A Simple Tube Escape Assay to Test Learning and Memory in Zebrafish with Minimized Habituation.

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 influence of acute dopamine transporter inhibition on manic-, depressive-like phenotypes, and brain oxidative status in adult 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.

A search for effective reinforcers in appetitive conditioning for adult zebrafish: Ecologically relevant unconditioned stimuli.

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.

A simple semi-automated home-tank method and procedure to explore classical associative learning in adult zebrafish.

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.

A perspective on psychedelic teratogenicity: the utility of zebrafish models.

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.

The zebrafish for preclinical psilocybin research.

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.

The ram cichlid (Mikrogeophagus ramirezi) learns an associative task: a new fish species for memory research.

Fish are the most species rich and evolutionarily oldest vertebrate taxon. This represents opportunities for biologists who intend to employ laboratory animals in their comparative or translational research. Yet, the overwhelming majority of such studies use a single fish species, the zebrafish, a suboptimal strategy from the comparative standpoint. Neuronal plasticity (learning and memory) is perhaps one of the most complex biological phenomena from a mechanistic standpoint, and thus its analysis could benefit from the use of evolutionarily ancient and simple vertebrate model organisms, i.e., fish species. However, learning & memory research with the zebrafish has been replete with problems. Here, we employ a novel fish species, the ram cichlid, we argue will be particularly appropriate for this purpose for practical as well as ethological/ecological reasons. First, we investigate whether the ram cichlid exhibits innate preference for certain colours (red, blue, yellow or green) in a four-choice task, the plus maze. Subsequently, we pair the apparently least preferred colour (green, the conditioned stimulus or CS) with food reward (the unconditioned stimulus, US) in the plus maze, a CS-US associative learning task. After eight pairing trials, we run a probe trial during which only the CS is presented. At this trial, we find significant preference to the CS, i.e., acquisition of memory of CS-US association. We argue that our proof-of-concept study demonstrating fast acquisition of CS-US association in the ram cichlid, coupled with the universal utility of some genome editing methods, will facilitate the mechanistic analysis of learning and memory.

Acute administration of a dopamine D2/D3 receptor agonist alters behavioral and neural parameters in adult zebrafish.

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.

Effects of different handling methods on the behavior of adult zebrafish.

The zebrafish is an important biomedical research organism. In most research, zebrafish are removed from their home tank and subsequently their phenotype is measured. The method of handling the fish, however, may significantly affect a variety of phenotypes. This is particularly problematic for studies of brain function that measure behavioral or neuronal responses. Nevertheless, the potential effects of handling have not been analyzed, and in fact are usually ignored. Here, we explore the effects of two usual and two rarely or never-before employed handling methods on the behavior of adult zebrafish. We exposed each fish to one of four handling methods, a between subject experimental design: (1) net chasing followed by air-suspension, (2) gentle net catching (without chasing) followed by air-suspension, (3) gentle net catching followed by being placed in a beaker (no chasing and very short air-suspension), (4) transportation in home tank and pouring the fish directly into the test tank (no chasing, netting or air-suspension). With these handling methods, the fish were placed in a test tank and their swim path was videorecorded and analyzed. Handling significantly affected swim path parameters, duration and frequency of immobility, absolute turn angle and its temporal variance and velocity, but not the distance to bottom. The behavioral effects confirmed that chasing and netting induce robust behavioral changes, and that pouring the fish from its home to its test tank is least aversive for zebrafish. We recommend using this latter method to reduce experimental error variation and increase reproducibility of results.

Zebrafish (Danio rerio): A newcomer with great promise in behavioral neuroscience.

Behavioral neuroscience is an interdisciplinary field aimed at understanding the neurobiology of behavior. Numerous investigators turn to animals to model and understand the mechanisms underlying vertebrate brain function including human brain disorders, species that share evolutionary history with us. The zebrafish is a relatively new study species for such purposes. However, as this review attempts to demonstrate, it will likely have a good future in behavioral neuroscience. It is a simple vertebrate that is small and cheap to keep and study in the laboratory. Yet, it is similar enough to our own species, thus, we are able to use it for both translational as well as basic research. In this invited review, I will discuss its advantages and some of its disadvantages, the reasons and counterarguments why it should or should not be employed in research. I will focus on its utility in behavioral neuroscience, and will also provide a brief historical account of the evolution between zebrafish research and the science represented by the International Behavioral Neuroscience Society.

Common Aquarium Plants as an Enrichment Strategy in Zebrafish Facilities.

An increasing number of laboratories utilize zebrafish as this species is now represented in practically every subfield of biology research. Environmental enrichment has been shown to improve welfare and health of a large number of species of animals studied in laboratories, kept in zoos, or used in agriculture. However, most laboratories keep zebrafish in barren tanks. Artificial and live aquatic plants have been used in a variety of contexts for aquarium fish, and they have also been suggested as a potential enrichment strategy for the zebrafish. However, no systematic studies have been conducted to investigate their beneficial effects in zebrafish husbandry and biology research. In this study, we review some of the potential benefits of using live plants, and argue that systematic analyses for proper choice and use of live plants in zebrafish husbandry are sorely needed. We provide a few examples of aquatic plant species out of the large variety produced for the aquarium hobby that could be adopted to the zebrafish laboratory setting as environmental enrichment. We speculate that once systematic analyses have been conducted, they will show benefits of this ethologically/ecologically relevant enrichment method, one of which will be healthier and less stressed fish leading to increased reproducibility of results in zebrafish research.

