AB Zebrafish Research Articles

Cardiovascular toxicity assessment of polyethylene nanoplastics on developing zebrafish embryos

Environmental exposure to nanoplastics is inevitable as the application of nanoplastics in our daily life is more and more extensively. So, the adverse effects of nanoplastics on human health are also gaining greater concerns. However, the subsequent toxicological response to nanoplastics, especially on cardiovascular damage was still largely unknown. In this regard, the evaluation of cardiovascular effects of nanoplastics was performed in zebrafish embryos. The results indicated that the no observed adverse effect level (NOAEL) of nanoplastics is 50 μg/mL. The pericardial toxicity and hemodynamic changes were assessed by Albino (melanin allele) mutant zebrafish line. Severe pericardial edema was observed in zebrafish embryos after exposure to nanoplastics. At the concentration higher than NOAEL, nanoplastics significantly decreased the cardiac output (CO) and blood flow velocity. The fluorescence images manifested that the nanoplastics could inhibit the subintestinal angiogenesis of transgenic zebrafish embryos line Tg (fli-1: EGFP), which might disturb the cardiovascular formation and development. The resulting vascular endothelial dysfunction and hypercoagulable state of circulating blood further accelerated thrombosis. Reactive oxidative stress (ROS) and systemic inflammation were also found in Wild AB and Tg (mpo: GFP) zebrafish embryos, respectively. We also found many neutrophils recruiting in the tail vein where the zebrafish embryo thrombosis occurred. Our data suggested that nanoplastics could trigger the cardiovascular toxicity in zebrafish embryos, which could provide an essential clue for the safety assessment of nanoplastics.

Compromised N-Glycosylation Processing of Kv3.1b Correlates with Perturbed Motor Neuron Structure and Locomotor Activity.

Simple SummaryModifications in the repertoire of N-glycans at the cell surface are associated with neurological complications. However, knowledge of specific N-glycans as to their impact on neuronal excitability is lacking. Our proposed studies will elucidate the roles of distinct N-glycan structures in modulating potassium channel function in highly repetitive firing neurons. This study is instrumental in understanding the development and progression of neurological diseases, thus, opening the door for potential therapeutic options.Neurological difficulties commonly accompany individuals suffering from congenital disorders of glycosylation, resulting from defects in the N-glycosylation pathway. Vacant N-glycosylation sites (N220 and N229) of Kv3, voltage-gated K+ channels of high-firing neurons, deeply perturb channel activity in neuroblastoma (NB) cells. Here we examined neuron development, localization, and activity of Kv3 channels in wildtype AB zebrafish and CRISPR/Cas9 engineered NB cells, due to perturbations in N-glycosylation processing of Kv3.1b. We showed that caudal primary (CaP) motor neurons of zebrafish spinal cord transiently expressing fully glycosylated (WT) Kv3.1b have stereotypical morphology, while CaP neurons expressing partially glycosylated (N220Q) Kv3.1b showed severe maldevelopment with incomplete axonal branching and extension around the ventral musculature. Consequently, larvae expressing N220Q in CaP neurons had impaired swimming locomotor activity. We showed that replacement of complex N-glycans with oligomannose attached to Kv3.1b and at cell surface lessened Kv3.1b dispersal to outgrowths by altering the number, size, and density of Kv3.1b-containing particles in membranes of rat neuroblastoma cells. Opening and closing rates were slowed in Kv3 channels containing Kv3.1b with oligomannose, instead of complex N-glycans, which suggested a reduction in the intrinsic dynamics of the Kv3.1b α-subunit. Thus, N-glycosylation processing of Kv3.1b regulates neuronal development and excitability, thereby controlling motor activity.

Investigating Opioid‐induced Respiratory Depression and Analgesia Using Larval Zebrafish

