Reproducibility and replicability remain a challenge in behavioural neuroscience, including zebrafish research. One source of this issue may stem from differences in how we raise and keep these fish in the laboratory. While there are efforts to standardize husbandry conditions, one potentially important source of variability is often ignored: human-animal interaction. In this opinion piece, we argue that routine husbandry procedures such as netting, feeding, tank cleaning and water changes can constitute repeated acute stressors that can lead to chronic alteration of zebrafish physiology and behaviour. These human-induced stress effects are known to interact with experimental treatments in other species, and thus are expected to confound results obtained with zebrafish too. Furthermore, timing of maintenance tasks, including human handling, and other temporal factors can introduce systematic facility-, study- or handler-specific effects. We suggest that these unreported human-animal interactions and unstandardized temporal maintenance patterns represent hidden variables that can undermine reproducibility of data, and argue that recognition and documentation of these factors are necessary for improving the reliability of zebrafish research.

The zebrafish is a frequently employed model organism in neuroscience. Behavioural analysis is often a critical component of neuroscience studies. Issues with replicability and reproducibility of experimental results have been in the focus in several fields of biology. In behavioural studies, one of the possible reasons for such issues is experimenter experience, i.e., the ability of the experimenter to properly quantify behaviour of their experimental animals. In this proof-of-concept study, we investigate how different levels and types of experimenter experience may influence results when the experimenter employs automated video-tracking or observation-based software to quantify behaviour of zebrafish. For example, we investigated whether experience with the particular software application, or with maintenance of zebrafish, or with zebrafish behaviour makes a difference. To answer these questions, multiple students, with different levels and types of prior experience, analyzed the video-recordings of the same 18 zebrafish. Subsequently, we analyzed the correlation coefficients between distinct experience types/levels and a variety of behavioural measures. We found that experience in zebrafish maintenance or behaviour did not correlate significantly with any behavioural measures quantified using video-tracking, but level of experience with the video-tracking software did with some measures. Conversely, experience with zebrafish maintenance and with zebrafish behaviour significantly correlated with some behaviours measured using the observation-based method, whereas level of experience with the software employed for this method did not. We conclude that the effect of the level and type of experience the experimenter has may affect results in a quantification method-dependent and behaviour-specific manner in the analysis of zebrafish behaviour.

Fetal alcohol spectrum disorders (FASD) result from exposure to alcohol (ethanol) during embryonic development. These diseases cause lifelong struggle for the affected patients. Due to the complex nature of how alcohol affects embryonic development, understanding of underlying mechanisms is lacking and treatment options are limited. Reliable diagnostic markers are also unavailable. As a start to bridge this hiatus, animal models have been proposed. One of the most recent ones among these animal models is the zebrafish. In this review, I focus on our own efforts that attempted to model the milder and most prevalent end of the spectrum of this disorder using zebrafish. We discovered that a short period (2 h-long) exposure of the zebrafish embryo to low doses of alcohol (up to 1% vol/vol external bath) at 24th hour post-fertilization led to a lifelong and dose-dependent impairment of social behavior (shoaling) in zebrafish, associated with an apparently selective disruption of dopaminergic neurotransmitter system responses. Here I review these findings and, for example, discuss how analysis of the neurochemistry of the zebrafish brain may aid our understanding of the mechanisms underlying embryonic alcohol-induced abnormalities. I theorize about how a non-selective and pharmacologically complex drug like alcohol may lead to the apparently selective impairment in shoaling and dopaminergic responses in zebrafish. Last, I briefly delineate future plans that may address questions including what specific brain areas, synaptic and molecular mechanisms may underlie the behavioral and neurochemical effects of embryonic alcohol exposure we have observed in zebrafish.

