Dpf Zebrafish Larvae Research Articles

Ammonia exposure impairs bone mineralization in zebrafish (Danio rerio) larvae

Ammonia is a major pollutant of freshwater environments. Previous studies have indicated that ammonia exposure adversely affects the physiology of freshwater fish. However, its effect on bone mineralization in freshwater fish larvae remains unclear. In this study, zebrafish larvae were used as a model to investigate the effects of different ammonia levels (0, 2.5, 5, and 10 mM NH4Cl) on the survival rate, body length, and bone mineralization of fish. The survival rate of zebrafish embryos exposed to different NH4Cl concentrations for 8 days was not affected. In contrast, the body length and bone mineralization of zebrafish larvae at 8 days post fertilization (dpf) were significantly reduced at 5 and 10 mM NH4Cl exposure. Further investigations revealed that ammonia exposure decreased the mRNA expression of osteoblast-related genes and increased that of osteoclast-related genes. Additionally, exposure to 5 mM and 10 mM NH4Cl induced the production of reactive oxygen species (ROS). 10 mM—but not 5 mM—NH4Cl exposure reduced the calcium and phosphorus content in 8 dpf zebrafish larvae. In conclusion, ammonia exposure induces bone resorption, while decreasing the calcium and phosphorus content of the whole body and bone formation, resulting in impaired bone mineralization in fish larvae.

Looks like home: numerosity, but not spatial frequency guides preference in zebrafish larvae (Danio rerio)

Despite their young age, zebrafish larvae have a well-developed visual system and can distinguish between different visual stimuli. First, we investigated if the first visual surroundings the larvae experience during the first days after hatching shape their habitat preference. Indeed, these animals seem to “imprint” on the first surroundings they see and select visual stimuli accordingly at 7 days post fertilization (dpf). In particular, if zebrafish larvae experience a bar background just after hatching, they later on prefer bars over white stimuli, and vice versa. We then used this acquired preference for bars to investigate innate numerical abilities. We wanted to specifically test if the zebrafish larvae show real numerical abilities or if they rely on a lower-level mechanism—i.e. spatial frequency—to discriminate between two different numerosities. When we matched the spatial frequency in stimuli with different numbers of bars, the larvae reliably selected the higher numerosity. A previous study has ruled out that 7 dpf zebrafish larvae use convex hull, cumulative surface area and density to choose between two numerosities. Therefore, our results indicate that zebrafish larvae rely on real numerical abilities rather than other cues, including spatial frequency, when spontaneously comparing two sets with different numbers of bars.

Unveiling the ototoxic effects of paraquat on zebrafish larva

Paraquat is a widely utilized herbicide in agricultural fields posing a significant impact on human health and the environment due to its potent oxidant properties. Rampant paraquat usage leads to serious health hazards to farmers and the ecosystem, particularly the water bodies. Paraquat exposure can damage dopaminergic neurons causing Parkinson's disease in humans and other animal models. Extensive research has been done regarding the mode of action, pathophysiology and molecular mechanisms of paraquat-induced Parkinson's disease. Meanwhile, the ototoxic effect of paraquat remains poorly understood. Potential ototoxins can cause sensorineural hearing loss, one of the most common sensory disabilities in humans. In this study, we investigated the harmful effects of paraquat on neuromast hair cells in zebrafish larvae, a powerful model organism for auditory research. We treated sub-lethal concentrations (125 μM to 1000 μM) of paraquat to 3 and 4 dpf zebrafish larvae to investigate its ototoxic effects via rheotaxis behavioral assay, neuromast staining and scanning electron microscopy. The behavioral assay findings showed a drastic decline in the rheotaxis behavior in all the concentrations of paraquat-treated larvae. Furthermore, DASPEI neuromast vital staining displayed a dose-dependent reduction in the neuromast hair cells as we increased the paraquat concentration. The scanning electron microscope data revealed the significant shortening of kinociliary length, a decrease in stereociliary density and changes in semilunar peridermal cell morphology signifying the damaging effects of paraquat at the cellular level. Collectively, the behavioral, anatomical and morphological studies highlight the potential ototoxic effects of paraquat on zebrafish neuromast hair cells, further signifying its potential role in causing hearing loss in humans.