Introduction Opioid drugs produce both analgesia and severe respiratory depression which can be lethal. In 2019, over 50,000 North Americans died of an opioid overdose. Our understanding on how to differentiate between these two effects is limited due to a lack of simple animal models. Therefore, we aim to understand respiratory depression and analgesia by opioids using novel zebrafish models, allowing for drug screening. Using these models, we tested a variety of drugs including serotoninergic agonists, glutamatergic modulators, and calcium channel activators, for their ability to reverse opioid-induced respiratory depression. Methods We have established phenotype-based approaches using in-vivo zebrafish models of respiratory depression and analgesia by fentanyl. To quantify respiratory depression, we measured respiratory activity by quantifying mandible movements in 12-14 day post-fertilization larvae. To quantify analgesia, we measured the escape swimming response to nociceptive stimuli such as formalin or allyl isothiocyanate (AITC) combined with fentanyl to induce analgesia. We then introduced various candidate drugs to measure their effects on respiratory depression and analgesia. Results Respiratory rate was normalized to each fish's baseline (baseline=100%). Zebrafish strains were found to be differentially sensitive to opioids, where Tübingen (TU) fish did not respond to fentanyl (1µM, p=0.4240, n=9) and AB fish showed significant respiratory rate depression (p<0.001, n=11). AB and TU crosses did not show respiratory depression (n=7). In AB zebrafish, a dose-dependent decrease in respiratory rate was observed with 1μM and 3µM fentanyl (p=0.01, n=11 and p<0.001, n=27, respectively). The opioid antagonist naloxone (20µM) and selective antagonist CTAP (4μM) reversed respiratory depression by fentanyl (p<0.001, n=9 and p=0.049, n=11, respectively). Respiratory depression by 1μM fentanyl was reversed by the AMPA receptor positive allosteric modulator CX614 (5μM, p<0.001, n=9) but not the serotoninergic 5-HT4 agonist BIMU8 (10μM, n=21). Calcium channel activators nefiracetam (1µM) and FLP-64176 (1µM) reversed respiratory depression by fentanyl (1µM, p=0.005, n=11 and3µM, p<0.001, n=15, respectively). Formalin (0.075%) and AITC (100µM) significantly increased swimming velocity (p=0.005, n=21 and p<0.001, n=23, respectively), which was reduced by fentanyl (4μM, p<0.001, n=12and 6µM, p=0.004, n=14, respectively). FLP-64176 reversed fentanyl analgesia when administered with formalin (p<0.001, n=10). Discussion: Our models show that respiratory depression and analgesia by opioids can be measured in zebrafish larvae. The strain differences point to a potential genetic protection which we are investigating using quantitative real-time polymerase chain reaction (qPCR). Our novel zebrafish models can be used to investigate the effects of opioids, as well as for drug screening to find ways to treat respiratory depression while preserving analgesia.

Developmental atrazine exposure in zebrafish produces the same major metabolites as mammals along with altered behavioral outcomes

Atrazine (ATZ) is the second most commonly applied agricultural herbicide in the United States. Due to contamination concerns, the U.S. EPA has set the maximum contaminant level in potable water sources at 3 parts per billion (ppb; μg/l). Depending on the time of year and sampling location, water sources often exceed this limit. ATZ is an endocrine disrupting chemical in multiple species observed to target the neuroendocrine system. In this study the zebrafish vertebrate model was used to test the hypothesis that a developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in developing larvae. Adult AB zebrafish were bred, embryos were collected, and exposed to 0, 0.3, 3, or 30 ppb ATZ from 1 to 120 h post fertilization (hpf). Targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals: desethyl atrazine (DEA), deisopropyl atrazine (DIA), and diaminochloroatrazine (DACT). The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group (p < 0.05). Further analysis into behavior during the dark and light phases showed zebrafish larvae exposed to 0.3 ppb ATZ had an increase in total distance moved in the first light phase and time spent moving in the first dark and light phases (p < 0.05). Alternatively, a decrease in total distance moved was observed in the second and third dark phases in zebrafish exposed to 30 ppb ATZ (p < 0.05). No significant differences were observed for any of the morphological measurements following ATZ exposure from 1 to 120 hpf (p > 0.05). These findings suggest that a ATZ exposure during early development generates metabolite profiles similar to mammals and leads to behavioral alterations supporting ATZ as a neurodevelopmental toxicant.

Cisplatin Cytotoxicity in Human Testicular Germ Cell Tumor Cell Lines Is Enhanced by the CDK4/6 Inhibitor Palbociclib.

