An introduction to high-throughput behavioral screening

PUBLICATIONS

Mitigating Human IAPP Amyloidogenesis In Vivo with Chiral Silica Nanoribbons

2013-01-01

Abstract

Amyloid fibrils generally display chirality, a feature which has rarely been exploited in the development of therapeutics against amyloid diseases. This study reports, for the first time, the use of mesoscopic chiral silica nanoribbons against the in vivo amyloidogenesis of human islet amyloid polypeptide (IAPP), the peptide whose aggregation is implicated in type 2 diabetes. The thioflavin T assay and transmission electron microscopy show accelerated IAPP fibrillization through elimination of the nucleation phase and shortening of the elongation phase by the nanostructures. Coarse‐grained simulations offer complementary molecular insights into the acceleration of amyloid aggregation through their nonspecific binding and directional seeding with the nanostructures. This accelerated IAPP fibrillization translates to reduced toxicity, especially for the right‐handed silica nanoribbons, as revealed by cell viability, helium ion microscopy, as well as zebrafish embryo survival, developmental, and behavioral assays. This study has implicated the potential of employing chiral nanotechnologies against the mesoscopic enantioselectivity of amyloid proteins and their associated diseases.

 

Link to the publication :

https://onlinelibrary.wiley.com/doi/abs/10.1002/smll.201802825

Neurobehavioral effects of two metabolites of BDE-47 (6-OH-BDE-47 and 6-MeO-BDE-47) on zebrafish larvae

Chemosphere

2013-01-01

Highlights

The neurobehavioral toxicity of 6-OH/MeO-BDE47 were evaluated with zebrafish larvae.

6-OH-BDE47 performed inhibiting effects on routine and average turns.

Different from 6-OH-BDE-47, 6-MeO-BDE47 mainly promoted responsive turns.

6-MeO-BDE-47 performed more adverse effects on social activity.


Abstract

Two metabolites, OH-BDEs and MeO-BDEs, of polybrominated diphenyl ethers (PBDEs) were ubiquitously detected in animal tissues and environmental samples, drawing a widely public concern to their toxicity. The comparison of toxicity between PBDEs and their metabolites has been a focus in recent years, however, comparisons seldom involve neurobehavioral toxicity of PBDEs metabolites in published works. In this study, zebrafish larvae were exposed to 6-OH-BDE-47 and 6-MeO-BDE-47 and their neurobehavioral traits (including locomotion, path angle, and social activity) were recorded using the instrument Zebrabox; meanwhile, light illumination was used as stimuli in the test duration. The results showed larvae were more active in dark periods than light periods, and preferred turning right (+) to left (−). Effects of the two metabolites varied in different behavioral indicators. They induced different effects on path angle but did not reverse the left-right asymmetry. 6-OH-BDE-47 did not induce the effects on larval locomotion and social activity, but mainly decreased average and routine turn numbers; 6-MeO-BDE-47 promoted larvae responsive turns but inhibited social activity. This study offered new experimental means to the neurobehavioral toxicity of various PBDE metabolites. Further studies may focus on the toxic mechanisms of specific neurobehavioral traits.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653518302662

Protecting the environment from psychoactive drugs: Problems for regulators illustrated by the possible effects of tramadol on fish behaviour

Science of the total environment

2014-01-01

Highlights

• Fish were exposed to the psychoactive drugs tramadol and fluoxetine.
• Alteration of fish behaviour was assessed in a novel tank diving test.
• The results were not easy to interpret with confidence.
• Ecotoxicological experts reached different conclusions based on the same results.
• Determining whether or not psychoactive drugs alter the behaviour of fish is difficult.