A unified approach to investigating 4 dpf zebrafish larval behaviour through a standardised light/dark assay

Zebrafish are a dynamic research model in the domains of neuropsychopharmacology, biological psychiatry and behaviour. Working with larvae ≤4 days post-fertilisation (dpf) offers an avenue for high-throughput investigation whilst aligning with the 3Rs principles of animal research. The light/dark assay, which is the most widely used behavioural assay for larval neuropharmacology research, lacks experimental reliability and standardisation. This study aimed to formulate a robust, reproducible and standardised light/dark behavioural assay using 4 dpf zebrafish larvae. Considerable between-batch and inter-individual variability was found, which we rectified with a normalisation approach to ensure a reliable foundation for analysis. We then identified that 5-min light/dark transition periods are optimal for locomotor activity. We also found that a 30-min acclimation in the light was found to produce significantly increased dark phase larval locomotion. Next, we confirmed the pharmacological predictivity of the standardised assay using ethanol which, as predicted, caused hyperlocomotion at low concentrations and hypolocomotion at high concentrations. Finally, the assay was validated by assessing the behavioural phenotype of hyperactive transgenic (adgrl3.1−/−) larvae, which was rescued with psychostimulant medications. Our standardised assay not only provides a clear experimental and analytical framework to work with 4 dpf larvae, but also facilitates between-laboratory collaboration using our normalisation approach.

A Simple Analysis and Scoring of Zebrafish Larvae Cardiac Performance in Teratogenicity Study using Light Microscopy and ImageJ Software

Background: Zebrafish is one of the suitable model organisms for teratogenicity study. One of the organs assessed during the study is the heart. There has been an established scoring system for the cardiac morphology, but not cardiac performance. Furthermore, the assessment of cardiac function in zebrafish often use expensive instruments and complicated methods. Materials and Methods: Cardiac performance scoring method and system was proposed for zebrafish larvae aged 5 dpf. Observation was done using light microscope and the video of the observation was taken using smartphone or camera. The videos were then converted and rendered to be compatible with the ImageJ program using the measure function and time series analyzer plugin. The assessed parameters were the heart rate and rhythm, strength of heart contraction, and synchronicity between atrium and ventricle. Results: The scoring system was formulated based on the assessments towards healthy control 5 dpf zebrafish larvae and references. The score for cardiac performance ranged from 3 – 6, with score 3 representing arrhythmic, weak, and asynchronous contractions, while score 6 representing rhythmic, strong, and synchronous contraction. The scoring system was also valid to point out the difference in cardiac performance between healthy control zebrafish larvae and larvae that previously exposed to lithium as teratogenic agent (p = 0.000). Conclusions: The scoring methods and system developed in this study allowed for a non – expensive and simple assessment of cardiac performance in zebrafish larvae aged 5 dpf, especially for a teratogenicity study.

Xanthohumol, a prenylated flavonoid from hops (Humulus lupulus L.) exerts multidirectional pro-healing properties towards damaged zebrafish hair cells by regulating the innate immune response

Xanthohumol (XN) is a prominent prenylated flavonoid present in the hop plant (Humulus lupulus L.). Despite undoubted pro-healing properties of hop plant, there is still a need for clinical investigations confirming these effects as well as the underlying molecular mechanisms. The present study was designed to (1) establish the role of XN in non-invasive inflammation induced by chemical damage to zebrafish hair cells, (2) clarify if it influences cell injury severity, neutrophil migration, macrophage activation, cell regeneration, and (3) find out whether it modulates the gene expression profile of chosen immune and stress response markers. All experiments were performed on 3 dpf zebrafish larvae. After fertilization the embryos were transferred to appropriate XN solutions (0.1 μM, 0.3 μM and 0.5 μM). The 40 min 10 μM CuSO4 exposure evoked severe damage to posterior lateral line hair cells triggering a robust acute inflammatory response. Four readouts were selected as the indicators of XN role in the process of inflammation: 1) hair cell death, 2) neutrophil migration towards damaged hair cells, 3) macrophage activation and recruitment to damaged hair cells, 4) hair cell regeneration. The assessments involved in vivo confocal microscopy imaging and qPCR based molecular analysis. It was demonstrated that XN (1) influences death pathway of damaged hair cells by redirecting their severe necrotic phenotype into apoptotic one, (2) impacts the immune response via regulating neutrophil migration, macrophage recruitment and activation (3) modulates gene expression of immune system markers and (4) accelerates hair cell regeneration.