Cisplatin-based chemotherapy is the mainstay of pharmacological treatment of testicular germ cell tumors (TGCTs) that, together with early diagnosis, surgery, and/or radiotherapy, has dramatically improved the prognosis. However, under the pressure of such pharmacological therapy (both classical cytotoxic drugs and targeted therapy), cancer cells may develop resistance. Thus, combination therapy that may include cytotoxic drugs and targeted therapy could offer an advantage to curing cancers. Here, we investigated the in vitro and in vivo antitumor activity of cisplatin, as a single-agent or in combination with palbociclib. The cell viability of Ntera-2/cl.D1 (NT2/D1) and 833K after exposure to palbociclib and/or cisplatin was evaluated by MTT dye reduction assay and by ATPLite Luminescence Assay. Gene and protein expression was evaluated by quantitative reverse transcription polymerase chain reaction and by western blot. Flow cytometric cell-cycle analysis was performed, as well. The in vivo experiments were conducted on NT2/D1 xenografts in AB zebrafish embryos exposed to the drugs. Palbociclib and cisplatin decreased TGCT cell viability both in vitro and in vivo. This effect was additive when cells were exposed to the drug combination. In the NT2/D1 cell lines, the drug combination also exerted a positive effect with regard to delaying cell recovery after the toxic insult. In the combination experiments, cisplatin-induced cell accumulation in G2/M was predominant compared with the palbociclib effect. These results could provide the rationale for developing further studies to improve the pharmacological treatment of TGCTs, but they must be demonstrated in a dedicated clinical trial.

Physical evidence on desmin-lamin B interaction.

Desmin is a muscle specific intermediate filament protein located in cytoplasm. Lamin B, on the other hand, is a nuclear intermediate filament protein. There are studies suggesting a possible interaction between desmin and lamin B yet there is no physical evidence. In the present study, we have shown for the first time a physical interaction between desmin and lamin B via reciprocal co-immunoprecipitation from muscle tissue of wild type AB zebrafish (Danio rerio, Hamilton). The interaction between desmin and lamin B might be a lead on a novel nucleocytoplasmic communication network.

Vitamin C Attenuates Oxidative Stress and Behavioral Abnormalities Triggered by Fipronil and Pyriproxyfen Insecticide Chronic Exposure on Zebrafish Juvenile.

Chronic exposure to synthetic insecticides in the early life of a child can lead to a series of disorders. Several causes as parental age, maternal smoking, birth complications, and exposure to toxins such as insecticides on childhood can lead to Autism spectrum disorder (ASD) occurrence. The aim of this study was to evaluate the potential protective role of vitamin C (Vit. C) from children’s supplements after 14 days chronic exposure to insecticide mixture fipronil (Fip) + pyriproxyfen (Pyr) on juvenile zebrafish for swimming performances, social behavior and oxidative stress associated with ASD model. Juvenile (14–17 mm) wild-type AB zebrafish (Danio rerio) (45 days) were exposed to relevant concentrations: vit. C (25 µg L−1), Fip (600 µg L−1/1.372 μM) + Pyr (600 µg L−1/1.89 μM), and [Fip (600 µg L−1/1.372 μM) + Pyr (600 µg L−1 /1.89 μM)] + vit. C (25 µg L−1). Our results showed that insecticides can disturb the social behavior of zebrafish during 14 days of the administration, decreased the swimming performances, and elevated the oxidative stress biomarkers of SOD (superoxide dismutase), GPx (glutathione peroxidase), and MDA (malondialdehyde). The vitamin C supplement significantly attenuated the neurotoxicity of insecticide mixture and oxidative stress. This study provides possible in vivo evidence to show that vitamin C supplements could attenuate oxidative stress and brain damage of fipronil and pyriproxyfen insecticide chronic exposure on zebrafish juvenile.

A comparison of behavioral and reproductive parameters between wild-type, transgenic and mutant zebrafish: Could they all be considered the same “zebrafish” for reglementary assays on endocrine disruption?

