Zebrafish Behavior Research Articles

Juvenile exposure to low-level 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavior and longitudinal morphometrics in zebrafish and F1 offspring.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental endocrine disruptor and model AhR agonist, is linked to skeletal abnormalities, cardiac edema, stunted growth rate, altered metabolism, and neurobehavioral deficits. We have previously reported transgenerational reproductive outcomes of developmental TCDD exposure in adult zebrafish (Danio rerio), an NIH-validated model for developmental and generational toxicology. Using the same paradigm of sublethal TCDD exposure (50 pg/ml) at both 3 and 7 weeks post fertilization (wpf), we investigated several novel endpoints, including longitudinal morphometrics and anxiety-linked behavior, in fish exposed as juveniles. We also assessed developmental abnormalities and neurobehavior in their F1 larval offspring. TCDD exposure induced timepoint-dependent decreases in several craniofacial and trunk morphometrics across juvenile development. In early adulthood, however, only exposed males underwent a transient period of compensatory growth, ending between 7 and 12 months post fertilization (mpf). At 12 mpf, exposed adult fish of both sexes displayed increased exploratory behaviors in a novel tank test. The F1 offspring of parents exposed at both 3 and 7 wpf were hyperactive, but neurobehavioral outcomes diverged depending on parental exposure window. F1 exposure-lineage larvae had increased rates of edema and skeletal abnormalities, but fewer unhatched larvae compared to controls. Parent- and timepoint-specific effects of exposure on abnormality rate were also evaluated; these outcomes were considerably less severe. Our novel behavioral findings expand current knowledge of the long-term and intergenerational consequences of early-life TCDD exposure in a zebrafish model, in addition to delineating minor longitudinal morphometric changes in exposed fish and abnormalities in larval offspring.

Acute clomipramine exposure elicits dose-dependent surfacing behavior in adult zebrafish (Danio rerio).

Chronic treatment with clomipramine, a tricyclic antidepressant drug, reduces symptoms of obsessive-compulsive disorder (OCD) and can influence the activity of the hypothalamic-pituitary-adrenal axis. However, little is known regarding the effects of acute clomipramine on the immediate expression of stress responses. Serotonergic drugs can elicit surfacing, a behavioral profile potentially related to toxicity in fish, although surfacing has not yet been observed after clomipramine exposure. The present study investigated the impact of acute exposure to clomipramine on basal and stress-induced behaviors in the novel tank test and cortisol levels in mixed-sex, wild-type, adult zebrafish (Danio rerio). The findings show clomipramine-exposed groups (regardless of stress exposure) spent much more time in the top of the novel tank and had significantly less overall motor activity in the behavioral task compared to the fish not exposed to the drug. Then, the dose-dependent effects of acute clomipramine on activity in the surface of the novel tank (top third of the top half) were investigated further. Clomipramine dose-dependently increased surface-dwelling and elicited a dose-dependent hypoactivity in overall motor behavior. There were no statistically significant differences in whole-body cortisol levels in either experiment. Like other serotonin-acting drugs, clomipramine strongly elicited surface-dwelling and depressed motor behavior in adult zebrafish. Additional testing is needed to elucidate whether surfacing represents a toxic state and how serotonin regulates surfacing.

Understanding the Intense Effects of Caloric Restriction and Overfeeding on Zebrafish (Danio Rerio) Behaviour and Biochemical Processes, Along With Their Implications for Offspring

Understanding the Intense Effects of Caloric Restriction and Overfeeding on Zebrafish (Danio Rerio) Behaviour and Biochemical Processes, Along With Their Implications for Offspring

Toxicity assessment of sub-acute silver nitrate exposure on adult zebrafish behaviour

Toxicity assessment of sub-acute silver nitrate exposure on adult zebrafish behaviour

VICR: A novel software for unbiased video and image analysis in scientific research.

In scientific research, objectivity and unbiased data analysis is crucial for the validity and reproducibility of outcomes. This is particularly important for studies involving video or image categorization. A common approach of decreasing the bias is delegating data analysis to researchers unfamiliar with the experimental settings. However, this requires additional personnel and is prone to cognitive biases. Here we describe the Video & Image Cutter & Randomizer (VICR) software (https://github.com/kkihnphd/VICR), designed for unbiased analysis by segmenting and then randomizing the segmented videos or still images. VICR allows a single researcher to conduct and analyze studies in a blinded manner, eliminating the bias in analysis and streamlining the research process. We describe the features of the VICR software and demonstrate its capabilities using zebrafish behavior studies. To our knowledge, VICR is the only software for the randomization of video and image segments capable of eliminating bias in data analysis in a variety of research fields.