Centella asiatica extract ameliorates deoxygenation-induced neurological dysfunction in zebrafish larvae

Background: Oxygen deprivation (OD) is a critical condition that can lead to brain damage and even death. Current hypoxia management approaches are limited in effectiveness. Centella asiatica, known for its neuroprotective properties, offers a potential alternative for OD treatment. Aim: This study aims to investigate the neuroprotective effects of Centella asiatica (CA) on the expression of Brain-Derived Neurotrophic Factor (BDNF) and Vesicular Glutamate Transporter 1 (VGLUT1) in zebrafish larvae under oxygen-deficient conditions. Methods: Zebrafish embryos were subjected to low oxygen levels (1.5 mg/L) 0-2 hours post-fertilization (hpf) until three days post-fertilization (dpf), simulating the early stages of OD. Subsequent treatment involved varying concentrations of CA (1.25-5 µg/ml) up to nine days post-fertilization. The expression levels of BDNF and VGLUT1 were measured using PCR methods. Statistical analysis was conducted using Two-way ANOVA to evaluate the impact of CA on the expression of BDNF and VGLUT1 in zebrafish larvae aged 3 and 9 dpf in oxygen-deprived conditions. Results: CA significantly influenced the expression of BDNF and VGLUT1 under oxygen deprivation (p<0.001). An increase in BDNF expression (p<0.001) and a decrease in VGLUT1 (p<0.01) were observed in zebrafish larvae experiencing OD and treated with CA. There was no significant difference in BDNF and VGLUT1 expression across age variations in zebrafish larvae at 3 dpf and 9 dpf in the treatment groups (p>0.05). CA concentration of 2.5 µg/ml effectively enhanced BDNF and reduced VGLUT1 in 3-9 dpf zebrafish larvae. Conclusion: CA demonstrates potential as a neuroprotective agent, modulating increased BDNF expression and reduced VGLUT1 under OD conditions. These findings lay a foundation for further research in developing therapies for oxygen deficiency.

Ontogeny of working memory and behavioural flexibility in the free movement pattern (FMP) Y-maze in zebrafish

The acquisition of executive skills such as working memory, decision-making and adaptive responding occur at different stages of central nervous system development. Zebrafish (Danio rerio) are increasingly used in behavioural neuroscience for complex behavioural tasks, and there is a critical need to understand the ontogeny of their executive functions. Zebrafish across developmental stages (4, 7, 14, 30 and 90 days post fertilisation (dpf)), were assessed to track development of working memory (WM) and behavioural flexibility (BF) using the free movement pattern Y-maze (FMP Y-maze). Several differences in both WM and BF were identified during the transition from yolk-dependent to independent feeding. Specifically, WM is evident in all age groups, even from 4 dpf. However, BF is not developed until larvae start free feeding, and show significant improvement thereafter, with young adults (90 dpf) demonstrating the most well-defined BF. We demonstrate, for the first time, objective WM processes in 4 dpf zebrafish larvae. This suggests that those wishing to study WM in zebrafish may be able to do so from 4 dpf, thus drastically increasing throughput. In addition, we show that zebrafish follow distinct stages of cognitive development and age-related changes during the early developmental period. Finally, our findings indicate distinct WM and BF mechanisms, which may be useful to study for translational purposes.

A New Bioassay for the Detection of Paralytic and Amnesic Biotoxins Based on Motor Behavior Impairments of Zebrafish Larvae.

The global concern about the increase of harmful algal bloom events and the possible impacts on food safety and aquatic ecosystems presents the necessity for the development of more accessible techniques for biotoxin detection for screening purposes. Considering the numerous advantages that zebrafish present as a biological model and particularly as a toxicants sentinel, we designed a sensitive and accessible test to determine the activity of paralytic and amnesic biotoxins using zebrafish larvae immersion. The ZebraBioTox bioassay is based on the automated recording of larval locomotor activity using an IR microbeam locomotion detector, and manual assessment of four complementary responses under a simple stereoscope: survival, periocular edema, body balance, and touch response. This 24 h acute static bioassay was set up in 96-well microplates using 5 dpf zebrafish larvae. For paralytic toxins, a significant decrease in locomotor activity and touch response of the larvae was detected, allowing a detection threshold of 0.1-0.2 µg/mL STXeq. In the case of the amnesic toxin the effect was reversed, detecting hyperactivity with a detection threshold of 10 µg/mL domoic acid. We propose that this assay might be used as a complementary tool for environmental safety monitoring.