Abstract

There is concern that psychoactive drugs present in the aquatic environment could affect the behaviour of fish, and other organisms, adversely. There is considerable experimental support for this concern, although the literature is not consistent. To investigate why, fish were exposed to three concentrations of the synthetic opiate tramadol for 23–24 days, and their anxiolytic behaviour in a novel tank diving test was assessed both before and after exposure. The results were difficult to interpret. The positive control drug, the anti-depressant fluoxetine, produced the expected results: exposed fish explored the novel tank more, and swam more slowly while doing so. An initial statistical analysis of the results provided relatively weak support for the conclusion that both the low and high concentrations of tramadol affected fish behaviour, but no evidence that the intermediate concentration did. To gain further insight, UK and Japanese experts in ecotoxicology were asked for their independent opinions on the data for tramadol. These were highly valuable. For example, about half the experts replied that a low concentration of a chemical can cause effects that higher concentrations do not, although a similar number did not believe this was possible. Based both on the inconclusive effects of tramadol on the behaviour of the fish and the very varied opinions of experts on the correct interpretation of those inconclusive data, it is obvious that more research on the behavioural effects of tramadol, and probably all other psychoactive drugs, on aquatic organisms is required before any meaningful risk assessments can be conducted. The relevance of these findings may apply much more widely than just the environmental risk assessment of psychoactive drugs. They suggest that much more rigorous training of research scientists and regulators is probably required if consensus decisions are to be reached that adequately protect the environment from chemicals.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0048969719305716

Advancing the zebrafish embryo test for endocrine disruptor screening using micro‐injection: ethinyl estradiol as a case study

Environmental Toxicology and chemistry

2014-01-01

ABSTRACT

Fish (embryo) toxicity test guidelines are mostly based on aquatic exposures. However, in some cases, other exposure routes can be more practical and relevant. Micro‐injection into the yolk of fish embryos could offer a particular advantage for administering hydrophobic compounds, such as many endocrine disruptors. Single dose micro‐injection was compared to continuous aquatic exposure in terms of compound accumulation and biological responses. 17α‐ethinyl estradiol (EE2) was used as a model compound. First, the optimal solvent and droplet size were optimized, and needle variation was assessed. Next, biological endpoints were evaluated. The accumulated internal dose of EE2 decreased over time in both exposure scenarios. Estrogen receptor (ER) activation was concentration/injected dose dependent, increased daily and was related to esr2b transcription. Vtg1 and cyp19a1b transcription was induced in both scenarios, but the cyp19a1b transcription pattern differed between routes. Injection caused an increase of cyp19a1b transcripts from 48 hours post fertilization (hpf) onwards, while after aquatic exposure the main increase occurred between 96 and 120 hpf. Some malformations only occurred after injection, while others were present for both scenarios. We conclude that responses can differ between exposure routes and therefore micro‐injection is not a direct substitute for, but can be complementary to aquatic exposure. Nevertheless, vtg1 and cyp19a1b transcription and ER activation are suitable biomarkers for endocrine disruptor screening in both scenarios.

Link to the publication :

https://setac.onlinelibrary.wiley.com/doi/abs/10.1002/etc.4343

Long-term effects of oxytetracycline exposure in zebrafish: A multi-level perspective

Chemosphere

2014-01-01

Highlights

• Zebrafish exposed to oxytetracycline present different feeding behavior pattern.
• Oxytetracycline induced fish boldness and hyperactivity in light periods.
• Energetic reserves and oxidative stress enzymes decrease after long-term exposure.
• Bacterial communities of fish gut and exposure water were affected by oxytetracycline.