Brain targeted luteolin-graphene oxide nanoparticle abrogates polyethylene terephthalate induced altered neurological response in zebrafish.

Polyethylene terephthalate (PET), a commonly used polymer in various food and plastic bag containers, has raised significant concerns regarding its environmental and human health risks. Despite its prevalent use, the impact of PET exposure on aquatic environments and its potential to induce neurotoxic conditions in species remain poorly understood. Furthermore, the mechanisms underlying amelioration through natural product intervention are not well-explored. In light of these gaps, our study aimed to elucidate the neurotoxic effects of PET in zebrafish through waterborne exposure, and to mitigate its neurological impact using luteolin-graphene oxide nanoparticles. Our investigation revealed that exposure to PET in water triggered adverse effects in zebrafish larvae, particularly in the head region. We observed heightened oxidative stress, lipid peroxidation, and cell death, accompanied by impaired antioxidant defense enzymes. Furthermore, abnormal levels of acetylcholine esterase and nitric oxide in the zebrafish brain indicated cognitive impairment. To address these issues, we explored the potential neuroprotective effects of luteolin-graphene oxide nanoparticles. These nanoparticles demonstrated efficacy in localizing within the zebrafish brain, enhancing their therapeutic impact against PET exposure. Treatment with luteolin-graphene oxide nanoparticles not only mitigated PET-induced neurological alterations but also exhibited a neuroprotective effect. This was evidenced by the regulation of pro-inflammatory cytokine gene expression in the zebrafish brain. Additionally, normalization of locomotory behavior in PET-exposed zebrafish following nanoparticle treatment underscored the potential effectiveness of luteolin-graphene oxide nanoparticles as a treatment against PET-induced neurotoxicity. In summary, our study emphasizes the urgent need to investigate the environmental and health risks associated with PET. We demonstrate the potential of luteolin-graphene oxide nanoparticles as an effective intervention against PET-induced neurotoxicity in zebrafish.

Translational neurobiology of zebrafish (<i>Danio rerio</i>)

The zebrafish (Danio rerio) is a small freshwater teleost fish species that is increasingly utilized in biomedical research, particularly in neuroscience and biological psychiatry. Currently, zebrafish is the second-most utilized model organism in biomedicine globally, after mice, based on the number of animals tested each year. The model’s significance results from its experimental ease of use, affordability, conservation of fish physiology, relatively high genetic homology with humans (70%), rapid development, and potential for high-throughput bioscreening of drugs and genetic mutations. Over the past 15 years, our laboratory has conducted extensive experimental work to establish the principles behind using zebrafish to study various brain pathologies, including acute and chronic stress, anxiety, and depression, as well as probing their molecular mechanisms. In addition, existing behavioral models to study the central nervous system (CNS) development and new data on the zebrafish’s critical role in memory research were reviewed. Furthermore, this study will illustrate the effectiveness of integrating zebrafish models with advanced biological research techniques, such as molecular biology, bioinformatics, omics technologies, and chemical biology methods. For example, adult zebrafish experiencing chronic stress exhibit behavioral affective syndromes along with changes in neurochemistry, specifically in the metabolism of serotonin and dopamine in the telencephalon. Moreover, alterations in the expression of genes regulating neurotransmitter receptors, glial biomarkers, cytoskeleton, and pro- and anti-inflammatory cytokines, occur in the brain. In particular, we will focus on neuroimmune and epigenetic mechanisms of CNS pathogenesis in zebrafish models, including changes in the expression of apoptotic genes in the brain. Additionally, our own findings on using artificial intelligence (AI) systems to study zebrafish behavior after administering various neurotropic drugs, such as anxiolytics, antidepressants, psychostimulants, and hallucinogens will be presented. In general, zebrafish is a strategic and promising model organism for translational neuroscience research, creating new models of CNS pathogenesis and finding new drugs to treat various human brain diseases. Studies of CNS pathogenesis in zebrafish are critical because of their evolutionary conservatism and ease of laboratory application, revealing novel brain disease biomarkers and potential remediation targets. Meanwhile, certain distinctive aspects of the biology and neurophysiology of zebrafish facilitate the resolution of supplementary experimental issues, thereby enhancing data and discoveries attained in typical animal models using rodents.