Keap1/Nrf2-independent antioxidative activity of Phyllanthus amarus extract in zebrafish

The Keap1 protein (Kelch-like ECH-related protein 1) and the Nrf2 transcription factor (NF-E2-related factor 2) are important systems for maintaining homeostasis, redox, and metabolism. Based on the Keap1/Nrf2 pathway, the antioxidative mechanism of P. amarus extract (PAE) was predicted. In this paper, we evaluated the protective effects of PAE on the oxidative toxicity induced by sodium arsenite (NaAsO2) and hydrogen peroxide (H2O2) in zebrafish larvae. We first determined that the LC50 values for NaAsO2, H2O2, and PAE at 3.5 days postfertilization (dpf) were 1 mM, 3 mM, and 200 μg/mL, respectively. Then, to assess the antioxidant effects of P. amarus, 3.5 dpf zebrafish larvae were pretreated with PAE at concentrations of 0, 50, 75, and 100 μg/ml for 12 h, and then the PAE solution was replaced with 1 mM NaAsO2 or 3 mM H2O2 to assess challenge survival within 48 h. Interestingly, all three concentrations, 50, 75, and 100 μg/mL PAE, increased the survival rate of zebrafish larvae compared to those larvae exposed to only 1 mM NaAsO2. Similarly, PAE at concentrations of 75 and 100 μg/mL protected zebrafish larvae after exposure to 3 mM H2O2. Real-time qPCR analysis was performed after 3.5 dpf zebrafish larvae were exposed to 100 μg/mL PAE for 12 h to verify whether the increasing antioxidative activity is depended on the Nrf2 pathway. The expression of the Nrf2 target genes glutathione-S-transferase Pi 1 (gstp1) and peroxidase 1 (prdx1) was assessed using real-time qPCR. However, the expression of this gene was not significantly different between control larvae and PAE-treated larvae. Thus, PAE induces antioxidant activity in zebrafish in a Keap1/Nrf2-independent manner.

Early developmental exposure to bisphenol A and bisphenol S disrupts socio-cognitive function, isotocin equilibrium, and excitation-inhibition balance in developing zebrafish

Dysregulation of the oxytocinergic system and excitation/inhibition (E/I) balance in synaptic transmission and neural circuits are common hallmarks of various neurodevelopmental disorders. Several experimental and epidemiological studies have shown that perinatal exposure to endocrine-disrupting chemicals bisphenol A (BPA) and bisphenol S (BPS) may contribute to a range of childhood neurodevelopmental disorders. However, the effects of BPA and BPS on social-cognitive development and the associated mechanisms remain largely unknown. In this study, we explored the impacts of early developmental exposure (2hpf-5dpf) to environmentally relevant concentrations of BPA, and its analog BPS (0.001, 0.01, and 0.1 μM), on anxiety, social behaviors, and memory performance in 21 dpf zebrafish larvae. Our results revealed that early-life exposure to low concentrations of BPA and BPS elevated anxiety-like behavior, while fish exposed to higher concentrations of these chemicals displayed social deficits and impaired object recognition memory. Additionally, we found that co-exposure with an aromatase inhibitor antagonized BPA- and BPS-induced effects on anxiety levels and social behaviors, while the co-exposure to an estrogen receptor antagonist restored recognition memory in zebrafish larvae. These results indicate that BPA and BPS may affect social-cognitive function through distinct mechanisms. On the other hand, exposure to low BPA/BPS concentrations increased both the mRNA and protein levels of isotocin (zebrafish oxytocin) in the zebrafish brain, whereas a reduction in its mRNA level was observed at higher concentrations. Further, alterations in the transcript abundance of chloride transporters, and molecular markers of gamma-aminobutyric acid (GABA) and glutamatergic systems, were observed in the zebrafish brain, suggesting possible E/I imbalance following BPA or BPS exposure. Collectively, the results of this study demonstrate that early-life exposure to low concentrations of the environmental contaminants BPA and BPS can interfere with the isotocinergic signaling pathway and disrupts the establishment of E/I balance in the developing brain, subsequently leading to the onset of a suite of behavioral deficits and neurodevelopmental disorders.