Abstract

Oxytetracycline (OTC) is a broad-spectrum antibiotic widely used in livestock production. Like many other pharmaceuticals, OTC is not completely metabolized by the organism and thus, increasing amounts of the compound are being detected in the aquatic environment. The assessment of the environmental risk of pharmaceuticals is hindered by their very low concentrations and specific modes of action and thus relevant exposure scenarios and sensitive endpoints are needed. Thus, this work aimed to study the long-term effect of OTC exposure in zebrafish (at behavior and biochemical levels) and associated bacterial communities (fish gut and water bacterial communities). Results revealed that at behavioral level, boldness increase (manifested by increased exploratory behavior of a new environment) was observed in fish exposed to low OTC concentrations. Moreover, changes in fish swimming pattern were observed in light periods (increased stress response: hyperactivity and freezing) probably due to photo-sensibility conferred by OTC exposure. Effects at biochemical level suggest that long-term exposure to OTC interfere with cellular energy allocation mainly by reducing lipids levels and increasing energy consumption. Moreover, evidences of oxidative damage were also observed (reduced levels of TG, GST and CAT). The analysis of water and gut microbiome revealed changes in the structure and diversity of bacterial communities potentially leading to changes in communities' biological function. Some of the effects were observed at the lowest concentration tested, 0.1 μg/L which is a concentration already detected in the environment and thus clearly demonstrating the need of a serious ecotoxicological assessment of OTC effects on non-target organisms.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653519301638

NEUROPROTECTIVE COMPOSITION , PREPARATION PROCESS THEREOF AND MEDICAL USES THEREOF

2014-01-01

ABSTRACT

The invention relates to a neuroprotective composition derived from mesenchymal stem cells , especially a neuro protective composition derived from the primary culture of dental pulp mesenchymal stem cells . The invention also relates to a process for preparing the neuroprotective com position , as well as the medical use of the neuroprotective composition in the treatment of Parkinson ' s disease .

 

Link to the publication :

https://patentimages.storage.googleapis.com/cf/7a/95/2fddaa73d87899/US20190167727A1.pdf

Neural signatures of sleep in zebrafish

Nature

2014-01-01

Abstract

Slow-wave sleep and rapid eye movement (or paradoxical) sleep have been found in mammals, birds and lizards, but it is unclear whether these neuronal signatures are found in non-amniotic vertebrates. Here we develop non-invasive fluorescence-based polysomnography for zebrafish, and show—using unbiased, brain-wide activity recording coupled with assessment of eye movement, muscle dynamics and heart rate—that there are at least two major sleep signatures in zebrafish. These signatures, which we term slow bursting sleep and propagating wave sleep, share commonalities with those of slow-wave sleep and paradoxical or rapid eye movement sleep, respectively. Further, we find that melanin-concentrating hormone signalling (which is involved in mammalian sleep) also regulates propagating wave sleep signatures and the overall amount of sleep in zebrafish, probably via activation of ependymal cells. These observations suggest that common neural signatures of sleep may have emerged in the vertebrate brain over 450 million years ago.

 

Link to the publication : https://www.nature.com/articles/s41586-019-1336-7

Identifcation of pathways that regulate circadian rhythms using a larval zebrafsh small molecule screen

Nature

2014-01-01

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

Introduction

Circadian rhythms help ensure that physiological processes and behaviors occur at appropriate times during the 24-hour day/night cycle. These rhythms are generated and sustained at the cellular level by a transcriptional-translational negative-feedback loop that cycles with a period of approximately 24 hours, and are entrained by environmental cues such as light, food availability and temperature1. Molecular circadian oscillations in cells in different tissues and brain regions cycle with distinct phases, suggesting that non-cell autonomous mechanisms transmit circadian information throughout an animal1. While the suprachiasmatic nucleus (SCN) acts as a central circadian pacemaker to regulate circadian rhythms in mammals, it is unclear how the SCN transmits circadian information and if additional non-cell autonomous mechanisms exist. It is also unclear whether some aspects of the vertebrate circadian clock, which has primarily been studied using nocturnal rodents, differ from those in diurnal vertebrates such as humans. Thus, despite extensive research, mechanisms that regulate circadian rhythms remain incompletely understood.

Attempts to identify these mechanisms have primarily used two approaches. Genetic screens in model organisms have identified many components of the molecular circadian clock1. However, this approach may fail to detect genes that regulate aspects of the clock that do not affect the phenotype studied, or genes that have functionally redundant paralogs. Genetic screens are also difficult to perform using vertebrate animals. More recently, luminescent reporters have been used in cell culture to screen for cell autonomous factors that regulate the mammalian molecular circadian oscillator2,3,4,5,6,7,8. However, this approach lacks in vivo relevance and will not detect mechanisms that act non-cell autonomously or do not operate in the cell types used. Thus, alternative approaches could reveal novel mechanisms that regulate the circadian clock.