Transgenic zebrafish models are efficiently used to study the effects of endocrine disrupting chemicals (EDC); thereby informing on their mechanisms of action. However, given the reported differences between zebrafish strains at the genetical, physiological and behavioral levels; care should be taken before using these transgenic models for EDC testing. In the present study, we undertook a set of experiments in different transgenic and/or mutant zebrafish strains of interest for EDC testing: casper, cyp19a1a-eGFP, cyp19a1a-eGFP-casper, cyp11c1-eGFP, cyp11c1-eGFP-casper. Some behavioral traits, and some biochemical and reproductive physiological endpoints commonly used in EDC testing were assessed and compared to those obtained in WT AB zebrafish to ensure that transgene insertion and/or mutations do not negatively modify basal reproductive physiology or behavior of the fish.Behavioral traits considered as anxiety and sociality have been monitored. Sociality was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the Novel tank diving test. No critical difference was observed between strains for either sociality or anxiety level. Concerning reproduction, no significant difference in the number of eggs laid per female, in the viability of eggs or in the female circulating VTG concentrations was noted between the 5 transgenic/mutants and the WT AB zebrafish studied.In summary, the transgene insertion and the mutations had no influence on the endpoints measured in basal conditions. These results were a prerequisite to the use of these transgenic/mutant models for EDC testing. Next step will be to determine the sensitivity of these biological models to chemical exposure to accurately validate their use in existing fish assays for EDC testing.

Investigating Opioid‐induced Respiratory Depression and Analgesia Using Larval Zebrafish

IntroductionOpioid drugs present the severe side‐effect of respiratory depression which can be lethal. In fact, every year, in North America, over 70,000 people die of opioid overdose. Our current understanding is limited due to a lack of simple and amenable animal models to study the effects of opioid drugs on the nervous system. We aim to understand respiratory depression and analgesia by opioid drugs using novel zebrafish models, allowing for high throughput drug screening and gene editing. Using these models, we will demonstrate whether respiratory depression can be reversed by serotonergic agonists, glutamatergic modulators and calcium channel activators.MethodsWe have established phenotype‐based approaches using in‐vivo zebrafish models of respiratory depression and analgesia by fentanyl, a clinically relevant opioid analgesic. To quantify respiratory depression, we measured respiratory activity by recording mandible movements in 12–14 day post‐fertilization larvae. To quantify analgesia, we measured the escape swimming response to nociceptive stimuli such as formalin and administered fentanyl to induce analgesia. To show that μ‐opioid receptors (MORs) are expressed in respiratory circuits in the brainstem, we used in‐situ hybridization for Oprm1 (mRNA of the MOR).ResultsIn comparing respiratory sensitivity to opioids between zebrafish strains, we showed that Tübingen (TU) fish did not respond to fentanyl (1μM, P=0.4240, n=9), whereas AB fish showed pronounced respiratory rate depression (54±7.7% decrease, P&lt;0.001, n=11). Zebrafish crossed between AB and TU did not show respiratory depression (P=0.104, n=7). In AB zebrafish, a dose‐dependent decrease in respiratory rate was observed, with 1μM fentanyl showing the most potent respiratory depression (P=0.01, n=11). The opioid receptor antagonist naloxone (5μM) or the selective MOR antagonist CTAP (4μM) reversed respiratory depression to baseline levels (P=0.002, n=8). Similarly, respiratory depression was reversed by the positive allosteric modulator of AMPA receptors CX614 (50μM, P=0.187, n=10) or the serotonergic 5‐HT4 agonist BIMU8 (10μM, P=0.024, n=6). To test the role of N‐type calcium channels, we showed that nefiracetam, a calcium channel activator, reversed respiratory depression by fentanyl (57±19%, P=0.002, n=9). Formalin (0.05%) significantly increased swimming velocity (320±313% of baseline, P=0.003, n=24–30), which was reduced by fentanyl (3μM, 117±151%, n=10). The effect of fentanyl was blocked by naloxone (5μM, P=0.001, n=16) and CTAP (4μM, P=0.029, n=16). MOR mRNA was expressed in the medulla where respiratory circuits are located.DiscussionOur models show that respiratory depression and analgesia by opioids can be mimicked in zebrafish larvae. The strain differences suggest a potential genetic protection against respiratory depression by opioids which can be uncovered using gene screening and knockout animals. Our novel zebrafish models are powerful models to investigate opioid‐induced respiratory depression and analgesia. Using them, we identified potential molecular targets to develop safe opioid pain therapies, such as calcium channels.Support or Funding InformationSt. Michael’s Hospital Foundation and the J.P. Bicknell Foundation Biomedical Grant

The Stability and Efficacy of Tricaine Methanesulfonate (MS222) Solution After Long-Term Storage.