Acute Administration of Ethanol and of a D1-Receptor Antagonist Affects the Behavior and Neurochemistry of Adult Zebrafish.

Alcohol abuse represents major societal problems, an unmet medical need resulting from our incomplete understanding of the mechanisms underlying alcohol's actions in the brain. To uncover these mechanisms, animal models have been proposed. Here, we explore the effects of acute alcohol administration in zebrafish, a promising animal model in alcohol research. One mechanism via which alcohol may influence behavior is the dopaminergic neurotransmitter system. As a proof-of-concept analysis, we study how D1 dopamine-receptor antagonism may alter the effects of acute alcohol on the behavior of adult zebrafish and on whole brain levels of neurochemicals. We conduct these analyses using a quasi-inbred strain, AB, and a genetically heterogeneous population SFWT. Our results uncover significant alcohol x D1-R antagonist interaction and main effects of these factors in shoaling, but only additive effects of these factors in measures of exploratory behavior. We also find interacting and main effects of alcohol and the D1-R antagonist on dopamine and DOPAC levels, but only alcohol effects on serotonin. We also uncover several strain dependent effects. These results demonstrate that acute alcohol may act through dopaminergic mechanisms for some but not all behavioral phenotypes, a novel discovery, and also suggest that strain differences may, in the future, help us identify molecular mechanisms underlying acute alcohol effects.

The effect of fish density and tank size on the behavior of adult zebrafish: A systematic analysis.

The zebrafish has been employed in several fields of biology due to its translational relevance and its simplicity and ease of maintenance. As a result, zebrafish are kept in thousands of laboratories around the world. Current industry standards favor keeping the largest possible number of fish in the smallest possible volume of water to increase efficiency and reduce costs. However, physiological and psychological stress resulting from such crowding may impact a variety of phenotypes, from brain function and behavior to cardiovascular function and cancer. Nevertheless, surprisingly little is known about what constitutes an optimal housing environment for the zebrafish, e.g., no systematic analyses have been performed to test the role of housing density and tank volume despite recent sporadic reports implying negative effects of the standard practice of crowding. Here, we conduct the first proof of concept analysis examining the potential impact of housing density and tank volume on the behavior of zebrafish. We randomly assigned adult zebrafish to one of three tank sizes (1.5, 10, or 50 L) with one of three housing densities (1, 2, or 4 fish/L), a 3 × 3 between subject experimental design, and maintained the fish in their corresponding condition for 2 weeks. Subsequently, we tested the behavior of the fish singly in a novel open tank for 12 min and quantified several of their swim path parameters using a video-tracking system. We found significant additive and interacting effects of tank size and/or housing density on swim path parameters including immobility, swim speed, turn angle, and distance to bottom and to stimulus. Although we had only three fish densities and three tank sizes and we did not explore the effects of more extreme conditions and although the interpretation of the above behavioral effects is speculative at this point, the results already demonstrate that both tank size and housing density exerts significant effects on the zebrafish and thus should be considered in zebrafish husbandry.

Researchers, animal support and regulatory staff: symbiosis or antagonism?

Animals are studied en masse by biologists around the world in a variety of biomedical and basic research studies. All this research benefits humankind and animals alike as it tackles a wide variety of problems ranging from those of conservation biology to medicine. Research with animal subjects is a complex endeavor that requires the cooperation and collaboration of a large number of experts, from the principal investigator through technicians and vivarium staff to regulatory experts. The research must be conducted in a humane manner that adheres to acceptable practices regulated by local, state and federal guidelines, rules and the law. In this short opinion article, we examine the current state of affairs regarding how researchers, animal support staff and regulatory experts work together. We pay particular attention to potential conflicts that may arise from the occasionally distinct roles played by those involved in animal research, and we provide some suggestions as short- and long-term remedies that have not been previously discussed in the literature.

Age-dependent effects of embryonic ethanol exposure on anxiety-like behaviours in young zebrafish: A genotype comparison study.