Most small molecule screens use in vitro or cell culture assays to identify drugs that bind a specific target or affect a specific process. However, these screens do not recreate the complex environment of whole animals and likely fail to identify some mechanisms that regulate the process under study. To overcome these limitations, we and others have used intact zebrafish as a vertebrate model system for small molecule screens9. This approach combines the in vivo relevance of whole-animal assays with moderate-throughput, low-cost drug screening. It also exploits several features of zebrafish larvae, including a relatively simple yet conserved vertebrate brain that lacks a mature blood-brain-barrier10, a small size that allows for screening in multi-well plates, and optical transparency that facilitates the use of luminescent reporters. Importantly, for the purposes of circadian research, the zebrafish molecular circadian oscillator closely resembles that of mammals11.

Here we describe a screen for small molecules that affect molecular circadian rhythms using a luminescent reporter in zebrafish larvae. We also monitor behavioral circadian rhythms using an assay that we previously used to identify drugs that regulate larval zebrafish locomotor behaviors12. We show that small molecules targeting pathways known to affect the circadian clock induce the expected circadian phenotypes in intact zebrafish. We also identify drugs that implicate novel pathways in regulating circadian rhythms in vivo that are absent in cultured cells. Finally, we show that inflammatory state affects circadian amplitude using both drugs and xpr1b mutant zebrafish, which lack microglia. These results reveal an unexpected role for the immune system in regulating the circadian clock.

Results

A screen for small molecules that affect molecular circadian rhythms in zebrafish larvae

A previous study described transgenic zebrafish in which the promoter for the period3 gene regulates expression of firefly luciferase (Tg(per3:luc)), and showed that this line accurately reports molecular circadian rhythms in zebrafish larvae13. To test whether this line could be used to screen for small molecules that affect molecular circadian rhythms, we asked whether compounds that affect the circadian clock in cell culture induce similar effects in zebrafish larvae. We entrained Tg(per3:luc) larvae in 14:10 hour light:dark (LD) conditions for 6 days at 22 °C13. We then placed individual larvae into each well of a 96-well plate, added small molecules or DMSO vehicle control to each well, and monitored luminescence for 72 hours in constant darkness (DD) (Fig. 1A). To validate our assay, we first tested a drug that targets a pathway known to affect circadian period length. Pharmacological inhibition of casein kinase 1 (CK1) increases period length in mammalian cell culture3,5,14, rodents5,15 and zebrafish5,15,16, similar to some ck1 mutant animals17,18,19,20. We tested a compound, A002195858, that inhibits CK1 in vitro (IC50 = 23 nM) and dose-dependently increases period length in mammalian cells (Fig. S2F), and found that it also dose-dependently increases period length in our larval zebrafish assay (Fig. 1B). We also found that the Src kinase inhibitor SU-665621 dose-dependently increases circadian amplitude in our assay (Fig. 1C). These results indicate that Tg(per3:luc) larvae can be used to report drug-induced changes in molecular circadian rhythms, and that phenotypes observed in mammalian cells can also be observed in zebrafish larvae.

Interleukin 6 deficiency affects spontaneous activity of mice in age- and sex-dependent manner

2014-04-01

Abstract

We analyzed the role of interleukin 6 (IL-6) in modulation of the pattern of mice spontaneous activity. Wild type (WT) and IL-6 deficient mice of both sexes, young and aging, were housed individually and various types of their activity were recorded and analyzed with the Phenorack system in their home cages during 72 hours-long sessions. All investigated groups of mice were active mainly during the dark phase of the 24-hours cycle. Generally, the IL-6 deficient animals were more active than their WT controls and females of both genotypes more active than males. Aging mice were less active than the sex and genotype-matched young animals. The independent variables (age, sex and genotype) strongly interacted, which suggests that the modulatory influence of IL-6 on mice behavior may be different in males and females and that it changes during aging. We conclude that under normal physiological conditions signaling of IL-6 via its receptor participates in modulation of the basic pattern of activity. This modulation differs in males and females and changes with aging.