Loss of Protein C in Zebrafish Results in Impaired Neutrophil Migration after Tissue Injury

Hemostasis is a natural protective process that developed to retain a circulating blood system, conferred by a complicated yet sophisticated balance of factors. Disturbances of this network result in thrombosis or hemorrhage. Among many well-characterized coagulation factors, protein C (PC) exhibits multifunctional roles including anticoagulant, cytoprotective, and anti-inflammatory activities. The importance of PC has been demonstrated not only by the increased risk of venous thrombosis in individuals with heterozygous deficiency, but also the observed neonatal lethality in patients. Knockout mice exhibit similar neonatal lethality, which has made it difficult to further study complete deficiency. The zebrafish is a vertebrate organism that is characterized by a powerful genetic system, prolific breeding, rapid and transparent development, and a well described and highly conserved coagulation cascade. Here we utilize genome editing to generate a null allele of the PC gene (proc) in zebrafish and discover that its loss not only impairs hemostatic balance, but also affects neutrophil recruitment to sites of tissue injury. Through examination of publicly available zebrafish genome sequence, we determined that the proc locus is duplicated in tandem, resulting in two closely adjacent copies with nearly identical sequences. We used CRISPR/Cas9 with two single guide RNAs flanking the entire locus to produce a 17.3 kilobase deletion that knocks out both copies of proc to produce a complete null mutation, verified by sequencing and quantitative PCR. proc-/- mutants survived well into adulthood, with ~50% lethality by seven months of age. The embryonic survival and accessibility enabled us to perform intravital microscopy to evaluate the hemostatic effects of PC deficiency. We used laser-induced endothelial injury on the posterior cardinal vein (PCV) at 3 days post fertilization (dpf), which typically results in rapid formation of an occlusive fibrin-rich thrombus. proc-/- mutants had an average time to occlusion of 60 seconds versus 13 seconds in controls (p < 0.0001), consistent with a consumptive coagulopathy, as previously seen in antithrombin III (at3) mutants. A transgenic background with fluorescently labeled fibrinogen showed that more than 95% of proc-/- mutants had spontaneous thrombi in the PCV, which was not present in controls. To assess the role of PC in inflammation, we used two different injury strategies, non-vascular tail transection and chemical treatment (copper sulfate), on 3 dpf zebrafish larvae. Staining for neutrophil granules revealed homing to the site of injury within 60-75 minutes. In proc-/- mutants we found an average 50% reduction in the number of neutrophils recruited to the site of injury yet counts in the caudal hematopoietic tissue (the site of larval hematopoiesis) were unchanged. Since protein S (PS) is a cofactor for PC anticoagulant function, we hypothesized that the consumptive coagulopathy, but not the neutrophil recruitment, would be PS-dependent. We used genome editing to disrupt the PS gene (pros1) and found that loss of PS also results in a mild consumptive coagulopathy, but spontaneous thrombus formation was less common in the PCV (25%) and was often in the heart instead (80%). Neutrophil recruitment was unaffected in pros1 mutants, and evaluation of double proc/pros1 mutants revealed no synergy in any of the phenotypes. In conclusion, PC and PS deficiency in zebrafish show some similarity to our previously reported model of AT3 deficiency, but the effects are less potent, allowing robust survival that enables in vivo analyses. Our data suggest that the thrombotic phenotypes of PC and PS deficiency are not identical, and display tissue-specific phenotypes. We also found evidence for PS-independent functions of PC in neutrophil migration. We speculate this is due to the role that PC plays in inflammation and signaling but cannot exclude a role in neutrophil extracellular trap (NET) formation. This model of complete proc-/- deficiency in an accessible organism will facilitate further in vivo study of PS-dependent and independent functions of PC, as well as interplay between the two factors. Disclosures Shavit: Bayer: Consultancy; Taked: Consultancy.

