in dimmer light and did not require the mineralocorticoid receptor (nr3c2). Our findings show that the HPA axis contributes to the SR at various stages, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPA axis activity.

The hypothalamic-pituitary-adrenal (HPA) axis and its effector molecules—glucocorticoids—modulate diverse aspects of physiology in vertebrates ranging from metabolism to immune function, behavior, and circadian cycle. While the glucocorticoid receptor ( nr3c1 ) is known to be involved in light adaptation (photoadaptation) of the retinal cells, the role of nr3c1 and other HPA axis signaling molecules in the behavioral phenotypes observed during photoadaptation have not been delineated. Therefore, we investigated locomotor adaptation to various light/dark durations using larval zebrafish that carry a mutated allele in key HPA axis receptors. First, we established baseline locomotion and compared WT and nr3c1 mutant larvae in constantly lit and dark conditions (for 12-hrs). WT larval zebrafish showed highest locomotor activity in the middle of the day and low activity during the early and later parts of the day, which was generally higher in the light. The baseline locomotion was depressed in nr3c1 mutants throughout the day in both environments with a more significant change noted in the light. Next, groups of larvae mutant in nr3c1, nr3c2 (mineralocorticoid receptor), or mc2r (melanocortin receptor type 2; adrenocorticotropic hormone (ACTH) receptor) along with their wildtype (WT) siblings were acclimated in the dark and underwent four cycles of dark-light illumination changes with different durations of illumination: 7.5, 6, 4, or 2 min. The nr3c1 and mc2r mutant fish showed significantly decreased locomotion during the dark phase when the illumination was provided for 4 or 2 min. However, with 4 min or longer illumination, if locomotor deficits were observed in mutant larvae, they demonstrated a “catch-up” phenotype with increasing locomotion during the later dark phases of the assay and ultimately reaching the swimming distances indistinguishable from their WT siblings in the assays with 7.5-min illumination. Finally, we looked at effects of light intensity, beginning with our short light exposure. A lower intensity of light failed to elicit any response even from WT fish after 1-min illumination. However, this dim light still evoked a robust response from nr3c1 mutants and their WT siblings after 7.5-min illumination. The nr3c2 mutant larvae consistently showed locomotor response similar to their WT siblings regardless of the length of the light phase. Thus, activation of the canonical HPA axis (i.e. nr3c1, mc2r ) was necessary to induce a rapid locomotor adaptation following after light to dark transition with shorter light exposure times (~≤4 min), but it was not needed for locomotor responses observed after longer exposure to light (~> 4 min). Moreover, the locomotor response after transitioning to dark from a short (1 min) exposure was dependent on the intensity of light, whereas the longer exposure was not. Together, these findings suggest that locomotion after longer exposure to light is not dependent on the HPA axis, suggesting that either parallel independent pathway(s) are responsible for the locomotor response in these cases or that the HPA axis facilitates a primary pathway to increase sensitivity to light exposure.

Abstract Social isolation is a well-established technique for inducing early adversity but, in rodent models, the need of parental care makes it difficult to distinguish the effects of social deprivation from the consequences of nutritional deficiencies. Zebrafish do not require parental care, allowing separation of social deprivation from nutritional deprivation, and have emerged as a promising model to study ontogeny of normal and pathological behaviors relevant for human neuropsychological disorders. Previous reports of life-long isolation in zebrafish showed some consistency with mammalian literature, depicting later social deficits and locomotor hyperactivity. However, unlike reports of higher anxiety and stress behavior in isolated rodents and primates, behavioral responses were tapered in isolated fish. To examine whether life-long developmental isolation has a dampening effect on zebrafish endocrine stress response, we applied stressors to zebrafish siblings that were either isolated or socialized, and compared their whole-body cortisol levels with non-stressed control siblings kept in low-housing densities. Utilizing previously validated paradigms (exposure to novel tank and unpredictable chronic stress), we exposed separate groups (n = 9–14, mixed-sex) of social and isolated zebrafish to acute and chronic stressors and measured their cortisol levels. A univariate ANOVA and post-hoc Tukey’s HSD tests confirmed that compared to socially raised control fish, developmental isolation did not increase baseline cortisol levels in zebrafish. Additionally, compared to the non-stressed condition, application of both acute and chronic stressors significantly increased cortisol levels in isolated fish and, to a similar degree, to socially raised fish. Our findings suggest that zebrafish isolation studies may help separate effects of social deprivation from nonsocial aspects of early adversity. These studies further substantiate the use of developmental isolation in zebrafish, particularly with acute and chronic stress paradigms, for modeling neuropsychological disorders. LAY SUMMARY A difficult childhood can make humans react more frequently or severely to later stress and modeling this effect in animals can help explain how and why early stress affects subsequent mental and physical health. Early social isolation does not affect later response to stressful situations in adult zebrafish, providing us with a model of psychiatric disorders that allows separation of effects of poor physical environments (lacking food, shelter, etc.) from poor social environments (lack of appropriate socialization).