Automated, high-throughput, in vivo analysis of visual function using the zebrafish

Developmental Dynamics

2016-03-06

Abstract

Background

Modern genomics has enabled the identification of an unprecedented number of genetic variants, which in many cases are extremely rare, associated with blinding disorders. A significant challenge will be determining the pathophysiology of each new variant. The zebrafish is an excellent model for the study of inherited diseases of the eye. By 5 days-post-fertilization (dpf) they have quantifiable behavioral responses to visual stimuli. However, visual behavior assays can take several hours to perform or can only be assessed one fish at a time.

Results

To increase the throughput for vision assays, we used the Viewpoint Zebrabox to automate the visual startle response and created software, Visual Interrogation of Zebrafish Manipulations (VIZN), to automate data analysis. This process allows 96 zebrafish larvae to be tested and resultant data to be analyzed in under 35 minutes. We validated this system by disrupting function of a gene necessary for photoreceptor differentiation and observing decreased response to visual stimuli.

Conclusions

This automated method along with VIZN allows rapid, high-throughput, in vivo testing of zebrafish’s ability to respond to light/dark stimuli. This allows the rapid analysis of novel genes involved in visual function by morpholino, CRISPRS, or small molecule drug screens.

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

2016-10-31

ABSTRACT

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To  analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days postfertilization (dpf) typically exhibit a powerful burst swim,  accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by
muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

 

Link to the publication :

https://www.jove.com/video/54431/dokunmatik-uyarlm-yant-ve-hareket-deneyleri-kullanarak-zebra-bal-kas?language=Turkish

A New Statistical Approach to Characterize Chemical-Elicited Behavioral Effects in High-Throughput Studies Using Zebrafish

2017-01-18

Abstract

Zebrafish have become an important alternative model for characterizing chemical bioactivity, partly due to the efficiency at which systematic, high-dimensional data can be generated. However, these new data present analytical challenges associated with scale and diversity. We developed a novel, robust statistical approach to characterize chemical-elicited effects in behavioral data from high-throughput screening (HTS) of all 1,060 Toxicity Forecaster (ToxCast™) chemicals across 5 concentrations at 120 hours post-fertilization (hpf). Taking advantage of the immense scale of data for a global view, we show that this new approach reduces bias introduced by extreme values yet allows for diverse response patterns that confound the application of traditional statistics. We have also shown that, as a summary measure of response for local tests of chemical-associated behavioral effects, it achieves a significant reduction in coefficient of variation compared to many traditional statistical modeling methods. This effective increase in signal-to-noise ratio augments statistical power and is observed across experimental periods (light/dark conditions) that display varied distributional response patterns. Finally, we integrated results with data from concomitant developmental endpoint measurements to show that appropriate statistical handling of HTS behavioral data can add important biological context that informs mechanistic hypotheses.

 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169408

Vitamin E deficiency during embryogenesis in zebrafish causes lasting metabolic and cognitive impairments despite refeeding adequate diets