Environmental levels of azoxystrobin disturb male zebrafish behavior: Possible roles of oxidative stress, cholinergic system, and dopaminergic system

A widely applied pesticide of azoxystrobin, is increasingly detected in the water environment. Concern has been raised against its potential detriment to aquatic ecosystems. It has been shown that exposure to azoxystrobin interfere with the locomotor behavior of zebrafish larvae. This study aims to investigate whether exposure to environmental levels of azoxystrobin (2 μg/L, 20 μg/L, and 200 μg/L) changes the behavior of male adult zebrafish. Herein, we evaluated behavioral response (locomotor, anxiety-like, and exploratory behaviors), histopathology, biochemical indicators, and gene expression in male adult zebrafish upon azoxystrobin exposure. The study showed that exposure to azoxystrobin for 42 days remarkably increased the locomotor ability of male zebrafish, resulted in anxiety-like behavior, and inhibited exploratory behavior. After treatment with 200 μg/L azoxystrobin, vasodilatation, and congestion were observed in male zebrafish brains. Exposure to 200 μg/L azoxystrobin notably elevated ROS level, MDA concentration, CAT activity, and AChE activity, while inhibiting SOD activity, GPx activity, ACh concentration, and DA concentration in male zebrafish brains. Moreover, the expression levels of genes related to the antioxidant, cholinergic, and dopaminergic systems were significantly changed. This suggests that azoxystrobin may interfere with the homeostasis of neurotransmitters by causing oxidative stress in male zebrafish brains, thus affecting the behavioral response of male zebrafish.

Maternal exposure to bisphenol S reduces anxiety and impairs collective antipredator behavior of male zebrafish (Danio rerio) offspring through dysregulation of their serotonergic system

Bisphenol S (BPS) is a common endocrine-disrupting chemical globally used in several consumer and industrial products. Although previous studies suggested that BPS induces multiple effects in exposed organisms, very little is known about its intergenerational effect on offspring behavior and/or the potential underlying mechanisms. To this end, adult female zebrafish Danio rerio were exposed to BPS (0, 10, 30 µg/L) and 1 µg/L of 17-β-estradiol (E2) as a positive control for 60 days. Afterwards, female fish were bred with untreated males, and their offspring were raised to 6 months old in control water. Maternal exposure to BPS decreased male offspring anxiety and antipredator behaviors while boldness remained unaffected. Specifically, maternal exposure to 10 and 30 µg/L BPS and 1 µg/L E2 were found to impact male offspring anxiety levels as they decreased the total time that individuals spent in the dark zone in the light/dark box test and increased the total track length in the center of the open field test. In addition, maternal exposure to all concentrations of BPS and E2 disrupted antipredator responses of male offspring by decreasing shoal cohesion in the presence of chemical alarm cues derived from conspecifics, which communicated high risk. To elucidate the possible molecular mechanism underlying these neuro-behavioral effects of BPS, we assessed the serotonergic system via changes in mRNA expression of serotonin receptors, including the 5-HT1A, 5-HT1B, and 5-HT1D subtypes, the serotonin transporter and monoamine oxidase (MAO). The impaired anxiety and antipredator responses were associated with reduced levels of 5-HT1A subtype and MAO mRNA expression within the brain of adult male offspring. Collectively, the results of this study demonstrate that maternal exposure to environmental concentrations of BPS can interfere with the serotonergic signaling pathway in the developing brain, subsequently leading to the onset of a suite of behavioral deficits in adult offspring.

Indoor and Personal PM2.5 Samples Differ in Chemical Composition and Alter Zebrafish Behavior Based on Primary Fuel Source.