A key requirement of associative learning studies is the ability to motivate the subject to acquire memory of the conditioned stimulus-unconditioned stimulus (CS-US) association. Although zebrafish have been found capable of acquiring CS-US associative memory, in many studies, the fish failed to learn. One reason for the failure, I argue in this perspective article, is that we do not yet know how to motivate zebrafish. I illustrate this problem using examples, and offer some solutions, based upon results obtained in my own laboratory for appetitive associative learning tasks for zebrafish. I highlight the value of considering the ethology and ecology of the zebrafish. I discuss why food may have been an ineffective US for zebrafish. I provide examples for how to improve the rewarding properties of food based upon the foraging behaviour of zebrafish in nature. I discuss the efforts to identify alternative USs, including the sight of conspecifics or the presence of other ecologically relevant stimuli. I theorize about conflicting motivators in zebrafish learning studies, including the effect of human handling versus that of experimenter-controlled USs. I conclude that systematic analyses of different USs are needed, along with detailed studies on how they may be optimized for the analysis of learning and memory in zebrafish.

Despite decades of research with laboratory rodents, the mechanisms underlying learning and memory, and their impairment, are still not fully understood. The zebrafish is a newcomer in this research area, but it has shown great promise. Food is often employed as a reinforcer in learning tasks with rodents. However, for zebrafish, food has been a problematic reinforcer. Controlling timing and localization of its delivery is difficult. What food types zebrafish prefer is also rarely studied? Here, we describe a novel food delivery hardware and procedure. The apparatus is simple, cheap to manufacture, and easy to employ. Using this new method, we compare how zebrafish respond to three food types, artemia nauplii, crushed tropical fish flakes, and small zebrafish pellets. In binary choice tasks, we show that zebrafish spend significantly more time near the artemia delivery cylinder, swim closer to, and visit this cylinder more frequently compared to food cylinders delivering flakes or pellets, while responses to these latter two cylinders do not differ from each other. We conclude that the newly developed method allows the quantification of food preference in zebrafish, and that it will lead to the identification of highly rewarding food types for learning studies in this species.

This short review appears in a special issue assembled to celebrate the 90th birthday of a Hungarian ethologist, Professor Vilmos Csányi. As such, it includes some autobiographical details specific to that scientist and the author of this review. However, these details also serve an important general message. They exemplify how science, i.e., specifically the use of fish in the analysis of behaviour and brain function progressed from the mid-1970s to the current day. They illuminate how scientists choose their study species, and how this choice influences the research questions one may be able to pose. The review discusses why the zebrafish has become a popular research subject of biology, including behavioural neuroscience. It argues that behavioural analysis should be an integral part of research into the analysis of brain function. It considers the dichotomy between the historical effect of North American behaviourism vs. the legacy of European Nobel laureate ethologists. It demonstrates, through a theoretical example, why merging these two "schools" of thoughts is the appropriate way to conduct behavioural research. It provides a few examples for how combining knowledge of ethology and ecology of the species with systematic laboratory studies may be beneficial. And it presents a brief outlook for the future of fish in biology research.

Analysis of behaviour was the first approach with which the functioning of the brain could be studied in the distant past. The simple beginnings have led to one of the most sophisticated, fast-paced, burgeoning fields of biology research, neuroscience. Analysis of behaviour remains an integral part of neuroscience, and has led to the formation of the subfield of behavioural neuroscience. Here, I focus on this subfield, and specifically on the role of research with fish in it. I review why behavioural analysis is important for brain research and why studying this cluster of phenotypes with fish is particularly important and useful. I discuss the use of zebrafish for modeling human brain disorders including anxiety, addiction, neurodegenerative diseases and sleep disorders. I also review why extending behavioural neuroscience research to other fish species may benefit brain research. Last, I speculate about the near future, and discuss novel concepts and techniques, new directions behavioural neuroscience research with fish may take. The review is not comprehensive and reflects my own personal biases. Many of the examples are drawn from studies conducted in my own laboratory. Nevertheless, I hope that the examples I discuss will provide a reasonable snapshot of the current state-of-affairs and of the proximate future of behavioural neuroscience studies with fish, and that they will persuade the reader to use these simple vertebrates, and benefit from the reductionist approach they offer.

No, we are not talking about a new dish for dinner, but a study representing an emerging research field. Psilocybin research is gaining momentum, and zebrafish behavioral neuroscience research has been exponentially expanding. At the intersection of these two research fields is a recent paper that utilized high-tech video-tracking to detect behavioral changes induced by this psychoactive drug in larval zebrafish.