ScienceDirect

2017-06-20

Abstract

Vitamin E (α-tocopherol; VitE) is a lipophilic antioxidant required for normal embryonic development in vertebrates, but the long-term effects of embryonic VitE deficiency, and whether they are ameliorated by feeding VitE–adequate diets, remain unknown. We addressed these questions using a zebrafish (Danio rerio) model of developmental VitE deficiency followed by dietary remediation. Adult zebrafish maintained on VitE–deficient (E–) or sufficient (E+) diets were spawned to obtained E– and E+ embryos, respectively, which we evaluated up to 12 days post-fertilization (dpf). The E– group suffered significantly increased morbidity and mortality as well as altered DNA methylation status through 5 dpf when compared to E+ larvae, but upon feeding with a VitE-adequate diet from 5–12 dpf both the E– and E+ groups survived and grew normally; the DNA methylation profile also was similar between groups by 12 dpf. However, 12 dpf E– larvae still had behavioral defects. These observations coincided with sustained VitE deficiency in the E– vs. E+ larvae (p< 0.0001), despite adequate dietary supplementation. We also found in E– vs. E+ larvae continued docosahexaenoic acid (DHA) depletion (p< 0.0001) and significantly increased lipid peroxidation. Further, targeted metabolomics analyses revealed persistent dysregulation of the cellular antioxidant network, the CDP-choline pathway, and glucose metabolism. While anaerobic processes were increased, aerobic metabolism was decreased in the E– vs. E+ larvae, indicating mitochondrial damage. Taken together, these outcomes suggest embryonic VitE deficiency causes lasting behavioral impairments due to persistent lipid peroxidation and metabolic perturbations that are not resolved via later dietary VitE supplementation.

 

 

http://www.sciencedirect.com/science/article/pii/S0891584917306457

A Brain-Derived Neurotrophic Factor Mimetic Is Sufficient to Restore Cone Photoreceptor Visual Function in an Inherited Blindness Model

Scientific reports

2017-09-12

Abstract

Controversially, histone deacetylase inhibitors (HDACi) are in clinical trial for the treatment of inherited retinal degeneration. Utilizing the zebrafish dye ucd6 model, we determined if treatment with HDACi can rescue cone photoreceptor-mediated visual function. dye exhibit defective visual behaviour and retinal morphology including ciliary marginal zone (CMZ) cell death and decreased photoreceptor outer segment (OS) length, as well as gross morphological defects including hypopigmentation and pericardial oedema. HDACi treatment of dye results in significantly improved optokinetic (OKR) (~43 fold, p < 0.001) and visualmotor (VMR) (~3 fold, p < 0.05) responses. HDACi treatment rescued gross morphological defects and reduced CMZ cell death by 80%. Proteomic analysis of dye eye extracts suggested BDNF-TrkB and Akt signaling as mediators of HDACi rescue in our dataset. Co-treatment with the TrkB antagonist ANA-12 blocked HDACi rescue of visual function and associated Akt phosphorylation. Notably, sole treatment with a BDNF mimetic, 7,8-dihydroxyflavone hydrate, significantly rescued dye visual function (~58 fold increase in OKR, p < 0.001, ~3 fold increase in VMR, p < 0.05). In summary, HDACi and a BDNF mimetic are sufficient to rescue retinal cell death and visual function in a vertebrate model of inherited blindness.

 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595969/

Endocrine disruptors affect larval zebrafish behavior: Testing potential mechanisms and comparisons of behavioral sensitivity to alternative biomarkers

Aquatic Toxicology

2017-10-06

Abstract

Larval zebrafish (Danio rerio) are a tool for assessing endocrine disruption during early development. Here, we investigated the extent to which a simple light/dark behavioral test at five days post fertilization could compliment current methods within the field. We exposed fertilized embryos to hormones (17β-estradiol, testosterone, dihydrotestosterone, 11-ketotestosterone, thyroxine, triiodothyronine, progesterone, and hydrocortisone) and other relevant compounds (17α ethinylestradiol, bisphenol A, bisphenol S, nonylphenol, flutamide, nilutamide, linuron, drospirenone, potassium perchlorate, mifepristone, and fadrozole) to screen for behavioral effects between 96 and 118 hours post fertilization (hpf). With the exception of progesterone, all the hormones tested resulted in altered behaviors. However, some inconsistencies were observed regarding the age of the larvae at testing. For example, the xenoestrogens 17α- ethinylestradiol and nonylphenol had behavioral effects at 96 hpf, but not at 118 hpf. Furthermore, although thyroxine exposure had pronounced effects on behavior, the thyroid disruptor potassium perchlorate did not. Finally, we were unable to demonstrate a role of nuclear receptors following testosterone and 17α- ethinylestradiol exposure, as neither the androgen receptor antagonist flutamide nor the general estrogen receptor inhibitor fulvestrant (ICI) could rescue the observed behavioral effects, respectively. Similarly, molecular markers for androgen and estrogen disruption were upregulated at concentrations below which behavioral effects were observed. These results demonstrate hormones and endocrine disruptors can alter the behavior of larval zebrafish, but the mechanistic pathways remain unclear.