Fine particulate matter (PM2.5) exposure has been linked to diverse human health impacts. Little is known about the potential heterogeneous impacts of PM2.5 generated from different indoor fuel sources and how exposure differs between personal and indoor environments. Therefore, we used PM2.5 collected by one stationary sampler in a kitchen and personal samplers (female and male participants), in homes (n = 24) in Kheri, India, that used either biomass or liquified petroleum gas (LPG) as primary fuel sources. PM2.5 samples (pooled by fuel type and monitor placement) were analyzed for oxidative potential and chemical composition, including elements and 125 organic compounds. Zebrafish (Danio rerio) embryos were acutely exposed to varying concentrations of PM2.5 and behavioral analyses were conducted. We found relatively high PM2.5 concentrations (5-15 times above World Health Organization daily exposure guidelines) and varied human health-related chemical composition based on fuel type and monitor placement (up to 15% carcinogenic polycyclic aromatic hydrocarbon composition). Altered biological responses, including changes to mortality, morphology, and behavior, were elicited by exposure to all sample types. These findings reveal that although LPG is generally ranked the least harmful compared to biomass fuels, chemical characteristics and biological impacts were still present, highlighting the need for further research in determining the safety of indoor fuel sources.

Effets of Antihistamines in Adult Zebrafish in Novel Tank Test

Histamine receptors play pivotal roles in various physiological functions, ranging from allergic responses to memory and sleep regulation, making them important drug targets. While second-generation antihistamines have been successfully used in rodents and humans, investigating their effects in non-traditional animal models enhances our understanding and aids the development of novel drug candidates. In this study, we examined the impact of the first-generation drug chloropyramine and the second-generation drugs loratadine and cetirizine, at concentrations of 1, 5, and 10 mg/L, on adult zebrafish b-ehavior using the novel tank test. All three drugs significantly altered fish locomotor a-ctivity, decreasing distance traveled and average velocity while increasing low acceleration frequency. Chloropyramine at 5 and 10 mg/L and loratadine at 1, 5, and 10 mg/L significantly reduced top entries compared to the control. Additionally, 5 mg/L chloropyramine increased the total duration of top entries, whereas loratadine at 10 mg/L r-educed this behavior compared to controls. Overall, chloropyramine and loratadine e-xhibited a sedative effect typical of antihistamines, while cetirizine solely reduced locomotor activity without affecting other patterns of fish behavior. Thus, cetirizine demonstrated the least impact on the central nervous system among the studied drugs, making it the optimal and safest choice among antihistamines.

Use of acoustic stimulus to determine behavioral changes in zebrafish after Cd exposure in a water quality warning system

Behavioral changes in zebrafish are an effective early warning system to determine water quality. However, only a few studies have examined the response of zebrafish to non-chemical stimulus after exposure to a contaminant. Therefore, this study investigated the differences in the behavioral responses of zebrafish to acoustic stimuli before and after exposure to cadmium (Cd). Acoustic escape response sensitivity curves were obtained and analyzed, followed by the determination of sensitive stimulus conditions at 100 Hz and 97 dB with a duration of 30 s and an interval of 30 min. Zebrafish exhibit a significant acoustic escape response, which is significantly reduced after exposure to Cd. The results showed that zebrafish stop demonstrating acoustic escape responses when exposed to higher Cd concentrations or longer acoustic exposures. Based on these results, a novel method for detecting abnormal behavior in zebrafish by acoustic stimulation has been proposed, which is expected to reduce the false alarm rate of this type of water quality technology.

Vitamin D modulation of brain-gut-virome disorder caused by polystyrene nanoplastics exposure in zebrafish (Danio rerio)