Tricaine methanesulfonate (MS222) is widely used for the anesthesia and euthanasia of laboratory zebrafish. Fresh solutions have been recommended for each use; however, researchers often mix and store concentrated stock solutions for convenience and to reduce occupational exposure and environmental waste. While this is common practice, published guidelines are often inconsistent. Thus, the objective of this study was to evaluate the stability and anesthetic efficacy of MS222 after long-term storage and to develop specific storage parameters. Stock solutions (100 mg/mL MS222) were mixed and stored in amber jars at 4 °C and -20 °C for 2- and 6-mo. Stability of the solutions was analyzed using liquid chromatography-ion trap mass spectrometry and compared with fresh MS222. Fifty adult (30 male, 20 female) wildtype AB zebrafish (Danio rerio) were randomly anesthetized with 150 mg/L of one of the following MS222 solutions to evaluate anesthetic efficacy: 1) freshly prepared (0m); 2) 2 mo at 4 °C (2m4); 3) 2 mo at -20 °C (2m-20); 4) 6 mo at 4 °C (6m4); 5) 6 mo at -20 °C (6m-20). Time to cessation of swimming, loss of equilibrium, lack of response to von Frey (VF) stimulation, return of equilibrium, and resumption of swimming were compared between groups. Two fish from each group were euthanized at 24-h and 2-wk after anesthesia, and histopathology was performed. All solutions were determined to be stable under all storage conditions. No clinically significant differences were observed between the fresh and stored stock groups during anesthetic testing. No evidence of anesthetic-related histologic changes were noted in the gills, skin, kidneys, muscle, and central nervous system. Hepatic megalocytosis and a reduction in hepatic vacuolation were seen to varying degrees across all groups, but did not follow a treatment-related trend. Therefore, 100 mg/mL solutions of MS222 can be stored in amber jars at 4 °C or -20 °C for 6 mo and still used to effectively anesthetize zebrafish.

Adrenocortical Carcinoma Xenograft in Zebrafish Embryos as a Model To Study the In Vivo Cytotoxicity of Abiraterone Acetate.

Abiraterone acetate (AbiAc) inhibits tumor growth when administered to immunodeficient mice engrafted with the in vitro cell model of human adrenocortical carcinoma (ACC). Here, we developed and validated a zebrafish model engrafted with cortisol-secreting ACC cells to study the effects of AbiAc on tumor growth. The experimental conditions for AbiAc absorption in AB zebrafish embryos including embryo number, AbiAc concentration, and absorption time curve by liquid chromatography-tandem mass spectrometry were set up. The AbiAc effect on steroid production in AB zebrafish embryos was measured as well. ACC cells (the NCI-H295R cell line, the primary cell ACC29, and the negative control cell SW13) were treated with drug-induced liver injury fluorescent dye, and ∼240 cells per 4 nL was injected in the subperidermal space of the yolk sac of AB zebrafish embryos (n = 80 ± 10). The cell area was measured with Noldus DanioScopeTM software. AbiAc absorption in AB zebrafish embryos was stage dependent. Abiraterone (Abi) concentration decreased, whereas its main metabolite, Δ4A, increased. Accordingly, we demonstrated that zebrafish expressed mRNA encoding the enzyme 3β-hydroxysteroid dehydrogenase, which converts Abi in Δ4A. Furthermore, ABiAc reduced cortisol production and increased progesterone in zebrafish embryos. Three days after cell injection, the cortisol-secreting ACC cell area in solvent-treated embryos was significantly higher than that in 1 µM AbiAC‒treated embryos, whereas no AbiAc effect was observed in SW13 cells, which lack the Abi target enzyme CYP17A1.Zebrafish embryos xenografted with ACC tumor cells could be a useful, fast, and reproducible experimental model to preclinically test the activity of new drugs in human ACC.

The emerging contaminant 3,3′-dichlorobiphenyl (PCB-11) impedes Ahr activation and Cyp1a activity to modify embryotoxicity of Ahr ligands in the zebrafish embryo model (Danio rerio)

