Zebrafish ( Danio rerio ) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish ‘ do ’, and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.
Morpholino oligonucleotides are the most common anti-sense "knockdown'' technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.
Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo , whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.
The zebrafish, Danio rerio, has emerged as a major model organism for biomedical research, yet little is known about its natural history. We review the literature pertaining to the geographic range, biotic and abiotic habitats, and life cycle of the zebrafish. We also report our own field study to document several aspects of zebrafish natural history across sites in northeast India. We found zebrafish particularly abundant in silt-bottomed, well-vegetated pools and rice paddies adjacent to slow moving streams at a range of elevations. We further identified co-occurring fishes likely to be zebrafish competitors and predators. Finally, we present observations that indicate substantial habitat degradation and loss, and suggest guidelines for documenting and preserving natural zebrafish populations.
Zebrafish ( Danio rerio ) has been a widely used vertebrate animal model in developmental biology and behavioral neuroscience, but knowledge about some of its basic behaviors, for example, light/dark preference, is still controversial. Appropriate preference for light or dark environments can be crucial for an animal's survival, so we hypothesize that zebrafish may have its light/dark preference varied with the circadian clock. In this present work, we tested the hypothesis by recording the light/dark preference in a two-compartment tank continuously for over 60 h. We found that the light avoidance of fish generally increases with time from morning (8:00am) to midnight (2:00am), and then decreases with time from midnight (2:00am) to morning (8:00am), exhibiting a clear circadian-like trend in the light/dark preference. As melatonin can mediate circadian clock output and promote sleep in zebrafish, by adding extra melatonin at around 9:00am on the third day, the mean proportion of time fish spent in the dark area was increased significantly. Our results demonstrate that the circadian clock plays a significant role in regulating the light/dark preference in zebrafish, which provides valuable insights into understanding the metabolism mechanism underlying the neurobehavior, and facilitate further studies related to the neurobiology of normal and pathological behavior.
Wild zebrafish exhibit a wide range of behavior. We found abundant wild zebrafish in flowing rivers and still water, in large, tightly-knit groups of hundreds of individuals, as well as in small, loose shoals. In two still-water populations, zebrafish were quite small in body size, common, and in tight groups of up to 22 fish. As in earlier laboratory studies, these zebrafish exhibited very low levels of aggression. In slowly flowing water in central India, zebrafish were relatively rare and gathered in small shoals (4–12 fish), often with other small fish, such as Rasbora daniconius . These stream zebrafish were larger in body size (27 mm TL) and much more aggressive than those in still water. In a second river population with much faster flowing water, zebrafish were abundant and again relatively large (21 mm TL). These zebrafish occurred in very large (up to 300 individuals) and tightly-knit (nearest-neighbor distances up to 21 mm) groups that exhibited collective rheotaxis and almost no aggression. This remarkable variation in social behavior of wild zebrafish offers an opportunity for future studies of behavioral genetics, development, and neuroscience.
We are entering a new era in our ability to modify and edit the genomes of model organisms. Zinc finger nucleases (ZFNs) opened the door to the first custom nuclease-targeted genome engineering in the late 1990s. However, ZFNs remained out of reach for most research labs because of the difficulty of production, high costs, and modest efficacy in many applications. Transcription activator-like effector nucleases (TALENs) were built upon a DNA binding system discovered in a group of plant bacterial pathogens and broadened custom nuclease technology, showing significant improvements in both targeting flexibility and efficiency. Perhaps most importantly, TALENs are open source and easy to produce, providing zebrafish laboratories around the world with affordable tools that can be made in-house rapidly, at low cost, and with reliably high activity. Now a new system for targeted genome engineering derived from the CRISPR/Cas system in eubacteria and archaea promises to simplify this process further. Together, these tools will help overcome many of the bottlenecks that have constrained gene targeting in zebrafish, paving the way for advanced genome engineering applications in this model teleost.
The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish.
Wild-type (WT) zebrafish are commonly used in behavioral tests, but the term WT is not a precise description, and corresponds to many different strains (e.g., AB, TU, WIK, and others). Previous studies compared the physiological, behavioral, or metabolic characteristics of different zebrafish strains (indigenous WT populations versus laboratory WT strains). AB and TU are widely used, but at least one study has demonstrated behavioral differences between them. To choose the most appropriate strain for our experiments, we systematically screened behavioral responses of AB and TU fish in several assays. We analyzed the locomotion activity and responses to a light/dark challenge in adults and larvae, and exploratory behavior and color conditioning in adults. Differences were observed for all tests, the strains displaying particular behavior depending on the tests. As larvae, TU displayed a wider activity range than AB larvae at the onset of locomotor behavior; as adults, TU were more reactive to sudden light transitions and recovered the swimming activity faster in T-maze or homebase release in novel tank tests, whereas AB fish had more contrasted circadian rhythms and performed better in color learning. Strain-specific behavior should be considered when designing experiments using behavior.