The hypothalamic neuropeptides of the vasopressin-oxytocin family (and their homologs for non-mammalian species) are key modulators of the Social Brain Network, acting via specific receptors reported in all the nuclei of this network. Different conclusive examples have proven the context-dependency actions of hypothalamic nonapeptides on social behavior in several vertebrate taxa. Teleost fishes provide endless possibilities of experimental model systems to explore the underlying mechanisms of nonapeptide actions on social behavior given that they are the most diverse group of vertebrates. Although it has been difficult to identify commonalities of nonapeptide actions across species, indisputable evidence in many teleost species have demonstrated a clear role of vasotocin in the modulation of aggressive and sexual behaviors. Though Neotropical South American fish contribute an important percentage of teleost diversity, most native species remain unexplored as model systems for the study of the neuroendocrine bases of social behavior. In this review, we will revise recent data on the two model systems of Neotropical fish, South American cichlids and weakly electric fish that have contributed to this issue.

To further improve the search ability of the decomposition based many/multi-objective evolutionary algorithm (MOEA/D) in the tackling many-objective optimization problems (MaOPs) possessing complex characteristics (e.g., disconnected, degenerate, inverted, extremely convex or differently-scaled), we suggest an adaptive MOEA/D with better versatility, where the weight vector adaption and selection mechanism are improved. Firstly, a new niche-guided scheme by considering both the vector angle and Euclidean distance is proposed to leverage the search direction adaption upon different evolution phases, which is expected to be more robust for handling different types of irregular Pareto fronts (PFs). Secondly, in mating selection, a coordinated selection scheme aided by a multi-criterion decision procedure is utilized to enhance the effectiveness of recombination. Finally, in environmental selection, a steady state replacement strategy considering both the ensemble ranking of favorite subproblems with respect to solutions and improvement region restriction of subproblems is employed to alleviate misleading selection. Comparison experiments on benchmark MaOPs with diverse characteristics have been performed and the empirical results demonstrate the superiority of our proposal. The effects of direction vector adaption mechanism and other pertinent enhancements are also investigated.

The role of the gut microbiome and its enteric metabolites, such as short-chain fatty acids (SCFAs), in the etiology of autism spectrum disorders (ASDs) has recently received increased attention. Of particular interest has been the SCFA, propionic acid (PPA). Several different rodent models have been developed using PPA treatment to examine behaviors of relevance to ASD. The effects of systemic (intraperitoneal, i.p.) administration of PPA on social behavior, anxiety-related behavior, and locomotor activity in juvenile male rats (age 35 days) were examined in this study. Rats received seven i.p. injections of buffered PPA (500 mg/kg) or phosphate-buffered saline. Behavior was video-recorded during social interaction in a large open field (first four injections) or assessed in an automated activity system (individual animals, last three injections). PPA treatment significantly reduced social interaction, increased anxiety-related behavior, and produced hypoactivity and increased abnormal motor movements. These findings suggest that PPA alters behaviors of relevance to ASD in juvenile rats. These results contribute to the behavioral validity of the rodent model of ASD with systemic PPA treatment.

The zebrafish represents an excellent compromise between system complexity and practical simplicity, features that make it useful for modeling and mechanistic analysis of complex brain disorders. Also promising are screens for psychoactive drugs with effects on larval and adult zebrafish behavior. This review, based upon a recent symposium held at the 2016 IBNS Congress, provides different perspectives on how the zebrafish may be utilized to advance research into human central nervous system disorders. It starts with a discussion on an important bottleneck in zebrafish research, measuring the behavior of this species (specifically shoaling), and continues with examples on research on autism spectrum disorder in larval zebrafish, on screening natural products for compounds with psychoactive properties in adult zebrafish, and on the development of a zebrafish model of fetal alcohol spectrum disorders. By providing information on a broad spectrum of brain disorders, experimental methods, and scientific approaches using both larval and adult zebrafish, the review is intended to showcase this underutilized laboratory species for behavioral neuroscience and psychopharmacology research.