Fetal Alcohol Spectrum Disorder (FASD) is characterized by a variety of morphological, behavioural and cognitive deficits, ranging from mild to severe. Numerous animal models, including the zebrafish, have been employed to better understand the onset, expression and progression of this disorder. Embryonic ethanol-induced deficits in learning and memory, anxiety, social responses and elevated alcohol self-administration have been successfully demonstrated in zebrafish. Studies in zebrafish have also shown the expression of these behavioural deficits depends upon the developmental stage of ethanol exposure, the age of observation, as well as the genotype (strain or population origin) of the tested zebrafish. Here, we investigate how the genotype and age of observation may influence embryonic ethanol-induced alterations in anxiety-like responses in zebrafish. Zebrafish embryos exposed to either 0% or 1% (vol/vol) ethanol at 24hpf were tested in an open tank at one of three stages: larval (6-8 days post fertilization (dpf)), mid-larval (16-18dpf), or juvenile (26-28dpf). Two genotypes were tested in this manner, ABNS (a quasi-inbred strain) and ABSK (a mix of AB, TU and TL strains). We found embryonic ethanol induced behavioural changes to significantly differ depending on the genotype and age of observation. For example, significant differences between control and ethanol exposed zebrafish in both genotypes were observed in juvenile zebrafish, but few significant treatment effects were observed in larval zebrafish. Additionally, ethanol appeared to alter anxiety-like behaviours in the ABNS genotype but did not have as robust of an effect on the ABSK genotype. Lastly, there were significant behavioural differences between unexposed (control) zebrafish of the two genotypes, suggesting baseline behavioural differences despite a common AB genetic origin.

Behavioral Effects of Buspirone in Juvenile Zebrafish of Two Different Genetic Backgrounds.

Anxiety continues to represent a major unmet medical need. Despite the availability of numerous anxiolytic drugs, a large proportion of patients do not respond well to current pharmacotherapy, or their response diminishes with chronic drug application. To discover novel compounds and to investigate the mode of action of anxiolytic drugs, animal models have been proposed. The zebrafish is a novel animal model in this research. It is particularly appropriate, as it has evolutionarily conserved features, and drug administration can be employed in a non-invasive manner by immersing the fish into the drug solution. The first step in the analysis of anxiolytic drugs with zebrafish is to test reference compounds. Here, we investigate the effects of buspirone hydrochloride, an anxiolytic drug often employed in the human clinic. We utilize two genetically distinct populations of zebrafish, ABSK, derived from the quasi-inbred AB strain, and WT, a genetically heterogeneous wild-type population. We placed juvenile (10-13-day, post-fertilization, old) zebrafish singly in petri dishes containing one of four buspirone concentrations (0 mg/L control, 5 mg/L, 20 mg/L or 80 mg/L) for 1 h, with each fish receiving a single exposure to one concentration, a between subject experimental design. Subsequently, we recorded the behavior of the zebrafish for 30 min using video-tracking. Buspirone decreased distance moved, number of immobility episodes and thigmotaxis, and it increased immobility duration and turn angle in a quasi-linear dose dependent but genotype independent manner. Although it is unclear whether these changes represent anxiolysis in zebrafish, the results demonstrate that behavioral analysis of juvenile zebrafish may be a sensitive and simple way to quantify the effects of human anxiolytic drugs.

Behavioral effects of acute ethanol in larval zebrafish (D. rerio) depend on genotype and volume of experimental well.

Ethanol consumption is a worldwide problem. Sensitivity to acute effects of ethanol influences the development of chronic ethanol abuse and ethanol dependence. Environmental and genetic factors have been found to contribute to differential effects of acute ethanol. Animal models have been employed to investigate these factors. An increasingly frequently utilized animal model in ethanol research is the zebrafish. A large proportion of ethanol studies with zebrafish have been conducted with adult zebrafish. However, high throughput drug and mutation screens are particularly well adapted to larval zebrafish. These studies are often carried out using the 96-well-plate that allows monitoring large numbers of fish efficiently. Here, we investigate the effects of acute (30 min long) ethanol exposure in 8-day post-fertilization (dpf) old zebrafish. We compare four genetically distinct populations (strains) of zebrafish, measuring numerous parameters of their swim path in two well sizes, i.e., in the 96-well-plate (small volume wells) and in the 6-well-plate (large volume wells). In general, we found that the highest dose of ethanol (1% vol/vol) reduced swim speed, increased duration of immobility, increased turn angle, and increased intra-individual variance of turn angle, while the intermediate dose (0.5%) had a less strong effect, compared to control. However, we also found that these ethanol effects were strain dependent and, in general, were better detected in the larger volume well. We conclude that larval zebrafish are appropriate for quantification of acute ethanol effects and also for the analysis of environmental and genetic factors that influence these effects. We also speculate that using larger wells will likely increase sensitivity of detection and precision in screening applications.

The Tapping Assay: A Simple Method to Induce Fear Responses in Zebrafish.

The zebrafish is increasingly employed in behavioral neuroscience as a translationally relevant model organism for human central nervous system disorders. One of the most prevalent CNS disorders representing an unmet medical need is the disorder cluster defined under the umbrella term anxiety disorders. Zebrafish have been shown to respond to a variety of anxiety and fear inducing stimuli and have been suggested for modeling human anxiety. Here, we describe a simple method with which we intend to induce fear/anxiety responses in this species. The method allows us to deliver a visual and lateral line stimulus (vibration or "tapping") to the fish with the use of a moving object, a ball colliding with the side glass of the experimental tank. We describe the hardware construction of the apparatus and the procedure of the behavioral paradigm. We also present data on how zebrafish respond to the tapping. Our results demonstrate that the method induces significant fear/anxiety responses. We argue that the simplicity of the method and the efficiency of the paradigm should make it popular among those who plan to use zebrafish as a tool in anxiety research.