3,3′-dichlorobiphenyl (PCB-11) is an emerging PCB congener widely detected in environmental samples and human serum, but its toxicity potential is poorly understood. We assessed the effects of three concentrations of PCB-11 on embryotoxicity and Aryl hydrocarbon receptor (Ahr) pathway interactions in zebrafish embryos (Danio rerio). Wildtype AB or transgenic Tg(gut:GFP) strain zebrafish embryos were exposed to static concentrations of PCB-11 (0, 0.2, 2, or 20 μM) from 24 to 96 h post fertilization (hpf), and gross morphology, Cytochrome P4501a (Cyp1a) activity, and liver development were assessed via microscopy. Ahr interactions were probed via co-exposures with PCB-126 or beta-naphthoflavone (BNF). Embryos exposed to 20 μM PCB-11 were also collected for PCB-11 body burden, qRT-PCR, RNAseq, and histology. Zebrafish exposed to 20 μM PCB-11 absorbed 0.18% PCB-11 per embryo at 28 hpf and 0.61% by 96 hpf, and their media retained 1.36% PCB-11 at 28 hpf and 0.84% at 96 hpf. This concentration did not affect gross morphology, but altered the transcription of xenobiotic metabolism and liver development genes, impeded liver development, and increased hepatocyte vacuole formation. In co-exposures, 20 μM PCB-11 prevented deformities caused by PCB-126 but exacerbated deformities in co-exposures with BNF. This study suggests that PCB-11 can affect liver development, act as a partial agonist/antagonist of the Ahr pathway, and act as an antagonist of Cyp1a activity to modify the toxicity of compounds that interact with the Ahr pathway.

Novel diterpenoid-type activators of the Keap1/Nrf2/ARE signaling pathway and their regulation of redox homeostasis

Oxidative stress is involved in the onset and progression of many human diseases. Activators of the Keap1/Nrf2/ARE pathway effectively inhibit the progression of oxidative stress-induced diseases. Herein, a small library of diterpenoids was established by means of phytochemical isolation, and chemical modification on naturally occurring molecules. The diterpenoids were subjected to a NAD(P)H: quinone reductase (QR) assay to evaluate its potential inhibition against oxidative stress. Sixteen diterpenoids were found to be novel potential activators of Nrf2-mediated defensive response. Of which, an isopimarane-type diterpenoid, sphaeropsidin A (SA), was identified as a potent activator of the Keap1/Nrf2/ARE pathway, and displayed approximately 5-folds potency than that of sulforaphane (SF). SA activated Nrf2 and its downstream cytoprotective genes through enhancing the stabilization of Nrf2 in a process involving PI3K, PKC, and PERK, as well as potentially interrupting Nrf2-Keap1 protein-protein interaction. In addition, SA conferred protection against sodium arsenite [As(III)]- and cigarette smoke extract (CSE)-induced redox imbalance and cytotoxicity in human lung epithelial cells, as wells as inhibited metronidazole (MTZ)-induced oxidative insult in Tg (krt4: NTR-hKikGR)cy17 transgenic zebrafish and lipopolysaccharide (LPS)-induced oxidative damage in wild-type AB zebrafish. These results imply that SA is a lead compound for therapeutic agent against oxidative stress-induced diseases, and diterpenoid is a good resource for discovering drug candidates and leads of antioxidant therapy.

The aggressive spiegeldanio, carrying a mutation in the fgfr1a gene, has no advantage in dyadic fights with zebrafish of the AB strain

Zebrafish which carries a mutation in the fibroblast growth factor receptor 1A (fgfr1a), also known as spiegeldanio (spd), has previously been reported to be bolder and more aggressive than wildtype (AB) zebrafish. However, in previous studies aggression has been quantified in mirror tests. In dyadic fights the behavior of the combatants is modified by the behavior of their opponent, and fighting a mirror has been reported to have different effects on brain gene expression and brain monoaminergic systems. In the present study aggression was quantified in fgfr1a mutants and AB zebrafish using a mirror test after which the fish were allowed to interact in pairs, either consisting of two fgfr1a mutants or one AB and one fgfr1a mutant fish. Following dyadic interaction aggressive behavior was again quantified in individual fish in a second mirror test after which the fish were sacrificed and brain tissue analyzed for monoamines and monoamine metabolites. The results confirm that fgfr1a mutants are more aggressive than AB zebrafish in mirror tests. However, fgfr1a mutant fish did not have any advantage in fights for social dominance, and agonistic behavior of fgfr1a mutants did not differ from that of AB fish during dyadic interactions. Moreover, as the AB fish, fgfr1a mutant fish losing dyadic interactions showed a typical loser effect and social subordination resulted in an activation of the brain serotonergic system in fgfr1a mutants as well as in AB fish. Overall the effects of dyadic interaction were similar in fgfr1a mutant fish and zebrafish of the AB strain.