BackgroundMany studies have investigated how nanoplastics (NPs) exposure mediates nerve and intestinal toxicity through a dysregulated brain-gut axis interaction, but there are few studies aimed at alleviating those effects. To determine whether and how vitamin D can impact that toxicity, fish were supplemented with a vitamin D-low diet and vitamin D-high diet.ResultsTransmission electron microscopy (TEM) showed that polystyrene nanoplastics (PS-NPs) accumulated in zebrafish brain and intestine, resulting in brain blood–brain barrier basement membrane damage and the vacuolization of intestinal goblet cells and mitochondria. A high concentration of vitamin D reduced the accumulation of PS-NPs in zebrafish brain tissues by 20% and intestinal tissues by 58.8% and 52.2%, respectively, and alleviated the pathological damage induced by PS-NPs. Adequate vitamin D significantly increased the content of serotonin (5-HT) and reduced the anxiety-like behavior of zebrafish caused by PS-NPs exposure. Virus metagenome showed that PS-NPs exposure affected the composition and abundance of zebrafish intestinal viruses. Differentially expressed viruses in the vitamin D-low and vitamin D-high group affected the secretion of brain neurotransmitters in zebrafish. Virus AF191073 was negatively correlated with neurotransmitter 5-HT, whereas KT319643 was positively correlated with malondialdehyde (MDA) content and the expression of cytochrome 1a1 (cyp1a1) and cytochrome 1b1 (cyp1b1) in the intestine. This suggests that AF191073 and KT319643 may be key viruses that mediate the vitamin D reduction in neurotoxicity and immunotoxicity induced by PS-NPs.ConclusionVitamin D can alleviate neurotoxicity and immunotoxicity induced by PS-NPs exposure by directionally altering the gut virome. These findings highlight the potential of vitamin D to alleviate the brain-gut-virome disorder caused by PS-NPs exposure and suggest potential therapeutic strategies to reduce the risk of NPs toxicity in aquaculture, that is, adding adequate vitamin D to diet.A5myG_s1qHAhSjY9k6V2Q1Video

Imidacloprid affects the visual behavior of adult zebrafish (Danio rerio) by mediating the expression of opsin and phototransduction genes and altering the metabolism of neurotransmitters

Imidacloprid poses a significant threat to aquatic ecosystems. In this study, we investigated the visual toxicity of imidacloprid and the underlying molecular mechanisms in adult zebrafish. After exposure to imidacloprid at environmental relevant concentrations (10 and 100μg/L) for 21 days, the detectable contents of imidacloprid were 23.0 ± 0.80 and 121 ± 1.56 ng/mg in eyes of adult zebrafish, respectively. The visual behavior of adult zebrafish was impaired including a reduced ability to track smoothly visual stimuli and visually guided self-motion. The immunofluorescence experiment showed that the content of Rhodopsin (Rho) in the retina of zebrafish was changed significantly. The expression rhythm of genes played key roles in capturing photons in dim (rho) and bright (opn1mw3, opn1lw2 and opn1sw2) light, and in phototransduction (gnb3b, arr3a and rpe65a), was disrupted significantly throughout a 24-h period in adult zebrafish. Targeted metabolomics analysis showed that the content of 16 metabolites associated with neurotransmitter function changed significantly, and were enriched in top three metabolism pathways including Arginine biosynthesis, Alanine, aspartate and glutamate metabolism, and Tryptophan metabolism. These results indicated that imidacloprid exposure at environmentally relevant concentrations could cause optical toxicity through disturbing the expression of opsins and affecting the phototransduction in the retina of zebrafish adults.

Pi_tailtrack: A compact, inexpensive and open-source behaviour-tracking system for head-restrained zebrafish.

Quantifying animal behaviour during microscopy is crucial to associate optically recorded neural activity with behavioural outputs and states. Here, I describe an imaging and tracking system for head-restrained larval zebrafish compatible with functional microscopy. This system is based on the Raspberry Pi computer, Pi NoIR camera and open-source software for the real-time tail segmentation and skeletonization of the zebrafish tail at over 100 Hz. This allows for precise and long-term analyses of swimming behaviour, which can be related to functional signals recorded in individual neurons. This system offers a simple but performant solution for quantifying the behaviour of head-restrained larval zebrafish, which can be built for 340€.

Ephedrine and cocaine cause developmental neurotoxicity and abnormal behavior in zebrafish

Ephedrine (EPH) and cocaine (COC) are illegal stimulant drugs, and have been frequently detected in aquatic environments. EPH and COC have negative effects on the nervous system and cause abnormal behaviors in mammals and fish at high concentrations, but their mechanisms of neurotoxicity remain unclear in larvae fish at low concentrations. To address this issue, zebrafish embryos were exposed to EPH and COC for 14 days post-fertilization (dpf) at 10, 100, and 1000 ng L−1. The bioaccumulation, development, behavior, cell neurotransmitter levels and apoptosis were detected to investigate the developmental neurotoxicity (DNT) of EPH and COC. The results showed that EPH decreased heart rate, while COC increased heart rate. EPH caused cell apoptosis in the brain by AO staining. In addition, behavior analysis indicated that EPH and COC affected spontaneous movement, touch-response, swimming activity and anxiety-like behaviors. EPH and COC altered the levels of the neurotransmitters dopamine (DA) and γ-aminobutyric acid (GABA) with changes of the transcription of genes related to the DA and GABA pathways. These findings indicated that EPH and COC had noticeable DNT in the early stage of zebrafish at environmentally relevant concentrations.