Functional changes in dopamine transporter (DAT) are related to various psychiatric conditions, including bipolar disorder (BD) symptoms. In experimental research, the inhibition of DAT induces behavioral alterations that recapitulate symptoms found in BD patients, including mania and depressive mood. Thus, developing novel animal models that mimic BD-related conditions by pharmacologically modulating the dopaminergic signaling is relevant. The zebrafish (Danio rerio) has been considered a suitable vertebrate system for modeling BD-like responses, due to the well-characterized behavioral responses and evolutionarily conservation of the dopaminergic system of this species. Here, we investigate whether GBR 12909, a selective inhibitor of DAT, causes neurobehavioral alterations in zebrafish similar to those observed in BD patients. Behaviors were recorded after a single intraperitoneal (i.p.) administration of GBR 12909 at different doses (3.75, 7.5, 15 and 30 mg/kg). To observe temporal effects on behavior, swim path parameters were measured immediately after the administration period during 30 min. Locomotion, anxiety-like behavior, social preference, aggression, despair-like behavior, and oxidative stress-related biomarkers in the brain were measured 30 min post administration. GBR 12909 induced prominent effects on locomotor activity and vertical exploration during the 30-min period. Hyperactivity was observed in GBR 30 group after 25 min, while all doses markedly reduced vertical drifts. GBR 12909 elicited hyperlocomotion, anxiety-like behavior, decreased social preference, aggression, and induced depressive-like behavior in a behavioral despair task. Depending on the dose, GBR 12909 also decreased SOD activity and TBARS levels, as well as increased GR activity and NPSH content. Collectively, our novel findings show that a single GBR 12909 administration evokes neurobehavioral changes that recapitulate manic- and depressive-like states observed in rodents, fostering the use of zebrafish models to explore BD-like responses in translational neuroscience research.

Learning and memory related brain disorders represent a large unmet medical need. Laboratory studies with animals may model brain disorders and facilitate uncovering their mechanisms. The zebrafish has been proposed for such studies. However, numerous factors that influence performance in learning tasks have yet to be understood in zebrafish. One such factor is what motivates zebrafish. Here we introduce a novel reinforcer, an ecologically relevant unconditioned stimulus (US). We placed a photograph of gravel underneath quarter of the bottom of an experimental tank on one side and also positioned artificial plants there, the “natural” US. First, we showed that this stimulus was preferred by zebrafish. Next, we investigated whether this stimulus could serve as US for associative learning. We marked the walls of the tank on the side where the US was presented with red paper, the conditioned stimulus (CS+) we found neutral before, and we also marked the walls on the other side of the tank where no US was placed with blue paper (CS-). In addition to fish receiving this “paired” training, we also ran unpaired training with another group of zebrafish, in which the fish saw the US associated with blue and red in a random manner. After having trained the fish in this manner, we tested the performance of the paired and unpaired group of zebrafish in a memory probe trial during which no US was present, and only the CSs (blue and red walls) were shown. We found the paired group of zebrafish to show significant preference for the CS+, as they spent more time and swam closer to the red side compared to the unpaired group and compared to chance. We conclude that ecologically relevant stimuli can serve as efficient US in appetitive conditioning of zebrafish.

Although zebrafish (Danio rerio) neuroscience research is rapidly expanding, the fundamental question of how these fish should be maintained in research laboratories remains largely unstudied. This may explain the diverse practices and broad range of environmental parameters used in zebrafish facilities. Here, we provide examples of these parameters and practices, including housing density, tank size, and water chemistry. We discuss the principles of stochastic resonance versus homeostasis and provide hypothetical examples to explain why keeping zebrafish outside of their tolerated range of environmental parameters may increase phenotypical variance and reduce replicability. We call for systematic studies to establish the optimal maintenance conditions for zebrafish. Furthermore, we discuss why knowing more about the natural behavior and ecology of this species could be a guiding principle for these studies.