Zebrafish show great potential for behavioral neuroscience. Promising lines of research, however, require the development and validation of software tools that will allow automated and cost-effective behavioral analysis. Building on our previous work with the RealFishTracker (in-house-developed tracking system), we present Argus, a data extraction and analysis tool built in the open-source R language for behavioral researchers without any expertise in R. Argus includes a new, user-friendly, and efficient graphical user interface, instead of a command-line interface, and offers simplicity and flexibility in measuring complex zebrafish behavior through customizable parameters. In this article, we compare Argus with Noldus EthoVision and Noldus The Observer, to validate this new system. All three software applications were originally designed to quantify the behavior of a single subject. We first also performed an analysis of the movement of individual fish and compared the performance of the three software applications. Next we computed and quantified the behavioral variables that characterize dyadic interactions between zebrafish. We found that Argus and EthoVision extract similar absolute values and patterns of changes in these values for several behavioral measures, including speed, freezing, erratic movement, and interindividual distance. In contrast, the manual coding of behavior in The Observer showed weaker correlations with the two tracking methods (EthoVision and Argus). Thus, Argus is a novel, cost-effective, and customizable method for the analysis of adult zebrafish behavior that may be utilized for the behavioral quantification of both single and dyadic interacting subjects, but further sophistication will be needed for the proper identification of complex motor patterns, measures that a human observers can easily detect.

Social isolation can be used to study behavioral, neural, and hormonal mechanisms that regulate interactions in social animals. Although isolation effects have been reported in social mammals and various fish species, systematic studies with isolated zebrafish are rare. Here, the authors examined behavior (social and nonsocial), physiological stress (whole-body cortisol levels), and neurochemicals (serotonin, dopamine, and their metabolites), following acute and chronic social isolation in adult zebrafish. To observe how isolated fish respond behaviorally to social stimuli, they exposed zebrafish to live conspecifics or animated images after acute (24 hr) or chronic (6 months) social isolation. The authors observed that isolation did not affect locomotor activity, but acute isolation had weak nonsignificant anxiogenic effects in adult zebrafish. They also found that all isolated fish responded to both live and animated social stimuli, and the stress hormone, cortisol was lower in chronically isolated fish. Finally, neurochemical analyses showed that serotonin levels increased when fish were exposed to social stimulus after acute isolation, but its metabolite 5HIAA decreased in response to social stimulus following both acute and chronic isolation. Levels of both dopamine and its metabolite DOPAC were also reduced in fish exposed to social stimulus after acute and chronic isolation. Overall, these results show that isolation in zebrafish is an effective tool to study fundamental mechanisms controlling social interaction at behavioral and physiological levels.

Unpredictable chronic mild stress (UCMS) and developmental social isolation are often utilized in laboratory animals to mimic unpredictable life stressors and early life adversity that may contribute to the development of major depressive disorder in humans. Zebrafish (Danio rerio) have been used to examine the effects of both developmental social isolation and UCMS. However, anxiety-like behavioral responses, social behavior, and neurochemical changes induced by stressors have not been well characterized. Furthermore, the possible interaction between UCMS and developmental isolation remains unexplored. In this study, we analyzed the effect of UCMS on developmentally isolated and socially reared zebrafish. The UCMS procedure entailed delivering unpredictably varying mild stressors twice a day for 15 consecutive days. To quantify social and anxiety-like behaviors, we measured the zebrafish's behavioral and neurochemical (dopaminergic and serotonergic) responses to an animated image of conspecifics in a novel tank. Our results suggest that UCMS increased anxiety-like behavioral responses, whereas developmental isolation altered motor responses during stimulus presentation. We also found that UCMS diminished weight gain and reduced whole-brain levels of dopamine and serotonin's metabolite 5-HIAA in developmentally isolated, but not socially reared zebrafish. Our findings reinforce the utility of combining developmental isolation with UCMS in zebrafish to model depressive-like behavior in humans.

The zebrafish is a social vertebrate and an excellent translational model for a variety of human disorders. Abnormal social behavior is a hallmark of several human brain disorders. Social behavioral problems can arise as a result of adverse early social environment. Little is known about the effects of early social isolation in adult zebrafish. We compared zebrafish that were isolated for either short (7 days) or long duration (180 days) to socially housed zebrafish, testing their behavior across ontogenesis (ages 10, 30, 60, 90, 120, 180 days), and shoal cohesion and whole-brain monoamines and their metabolites in adulthood. Long social isolation increased locomotion and decreased shoal cohesion and anxiety in the open-field in adult. Additionally, both short and long social isolation reduced dopamine metabolite levels in response to social stimuli. Thus, early social isolation has lasting effects in zebrafish, and may be employed to generate zebrafish models of human neuropsychiatric conditions.