Long-term effects of embryonic exposure to benzophenone-3 on neurotoxicity and behavior of adult zebrafish

Benzophenone-3 (BP-3) is the most widely used ultraviolet filter (UV filter) in industries to avoid UV radiation damage. BP-3 is added to most sunscreens to protect the skin, hair, and lips from sun rays. It results in continuous discharge into aquatic environments, leading to aquatic biota and human's continuous exposure. Consequences of BP-3 exposure on the physiology and behavior of aquatic animals, mainly zebrafish, have been investigated, including their neurotoxic effects. However, little is known about its consequences in long-term developmental endpoints. This study aimed to investigate the long-term effects of embryonic BP-3 exposure on biomarkers of neurotoxicity in zebrafish. For this, we exposed embryos to 5, 10, and 20 μg∙L−1 BP-3 concentration and let fish grow to adulthood (5mpf). We evaluated anxiety-like behavior, social preference, aggressiveness, and enzymatic activity of the antioxidant defenses system and neurotoxic biomarkers (Glutathione S-transferase -GST, catalase -CAT, and acetylcholinesterase -AChE) in adult zebrafish. Enzymatic activities were also investigated in larvae immediately after BP-3 exposure. Animals early exposed to BP-3 presented anxiety-like behaviors and decreased social preference, but aggressiveness was not altered. In general, exposure to BP-3 leads to altered enzymatic activity, which persists into adulthood. GST activity increased in embryos and adults, while CAT activity decreased in both life stages. AChE activity enhanced only at the larval stage (96 hpf). The long-term behavioral and biochemical effects of BP-3 highlight the need for abolishing or restricting the compound from personal care products, which are continually disposed into the environment and threaten the biota and human health.

Feeding frequency does not interact with BPA exposure to influence metabolism or behaviour in zebrafish (Danio rerio)

Resource limitation can constrain energy (ATP) production, and thereby affect locomotion and behaviour such as exploration of novel environments and boldness. Consequently, ecological processes such as dispersal and interactions within and between species may be influenced by food availability. Energy metabolism, and behaviour are regulated by endocrine signalling, and may therefore be impacted by endocrine disrupting compounds (EDCs) including bisphenol A (BPA) derived from plastic manufacture and pollution. It is important to determine the impacts of these novel environmental contexts to understand how human activity alters individual physiology and behaviour and thereby populations. Our aim was to determine whether BPA exposure interacts with feeding frequency to alter metabolism and behaviour. In a fully factorial experiment, we show that low feeding frequency reduced zebrafish (Danio rerio) mass, condition, resting metabolic rates, total distance moved and speed in a novel arena, as well as anxiety indicated by the number of times fish returned to a dark shelter. However, feeding frequency did not significantly affect maximal metabolic rates, aerobic scope, swimming performance, latency to leave a shelter, or metabolic enzyme activities (citrate synthase and lactate dehydrogenase). Natural or anthropogenic fluctuation in food resources can therefore impact energetics and movement of animals with repercussions for ecological processes such as dispersal. BPA exposure reduced LDH activity and body mass, but did not interact with feeding frequency. Hence, behaviour of adult fish is relatively insensitive to disruption by BPA. However, alteration of LDH activity by BPA could disrupt lactate metabolism and signalling and together with reduction in body mass could affect size-dependent reproductive output. BPA released by plastic manufacture and pollution can thereby impact conservation and management of natural resources.

Short-term high-fat diet feeding induces cognitive decline, aggressiveness and anxiety-like behavior in adult zebrafish (Danio rerio)

Short-term high-fat diet feeding induces cognitive decline, aggressiveness and anxiety-like behavior in adult zebrafish (Danio rerio)

Polyethylene and polypropylene microplastics induce anxiety-like behavior and brainstem astrogliosis in zebrafish

Polyethylene and polypropylene microplastics induce anxiety-like behavior and brainstem astrogliosis in zebrafish