Quantification of computational fluid dynamics simulation assists the evaluation of protection by Gypenosides in a zebrafish pain model

In recent years, due to its rapid reproduction rate and the similarity of its genetic structure to that of human, the zebrafish has been widely used as a pain model to study chemical influences on behavior. Swimming behaviors are mediated by motoneurons in the spinal cord that drive muscle contractions, therefore a knowledge of internal muscle mechanics can assist the understanding of the effects of drugs on swimming activity. To demonstrate that the technique used in our study can supplement biological observations by quantifying the contribution of muscle effects to altered swimming behaviours, we have evaluated the pain/damage caused by 0.1% acetic acid to the muscle of 5 dpf zebrafish larvae and the effect of protection from this pain/damage with the saponin Gypenosides (GYP) extracted from Gynostemma pentaphyllum. We have quantified the parameters related to muscle such as muscle power and the resultant hydrodynamic force, proving that GYP could alleviate the detrimental effect of acetic acid on zebrafish larvae, in the form of alleviation from swimming debility, and that the muscle status could be quantified to represent the degree of muscle damage due to the acetic acid and the recovery due to GYP. We have also linked the behavioral changes to alteration of antioxidant and inflammation gene expression. The above results provide novel insights into the reasons for pain-related behavioral changes in fish larvae, especially from an internal muscle perspective, and have quantified these changes to help understand the protection of swimming behaviors and internal muscle by GYP from acetic acid-induced damage.

Garcinol pacifies acrylamide induced cognitive impairments, neuroinflammation and neuronal apoptosis by modulating GSK signaling and activation of pCREB by regulating cathepsin B in the brain of zebrafish larvae

The presence of acrylamide (ACR) in food results in evident cognitive decline, accumulation of misfolded proteins, neurotoxicity, neuroinflammation, and neuronal apoptosis leading to progressive neurodegeneration. Here, we used 4 dpf zebrafish larvae exposed to ACR (1mM/3days) as our model, and neuronal proteins were analyzed. Next, we tested the effect of garcinol (GAR), a natural histone-acetylation inhibitor, whose neuroprotection mechanism of action remains to be fully elucidated. Our result revealed that ACR exposure significantly impaired cognitive behavior, downregulated oxidative repair machinery, and enhanced microglia-induced neuronal apoptosis. Moreover, ACR mediated cathepsin-B (CAT-B) translocation acted as the intracellular secretase for the processing of amyloid precursor protein (APP) and served as an additional risk factor for tau hyper-phosphorylation. Here, GAR suppresses ACR mediated CATB translocation as similar with standard inhibitor CA-074. And, this pharmacological repression helped in inhibiting amyloidogenic APP processing and downstream tau hyper-phosphorylation. GAR neuroprotection was accompanied by CREB, ATF1, and BDNF activation promoting neuronal survival. At the same time, GAR subdued cdk5 and GSK3β, the link between APP processing and tau hyper-phosphorylation. Taken together, our findings indicate that GAR rescued from ACR mediated behavioral defects, oxidative injury, neuroinflammation, undesirable APP processing, tau hyper-phosphorylation which in turn found to be CATB dependent.

Quantification of the influence of drugs on zebrafish larvae swimming kinematics and energetics.

The use of zebrafish larvae has aroused wide interest in the medical field for its potential role in the development of new therapies. The larvae grow extremely quickly and the embryos are nearly transparent which allows easy examination of its internal structures using fluorescent imaging techniques. Medical treatment of zebrafish larvae can directly influence its swimming behaviours. These behaviour changes are related to functional changes of central nervous system and transformations of the zebrafish body such as muscle mechanical power and force variation, which cannot be measured directly by pure experiment observation. To quantify the influence of drugs on zebrafish larvae swimming behaviours and energetics, we have developed a novel methodology to exploit intravital changes based on observed zebrafish locomotion. Specifically, by using an in-house MATLAB code to process the recorded live zebrafish swimming video, the kinematic locomotion equation of a 3D zebrafish larvae was obtained, and a customised Computational Fluid Dynamics tool was used to solve the fluid flow around the fish model which was geometrically the same as experimentally tested zebrafish. The developed methodology was firstly verified against experiment, and further applied to quantify the fish internal body force, torque and power consumption associated with a group of normal zebrafish larvae vs. those immersed in acetic acid and two neuroactive drugs. As indicated by our results, zebrafish larvae immersed in 0.01% acetic acid display approximately 30% higher hydrodynamic power and 10% higher cost of transport than control group. In addition, 500 μM diphenylhydantoin significantly decreases the locomotion activity for approximately 50% lower hydrodynamic power, whereas 100 mg/L yohimbine has not caused any significant influences on 5 dpf zebrafish larvae locomotion. The approach has potential to evaluate the influence of drugs on the aquatic animal’s behaviour changes and thus support the development of new analgesic and neuroactive drugs.