BACKGROUND:Fetal alcohol spectrum disorders (FASD) may vary in symptoms and severity. In the milder and more prevalent forms of the disease, behavioral abnormalities may include impaired social behavior, for example, difficulty interpreting social cues. Patients with FASD remain often undiagnosed due to lack of biomarkers, and treatment is unavailable because the mechanisms of the disease are not yet understood. Animal models have been proposed to facilitate addressing these problems. More recently, short exposure of the zebrafish embryo to low concentrations of alcohol was shown to lead to significant and lasting impairment of behavior in response to social stimuli. The impairment may be the result of abnormal social behavior or altered fear/anxiety. The goal of the current study was to investigate the latter.METHODS:Here, we employed the alcohol exposure regimen used previously (exposure of 24th hour postfertilization embryos to 0.00, 0.25, 0.50, 0.75, or 1.00% vol/vol alcohol for 2 hours), allowed the fish to reach adulthood, and measured the behavioral responses of these adults to a novel tank (anxiety-related behaviors) as well as to an animated image of a sympatric predator of zebrafish (fear-related behaviors).RESULTS:We found behavioral responses of embryonic alcohol-exposed adult fish to remain statistically indistinguishable from those of controls, suggesting unaltered anxiety and fear in the embryonic alcohol-treated fish.CONCLUSIONS:Given that motor and perceptual function was previously shown to be also unaltered in the adults after embryonic alcohol exposure, our current results suggest that the impaired response of these fish to social stimuli may be the result of abnormal social behavior.

Background: The zebrafish has been a favorite of developmental biologists and geneticists for decades, however, recently, it has gained popularity among behavior researchers too. The reason for the popularity of this species is that while it is a simple vertebrate, it possesses numerous features that make it translationally relevant. Objective: Social interaction is an essential component of human as well as zebrafish behavior; yet, the biological mechanisms underlying social behavior and its dysfunction remain poorly understood in both species. This review focuses on pharmacological manipulation of social behavior in zebrafish, studies where the ultimate goal is to understand the psychopharmacology of human behavior. Methods: We focused specifically on quantification of social interaction, specifically shoaling, in zebrafish, and compared it to rodent literature where appropriate. If no social studies exist for specific classes of compounds in zebrafish or if other behaviors may help with interpretation of social responses, we also highlighted non-social behaviors (locomotion, anxiety, etc). Results: We discussed findings on the effect of exposure of zebrafish to alcohol, psychostimulants, depressants and other drugs of abuse. Comparison of studies that have quantified and manipulated zebrafish social behavior of either single zebrafish or groups of zebrafish high-light both growth and gaps in this rapidly evolving field. Conclusion: Although new in psychopharmacology research, the zebrafish will be an important tool with which analysis and modeling of human social behavior and disorders involving social abnormalities including Fetal Alcohol Spectrum Disorders, Autism spectrum disorders, and Schizophrenia may be facilitated. Keywords: Zebrafish, social behavior, alcohol, psychostimulants, antidepressants, anxiolytics, shoaling.

Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapidly, for example, in the brain in response to environmental stimuli. c-fos is an IEG often used as a marker of neuronal activation. c-fos mRNA expression has started to be quantified and localized in the zebrafish brain following environmental manipulations but analysis of the expression of c-fos protein in the zebrafish brain has rarely been attempted. Here, we describe an immunofluorescence staining method for quantifying c-fos protein expression in different regions of the zebrafish brain. In addition, we expose zebrafish to caffeine, a positive control for c-fos activation in the brain. To confirm cell nucleus specific binding of the c-fos antibody, we counterstained brain sections with the nuclear fluorescent stain DAPI. Furthermore, we describe a method for reducing background autofluorescence often observed in zebrafish brain tissue. Our analysis showed that exposure to caffeine increased the number of c-fos protein-positive cells in specific zebrafish brain regions detected by the immunofluorescence method. Our results demonstrate the feasibility of immunofluorescence-based methods in the analysis of neuronal activation in the zebrafish brain, and reinforce the utility of the zebrafish in behavioral neuroscience research.