Emotional disturbances constitute a major health issue affecting a considerable portion of the population in western countries. In this context, animal models offer a relevant tool to address the underlying biological determinants and to screen novel therapeutic strategies. While rodents have traditionally constituted the species of choice, zebrafish are now becoming a viable alternative. As zebrafish gain momentum in biomedical sciences, considerable efforts are being devoted to developing high-throughput behavioral tests. Here, we present a comparative study of zebrafish behavioral response to fear-evoking stimuli offered via three alternative methodologies. Specifically, in a binary-choice test, we exposed zebrafish to an allopatric predator Astronotus ocellatus , presented in the form of a live subject, a robotic replica, and a computer-animated image. The robot's design and operation were inspired by the morphology and tail-beat motion of its live counterpart, thereby offering a consistent three-dimensional stimulus to focal fish. The computer-animated image was also designed after the live subject to replicate its appearance. We observed that differently from computer-animated images, both the live predator and its robotic replica elicited robust avoidance response in zebrafish. In addition, in response to the robot, zebrafish exhibited increased thrashing behavior, which is considered a valid indicator of fear. Finally, inter-individual response to a robotic stimulus is more consistent than that shown in response to live stimuli and animated images, thereby increasing experimental statistical power. Our study supports the view that robotic stimuli can constitute a promising experimental tool to elicit targeted behavioral responses in zebrafish.
Astyanax mexicanus , a teleost fish comprising both sighted river-dwelling and blind cave-dwelling morphs, is becoming increasingly used in the field of developmental and evolutionary biology. Thus, new experimental and technological tools are needed on this emerging fish model by the expanding scientific community. Here, we describe Astyanax husbandry and egg spawning habits, a prerequisite to the successful establishment of Astyanax transgenic lines. We then compare two different transgenesis methods on both surface and cave Astyanax . Both meganuclease (I-SceI)- and transposase (Tol2)-mediated transgenesis are equivalently efficient, resulting in ∼40% mosaic transgenic fish in F0. Furthermore, the transmission rate was analyzed in F1 in the case of the I-SceI method and was found to be 16%. Finally, the transgene was found stable up the F3 generation, demonstrating the feasibility of generating stable transgenic lines in Astyanax and opening a wide range of possibilities for this fish model.
Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.
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.
For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals.
Zebrafish larvae ( Danio rerio ) are increasingly used to translate findings regarding drug efficacy and safety from in vitro -based assays to vertebrate species, including humans. However, the limited understanding of drug exposure in this species hampers its implementation in translational research. Using paracetamol as a paradigm compound, we present a novel method to characterize pharmacokinetic processes in zebrafish larvae, by combining sensitive bioanalytical methods and nonlinear mixed effects modeling. The developed method allowed quantification of paracetamol and its two major metabolites, paracetamol-sulfate and paracetamol-glucuronide in pooled samples of five lysed zebrafish larvae of 3 days post-fertilization. Paracetamol drug uptake was quantified to be 0.289 pmole/min and paracetamol clearance was quantified to be 1.7% of the total value of the larvae. With an average volume determined to be 0.290 μL, this yields an absolute clearance of 2.96 × 10 7 L/h, which scales reasonably well with clearance rates in higher vertebrates. The developed methodology will improve the success rate of drug screens in zebrafish larvae and the translation potential of findings, by allowing the establishment of accurate exposure profiles and thereby also the establishment of concentration–effect relationships.
The adult zebrafish has the potential to become an important model for diabetes-related research. To realize this potential, small-scale methods for analyzing pancreas function are required. The measurement of blood glucose level is a commonly used method for assessing beta-cell function, but the small size of the zebrafish presents challenges both for collecting blood samples and for measuring glucose. We have developed methods for collecting microsamples of whole blood and plasma for the measurement of hematocrit and blood glucose. We demonstrate that two hand-held glucose meters designed for use by human diabetics return valid results with zebrafish blood. Additionally, we present methods for fasting and for performing postprandial glucose and intraperitoneal glucose tolerance tests. We find that the dynamics of zebrafish blood glucose homeostasis are consistent with patterns reported for other omnivorous teleost fish.
To adequately connect zebrafish medical models to human biology, it is essential that gene nomenclature reflects gene orthology. Analysis of gene phylogenies and conserved syntenies shows that the zebrafish gene currently called wnt11 (ENSDARG00000004256, ZFIN ID: ZDB-GENE-990603-12) is not the ortholog of the human gene called WNT11 (ENSG00000085741); instead, the gene currently called wnt11r (ENSDARG00000014796, ZFIN ID: ZDB-GENE-980526-249) is the zebrafish ortholog of human WNT11. Genomic analysis of Wnt11- family genes suggests a model for the birth of Wnt11- family gene ohnologs in genome duplication events, provides a mechanism for the death of a Wnt11- family ohnolog in mammals after they diverged from birds, and suggests revised nomenclature to better connect teleost disease models to human biology.
Wertheimerinae is a small subfamily of thorny catfish composed of two species found in eastern Brazilian coastal drainages: Wertheimeria maculata and Kalyptodoras bahiensis . According to molecular phylogenetic analysis, Franciscodoras marmoratus an endemic species of the São Francisco River is also a member of this subfamily. Even though both phylogenetic approaches suggest that this group is one of the oldest lineages of the Doradidae, a disagreement remains about the constitution of Wertheimerinae. Hence, cytogenetic analysis is important to understand the karyotypic evolution of thorny catfish and can be a useful cytotaxonomic tool to clarify the relationships between these species. All Wertheimerinae species, and F. marmoratus here analyzed, shared 2n = 58 chromosomes, karyotypic formulas (24m+12sm +8st +14a), and nucleolus organizer region (NOR) pattern (terminal 18S rDNA sites on pair 22). Differences were noted in heterochromatin and 5S rDNA site distribution. The chromosomal markers here applied added to the molecular data, reinforcing that these three species actually represent a well-resolved taxonomic unit. Our results represent one more evidence of the ancient connectivity between eastern coastal drainages and São Francisco River, whose separation represented an important event for the allopatric speciation that produced the current forms of Wertheimerinae subfamily.