 

http://www.sciencedirect.com/science/article/pii/S0166445X17302801

Comorbid masticatory impairment delays recovery from acute cerebral ischemia and locomotor hypoactivity after subarachnoid hemorrhage in mice

Clinical and Experimental Pharmacology and Physiology

2017-10-17

Summary

Tooth loss and related changes in the functionality may lead to worse outcome of stroke patients, but the link involved in this pathway remains unclear. This study aimed to determine the effect of comorbid masticatory impairment on acute cerebral ischemia and neurobehavioral outcome after experimental subarachnoid hemorrhage (SAH). Thirty C57BL/6 mice with (molar-less) or without (control) prior treatment of cutting off the upper molars (separated by at least one week), were subjected to SAH by endovascular perforation. Grading of SAH and acute cerebral infarction were assessed by T2*- and diffusion-weighted MR images, respectively. Cerebral blood flow (CBF) by continuous arterial spin labeling and parameters related to locomotor activity and exploration ability by open-field test were analyzed serially until 2 weeks after SAH. In all mice, global CBF depression was notable immediately after SAH induction (P<0.001), which recovered close to the baseline levels until day 7. However, molar-less mice demonstrated a prolonged hypoperfusion (<50% of the baseline CBF) as compared to the control (3 ± 2 vs. 1 ± 1 days; P=0.037). The average distance traveled and the ratio of central-area distance/total traveled distance by open-field test between 1 and 2 weeks was significantly lower in molar-less mice than in the control mice (P<0.05), although the occurrence of post-SAH new infarction were not statistically different (P>0.05). These data suggest a possible link between comorbid masticatory impairment and early brain injury/ischemia to deteriorate neurobehavioral functional outcome in patients after SAH.

 

http://onlinelibrary.wiley.com/doi/10.1111/1440-1681.12874/full

Ablation of TFR1 in Purkinje cells inhibits mGlu1 trafficking and impairs motor coordination, but not autistic-like behaviors

Journal of Neuroscience

2017-10-20

Abstract

Group 1 metabotropic glutamate receptors (mGlu1/5) are critical to synapse formation and participate in synaptic long-term potentiation (LTP) and long-term depression (LTD) in the brain. mGlu1/5 signaling alterations have been documented in cognitive impairment, neurodegenerative disorders, and psychiatric diseases, but underlying mechanisms for its modulation are not clear. Here, we report that transferrin receptor 1 (TFR1), a trans-membrane protein of clathrin complex, modulates the trafficking of mGlu1 in cerebellar Purkinje cells (PCs) from male mice. We show that conditional knockout of TFR1 in PCs does not affect the cyto-architecture of PCs, but reduces mGlu1 expression at synapses. This regulation by TFR1 acts in concert with that by Rab8 and Rab11, which modulate the internalization and recycling of mGlu1, respectively. TFR1 can bind to Rab proteins and facilitate their expression at synapses. PC ablation of TFR1 inhibits parallel fiber-PC LTD, whereas parallel fiber-LTP and PC intrinsic excitability are not affected. Finally, we demonstrate that PC ablation of TFR1 impairs motor coordination, but does not affect social behaviors in mice. Together, these findings underscore the importance of TFR1 in regulating mGlu1 trafficking and suggest that mGlu1 and mGlu1-dependent parallel fiber-LTD are associated with regulation of motor coordination, but not autistic behaviors.