Identification of specific metabolic pathways as druggable targets regulating the sensitivity to cyanide poisoning.

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning.

Dissection of Larval Zebrafish Gonadal Tissue.

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Vision, Color Vision, and Visually Guided Behavior: The Novel Toxicological Targets of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47)

Our published studies have revealed that 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) could disrupt retina morphologies and related gene expressions of zebrafish larvae. Its possible effects on fish vision needed to be uncovered since sensory systems, especially eyes, are vital to wildlife. In this paper, two tests for vision development (opsin gene expression and photoreceptor immunostaining) and two tests for visually guided behaviors (optokinetic response and looming-evoked escape) were designed to investigate the potential visual impairments and subsequent ecological consequences caused by BDE-47 exposure in 6 dpf zebrafish larvae. The short wavelength sensitive cone opsins and rhodopsin were significantly inhibited by BDE-47 exposure. Meanwhile, BDE-47 exposure significantly reduced larval optokinetic responses with blue light stimuli and induced less larvae to exhibit escape response with looming stimuli, which confirmed the adverse consequences of visual impairments in zebrafish. Our results indicat...

Innate Color Preference of Zebrafish and Its Use in Behavioral Analyses.

Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for high-throughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique.

Novel perspective on field recordings in zebrafish models of epilepsy

Research in Epilepsy relies strongly on animal models, either for describing genetic conditions involved in the disease or for testing potential drugs that might alleviate the symptoms [1]. In this study, we looked at the differential effect of Pentylenetetrazole (PTZ), a well-known epileptogenic drug [2], on 5-6 dpf zebrafish larvae of two different genetic conditions: a mutant line, where a gene known to be involved in focal epilepsy was specifically knocked down, as well as control mismatch oligonucleotide-injected zebrafish. The zebrafish larvae were scored for phenotypic features, including hyperactivity and aberrant locomotion. These phenotypic features were present in all mutant fish recorded, but were absent in the mismatch control. We acquired electrophysiological field recordings from the optic tectum before and after PTZ application and selected a low frequency band of the signal (0.05 - 0.5 Hz). Troughs, representing epileptic events, were extracted and a wide range of analyses were applied: total event count, event time histogram, event duration distributions, inter-event-interval (IEI) distribution, classical and scaled autocorrelations on the field and event signals [3]. Traditional analysis provided a quantitative evaluation of PTZ-induced epileptic events: an increase in number of epileptic events was observed in mutant fish with respect to control, and the event time histogram showed a more abrupt increase at 32-37 min. after PTZ application, whereas in the control case, the increase was constant throughout the response period. Compared to controls, in mutant fish, PTZ application led to a larger increase in the number of short (0.5-1.5 s) events with small IEI (< 10s). Correlation analysis revealed qualitative information about the effect of PTZ on field recordings: The scaled autocorrelation (scale segment of 1s) on the field signal exhibited oscillatory components around 0.4 Hz in all conditions, but mutant fish exhibited frequency variability with time after PTZ application and variability across animals, leading to a low average oscillation power. In controls the frequency was robustly locked at 0.4 Hz. Autocorrelation histograms computed on the extracted events evidenced a wide refractory period (~ 4 s, see Figure ​Figure1A),1A), followed by baseline in mutant fish recordings, whereas control animals exhibited a narrow refractory period (~ 2 s, see Figure ​Figure1B),1B), followed by a secondary peak and a slow modulation (~ 35 s). Scaled autocorrelation histograms (scale segment of 1000s) showed constant correlation decrease with increased lag in the control fish, but not in mutant fish, where correlation for larger lags fluctuated around zero (see Figure ​Figure1C1C and ​and1D1D). Figure 1 Correlation histograms on events from zebrafish field recordings: Top: Autocorrelation of mutant (A) versus control (B) animals; Bottom: Scaled autocorrelation with scale segment of 1000s of mutant (C) versus control (D) animals. The characteristics revealed by correlation analysis suggest potentially different mechanisms underlying PTZ-induced epileptic events. We therefore propose that, in addition to the traditional statistics on epileptic events, looking at the temporal characteristics and correlation structure of field recordings may lead to a better classification and understanding of the mechanisms underlying epileptogenesis in various models of epilepsy.