OBJECTIVE:Intellectual disability is often the outcome of neurodevelopmental disorders and is characterized by significant impairments in intellectual and adaptive functioning. X-linked intellectual disability (XLID) is a subset of these disorders caused by genetic defects on the X chromosome, affecting about 2 out of 1,000 males. In syndromic form, it leads to a broad range of cognitive, behavioral, ocular, and physical disabilities. METHODS:Employing exome or genome sequencing, here we identified 4 missense variants (c.475C > G; p.H159D, c.1373C > A; p.T458N, and c.1585G > A; p.E529K, c.953C > T; p.S318L) and a putative truncating variant (c.1413_1414del; p.Y471*) in the SRPK3 gene in 9 XLID patients from 5 unrelated families. To validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish. RESULTS:The 8 patients ascertained postnatally shared common clinical features including intellectual disability, agenesis of the corpus callosum, abnormal eye movement, and ataxia. A ninth case, ascertained prenatally, had a complex structural brain phenotype. Together, these data indicate a pathological role of SRPK3 in neurodevelopmental disorders. In post-fertilization day 5 larvae (free swimming stage), KO zebrafish exhibited severe deficits in eye movement and swim bladder inflation, mimicking uncontrolled ocular movement and physical clumsiness observed in human patients. In adult KO zebrafish, cerebellar agenesis and behavioral abnormalities were observed, recapitulating human phenotypes of cerebellar atrophy and intellectual disability. INTERPRETATION:Overall, these results suggest a crucial role of SRPK3 in the pathogenesis of syndromic X-linked intellectual disability and provide new insights into brain development, cognitive and ocular dysfunction in both humans and zebrafish. ANN NEUROL 2024;96:914-931.

Various methods have been used in rodents to evaluate learning and memory. Although much less frequently used, the zebrafish emerges as an alternative model organism in this context. For example, it allows assessing potential behavioral deficits because of neurodevelopmental disorders or environmental neurotoxins. A variety of learning tasks have been employed in previous studies that required extensive habituation and training sessions. Here, we introduce a simpler and faster method to evaluate learning and memory of zebrafish with minimum habituation. A new apparatus, a transparent L-shaped tube, was developed in which we trained each zebrafish to swim through a long arm and measured the time to swim through this arm. We demonstrate that in this task, zebrafish could acquire both short-term (1 h) and long-term memory (4 days). We also studied learning and memory of a gene knockout (KO) zebrafish that showed social impairments related to autism. We found KO mutant zebrafish to show a quantitative impairment in habituation, learning, and memory performance compared with wild-type control fish. In conclusion, we established a novel learning apparatus and sensitive paradigm that allowed us to evaluate learning and memory of adult zebrafish that required only a brief habituation period and minimal training.

The zebrafish is a laboratory species that gained increasing popularity the last decade in a variety of subfields of biology, including toxicology, ecology, medicine, and the neurosciences. An important phenotype often measured in these fields is behaviour. Consequently, numerous new behavioural apparati and paradigms have been developed for the zebrafish, including methods for the analysis of learning and memory in adult zebrafish. Perhaps the biggest obstacle in these methods is that zebrafish is particularly sensitive to human handling. To overcome this confound, automated learning paradigms have been developed with varying success. In this manuscript, we present a semi-automated home tank-based learning/memory test paradigm utilizing visual cues, and show that it is capable of quantifying classical associative learning performance in zebrafish. We demonstrate that in this task, zebrafish successfully acquire the association between coloured-light and food reward. The hardware and software components of the task are easy and cheap to obtain and simple to assemble and set up. The procedures of the paradigm allow the test fish to remain completely undisturbed by the experimenter for several days in their home (test) tank, eliminating human handling or human interference induced stress. We demonstrate that the development of cheap and simple automated home-tank-based learning paradigms for the zebrafish is feasible. We argue that such tasks will allow us to better characterize numerous cognitive and mnemonic features of the zebrafish, including elemental as well as configural learning and memory, which will, in turn, also enhance our ability to study neurobiological mechanisms underlying learning and memory using this model organism.