Radioprotective effects of mistletoe extract in zebrafish embryos in vivo

Introduction: Radioprotectors can enhance the efficacy of cancer radiotherapy, but their clinical use remains uncommon. The present study aimed to assess the radioprotective potential of mistletoe extract (commercial name: Abnoba Viscum), a well-known complementary cancer medicine, in zebrafish larvae.Materials and methods: Wild-type AB zebrafish embryos at 4 h-post-fertilization were exposed to 5 Gy 9-MeV electron beam irradiation after being treated for 1 h with 4 mMl/L amifostine or 0.2 mg/ml Abnoba Viscum A, F, M, or Q. Primary endpoints were abnormality-free survival and abnormality-free rates among survivors at 5 days-post-fertilization.Results: The crude abnormality-free survival rates were 33.7%, 49.0%, 38.8%, 43.9%, 38.1%, and 52.6%, whereas abnormality-free rates among survivors were 36.4%, 49.6%, 37.8%, 45.6%, 52.0%, and 62.8% for the control (with no pharmacologic treatment), amifostine, Abnoba Viscum A, F, M, and Q groups, respectively. Abnormality-free survival rates in the amifostine and Abnoba Viscum Q groups were significantly different from those in the control (p = .040 and .012, respectively), with an odds ratio (OR) of 1.90 [95% confidence interval (CI): 1.03–3.51] and 2.20 (95% CI: 1.19–4.08), respectively. Abnormality-free rates among survivors in the amifostine and Abnoba Viscum M and Q groups were significantly different from those in the control group (p = .048, .042, and <.001, respectively), with an OR of 1.79 (95% CI: 1.00–3.20), 1.82 (95% CI: 1.02–3.26), and 2.98 (1.67–5.33), respectively.Conclusion: Abnoba Viscum Q has at least a similar radioprotective effect to that of amifostine. Mistletoe extracts have been clinically applied for a long time and their effectiveness and feasibility have been verified. Abnoba Viscum Q might be a new candidate radioprotectant to enhance cancer radiotherapy efficacy.

Loose social organisation of AB strain zebrafish groups in a two-patch environment.

We study the collective behaviour of zebrafish shoals of different numbers of individuals (1, 2, 3, 5, 7, 10 and 20 AB zebrafish Danio rerio) in a constraint environment composed of two identical square rooms connected by a corridor. This simple set-up is similar to a natural patchy environment. We track the positions and the identities of the fish and compute the metrics at the group and at the individual levels. First, we show that the number of fish affects the behaviour of each individual in a group, the cohesion of the groups, the preferential interactions and the transition dynamics between the two rooms. Second, during collective departures, we show that the rankings of exit correspond to the topological organisations of the fish prior to their collective departure. This spatial organisation appears in the group a few seconds before a collective departure. These results provide new evidences on the spatial organisation of the groups and the effect of the number of fish on individual and collective behaviours in a patchy environment.

Transplantation of Human-Induced Pluripotent Stem Cell-Derived Neural Precursors into Early-Stage Zebrafish Embryos.

Induced pluripotent stem cells (iPS cells) generated from somatic cells through reprogramming hold great promises for regenerative medicine. However, how reprogrammed cells survive, behave in vivo, and interact with host cells after transplantation still remains to be addressed. There is a significant need for animal models that allow in vivo tracking of transplanted cells in real time. In this regard, the zebrafish, a tropical freshwater fish, provides significant advantage as it is optically transparent and can be imaged in high resolution using confocal microscopy. The principal goal of this study was to optimize the protocol for successful short-term and immunosuppression-free transplantation of human iPS cell-derived neural progenitor cells into zebrafish and to test their ability to differentiate in this animal model. To address this aim, we isolated human iPS cell-derived neural progenitor cells from human fibroblasts and grafted them into (a) early (blastocyst)-stage wild-type AB zebrafish embryos or (b) 3-day-old Tg(gfap:GFP) zebrafish embryos (intracranial injection). We found that transplanted human neuronal progenitor cells can be effectively grafted and that they differentiate and survive in zebrafish for more than 2weeks, validating the model as an ideal platform for in vivo screening experiments. We conclude that zebrafish provides an excellent model for studying iPS cell-derived cells in vivo.