The dopaminergic neurotransmitter system is implicated in several brain functions and behavioral processes. Alterations in it are associated with the pathogenesis of several human neurological disorders. Pharmacological agents that interact with the dopaminergic system allow the investigation of dopamine-mediated cellular and molecular responses and may elucidate the biological bases of such disorders. Zebrafish, a translationally relevant biomedical research organism, has been successfully employed in prior psychopharmacology studies. Here, we evaluated the effects of quinpirole (dopamine D2/D3 receptor agonist) in adult zebrafish on behavioral parameters, brain-derived neurotrophic factor (BDNF) and neurotransmitter levels. Zebrafish received intraperitoneal injections of 0.5, 1.0, or 2.0 mg/kg quinpirole or saline (control group) twice with an inter-injection interval of 48 h. All tests were performed 24 h after the second injection. After this acute quinpirole administration, zebrafish exhibited decreased locomotor activity, increased anxiety-like behaviors and memory impairment. However, quinpirole did not affect social and aggressive behavior. Quinpirole-treated fish exhibited stereotypic swimming, characterized by repetitive behavior followed by immobile episodes. Moreover, quinpirole treatment also decreased the number of BDNF-immunoreactive cells in the zebrafish brain. Analysis of neurotransmitter levels demonstrated a significant increase in glutamate and a decrease in serotonin, while no alterations were observed in dopamine. These findings demonstrate that dopaminergic signaling altered by quinpirole administration results in significant behavioral and neuroplastic changes in the central nervous system of zebrafish. Thus, we conclude that the use of quinpirole administration in adult zebrafish may be an appropriate tool for the analysis of mechanisms underlying neurological disorders related to the dopaminergic system.

In this review, we discuss the possible utility of zebrafish in research on psilocybin, a psychedelic drug whose recreational use as well as possible clinical application are gaining increasing interest. First, we review behavioral tests with zebrafish, focussing on anxiety and social behavior, which have particular relevance in the context of psilocybin research. Next, we briefly consider methods of genetic manipulations with which psilocybin's phenotypical effects and underlying mechanisms may be investigated in zebrafish. We briefly review the known mechanisms of psilocybin, and also discuss what we know about its safety and toxicity profile. Last, we discuss examples of how psilocybin may be employed for testing treatment efficacy in preclinical research for affective disorders in zebrafish. We conclude that zebrafish has a promising future in preclinical research on psychedelic drugs.

Associative learning is often studied using food reward as the unconditioned stimulus (US). With warm-blooded species, to get the subject more motivated the solution has been to feed less, making the subject hungrier. Here we show the opposite with zebrafish. We randomly assigned zebrafish to two groups: a once-a-day-fed and a five -times-a-day-fed group, with the same amount of food fed per occasion for fish of both groups, a feeding regimen that lasted for three months. Subsequently, we trained fish by pairing food (US) with a red cue card (the conditioned stimulus, CS), which were placed together in one arm of a plus-maze across eight training sessions. We also ran unpaired training, in which the CS and US were presented in different arms. We found the previously once-a-day-fed zebrafish to consume less food throughout habituation and training sessions compared to the previously five-times-a-day-fed ones. Furthermore, five-times-a-day-fed fish in the paired group swam significantly closer to the CS during a post-training probe trial compared to the five-times-a-day-fed unpaired fish, a paired training effect that was absent in once-a-day-fed fish. Groups did not differ in health or general activity. In sum, elevated chronic feeding improved food consumption and enhanced learning and memory performance without affecting activity levels in adult zebrafish.

OPINION article Front. Behav. Neurosci., 10 January 2023Sec. Individual and Social Behaviors Volume 16 - 2022 | https://doi.org/10.3389/fnbeh.2022.1090248

Psychedelic drugs have experienced an unprecedented surge in recreational use within the past few years. Among recreational users, the risks of psychedelic use by pregnant and breastfeeding women are severely understudied and there is little information on the potential teratogenic effects of these drugs. We provide an overview of the previous data on psychedelic teratogenicity from rodent studies and human surveys, discuss their limitations, and propose the utility of the zebrafish as a potential effective model for investigating psychedelic teratogenicity. Recent years have validated the use of zebrafish in the study of fetal exposure and developmental biology; we highlight these properties of the zebrafish for its suitability in psychedelic toxicity research.

COPYRIGHT © 2023 Ortiz and Gerlai. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Editorial: Zebrafish as a tool for neurosciences: evolutionary conservation and translational relevance