Short exposure to low concentrations of alcohol during embryonic development has only subtle and strain- dependent effect on the levels of five amino acid neurotransmitters in zebrafish

The zebrafish has been successfully employed to model and study the effects of embryonic alcohol exposure. Short exposure to low alcohol concentrations during embryonic development has been shown to significantly disrupt social behavior as well as the dopaminergic and serotoninergic systems in zebrafish. However, analysis of potential effects of embryonic alcohol exposure on other amino acid neurotransmitter systems has not been performed. Here we analyzed neurochemicals obtained from adult AB and TU strain zebrafish that were immersed in 0.00% (control), 0.25%, 0.50%, 0.75% or 1.00% alcohol solution (vol/vol%) at 24 h post-fertilization for 2 h. From whole brain extracts, we quantified glutamate, aspartate, glycine, taurine and GABA levels using high performance liquid chromatography (HPLC). We found embryonic alcohol exposure not to have any significant effect on the levels of glutamate, aspartate, glycine and GABA in both AB and TU zebrafish. AB zebrafish showed a significant elevation of taurine levels, but only in the highest alcohol dose group compared to control. These results, albeit mainly negative, together with prior findings suggest that behavioral abnormalities resulting from embryonic alcohol exposure described before for AB zebrafish may primarily be due to altered dopaminergic and serotoninergic mechanisms. Furthermore, a Principal Component Analysis conducted with all neurochemicals tested in this and in our prior study, found a strain-dependent correlation structure response to embryonic alcohol treatment, confirming that embryonic alcohol effects may be genotype dependent.

A Novel Zebrafish (Danio rerio) Assay for Assessing Musk Ambrette-Induced Toxicity.

Musk ambrette (4-tert-butyl-3-methoxy-2,6-dinitrotoluene) is a nitro musk, a cheap substitute for natural musk and a potential environmental pollutant based on its persistence, accumulation in human organisms. We investigated the acute toxicity of musk ambrette using wild-type AB and transgenic Tg(fli1a:EGFP)y1 zebrafish. Different concentrations were delivered to zebrafish by direct soaking from 6 to 72h post-fertilization (hpf). The LC50 of musk ambrette was 76.4µgmL-1. As musk ambrette concentration increased, zebrafish embryos showed developmental delays (50µgmL-1, 22 hpf), pericardial edema (5µgmL-1, 48 hpf), circulatory disturbances, curved body axis (1µgmL-1, 72 hpf) and death (100µgmL-1, 22 hpf). Target organ toxicity was evaluated by a zebrafish angiogenesis model. Musk ambrette induced cardiovascular morphological changes, vessel permeability variation, angiogenic changes and cardiotoxicity (10µgmL-1, 48 hpf). The disappearance of caudal vein plexus confirmed the vascular development toxicity. Musk ambrette negatively affects early life-stage survival and demonstrates various toxicities in zebrafish.

Advanced age does not diminish phagocytic activity or NET production in zebrafish (Danio rerio) kidney leukocytes

Abstract The pace of growth in populations over the age of 65 is increasing in the U.S. and worldwide, posing serious challenges for health care systems. Studies in both humans and mice have revealed evidence of dysregulation of innate immunity in older individuals, although experimental systems and results of those studies vary widely. To explore whether the zebrafish (Danio rerio) model could be valuable in understanding the effects of aging on innate immune processes, we assessed phagocytic activity, respiratory burst response and neutrophil extracellular trap formation (NETosis) by anterior kidney phagocytes from healthy young (7–8 months old) and aged (&amp;gt;2.5 year old) zebrafish. Exposing leukocytes from transgenic mpx:GFP fish to opsonized pHrodo-labeled killed E. coli for 30 or 60 minutes revealed is no difference in the mean percentage of neutrophils that phagocytosed bacteria between the two ages at either time point. Interestingly, older fish displayed a wider range of responses and a higher level of individual variability than the younger population. Active neutrophils from the aged fish also exhibited a higher mean fluorescence intensity (MFI) than young fish at 30 min incubation, but fluorescence levels were similar in the two populations after 60 minutes of incubation. Leukocytes from older and younger wild type AB fish showed identical respiratory burst responses to PMA. When NET production was stimulated by calcium ionophore, neutrophils from aged AB zebrafish exhibited a higher NET index than the younger cohort. These initial results indicate that older zebrafish preserve a similar level of activity, if not hints of higher activity, in tested neutrophil functions compared to younger fish.