Significance statement

mGlu1/5 signaling alterations have been documented in cognitive impairment, neurodegenerative disorders and psychiatric diseases. Recent work suggests that altered mGlu1 signaling in PCs may be involved in not only motor learning, but also autistic-like behaviors. We find that conditional knockout of TFR1 in PCs reduces synaptic mGlu1 by tethering Rab8 and Rab11 in the cytosol. PC ablation of TFR1 inhibits parallel fiber-PC LTD, whereas parallel fiber-PC LTP and PC intrinsic excitability are intact. Motor coordination is impaired, but social behaviors are normal in TFR1flox/flox;pCP2-cre mice. Our data reveal a new regulator for trafficking and synaptic expression of mGlu1 and suggest that mGlu1-dependent LTD is associated with motor coordination, but not autistic-like behaviors.

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

PLoS ONE

2017-10-20

Abstract

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186645

Telmisartan Activates PPARδ to Improve Symptoms of Unpredictable Chronic Mild Stress-Induced Depression in Mice

Nature

2017-10-25

Abstract

Major depression is a common mental disorder that has been established to be associated with a decrease in serotonin and/or serotonin transporters in the brain. Peroxisome proliferator-activated receptor δ (PPARδ) has been introduced as a potential target for depression treatment. Telmisartan was recently shown to activate PPARδ expression; therefore, the effectiveness of telmisartan in treating depression was investigated. In unpredictable chronic mild stress (UCMS) model, treatment with telmisartan for five weeks notably decrease in the time spent in the central and the reduced frequency of grooming and rearing in open filed test (OFT) and the decreased sucrose consumption in sucrose preference test (SPT) compared with the paradigms. Telmisartan also reversed the decrease in PPARδ and 5-HTT levels in the hippocampus of depression-like mice. Administration of PPARδ antagonist GSK0660 and direct infusion of sh-PPARδ into the brain blocked the effects of telmisartan on the improvement of depression-like behavior in these mice. Moreover, telmisartan enhanced the expression of PPARδ and 5HTT in H19-7 cells. In conclusion, the obtained results suggest that telmisartan improves symptoms of stress-induced depression in animals under chronic stress through activation of PPARδ. Therefore, telmisartan may be developed as a potential anti-depressant in the future.

 

Full publication available on nature : https://www.nature.com/articles/s41598-017-14265-4

Role of neurexin2a in lead-induced locomotor defect in developing zebrafish

Aquatic Toxicology

2017-11-22

Abstract

Low-dose chronic lead (Pb) exposure interferes with the development of the nervous system, which may lead to learning disabilities, behavioral abnormalities, and mental retardation. Neurexins (Nrxns) are synaptic cell-adhesion molecules associated with neurological disorders. We hypothesized that Pb can affect the expression of nrxns during synapse formation and alter the phenotype behavior. Here, apoptosis, nrxns mRNA expression, and alterations of locomotion were examined after exposure to Pb in zebrafish embryos/larvae. To confirm the function of nrxn2a, rescue experiments were performed using β-nrxn2a mRNA microinjection. Pb exposure increased apoptosis and altered locomotor behavior in zebrafish larvae. Quantitative PCR showed that among several synaptic adhesion molecules, only nrxn2a were affected by Pb exposure. Moreover, exposure to Pb at 10 μmol/L upregulated mRNA expression of nrxn1a and nrxn3a at 24 hours post fertilization (hpf) and downregulated expression at 48 hpf, whereas the expression remained unchanged at 72 hpf. Only the two isoforms of nrxn2a were downregulated by Pb at 10 μmol/L at all three time points. Rescue experiments showed that nrxn2ab mRNA injection recovered the decreased locomotor activity and the increased apoptosis induced by Pb. In addition, overexpression of β-nrxn2a mRNA upregulated α-nrxn2a. These data indicated that Pb inhibited the expression of nrxn2a genes, which play a critical role in neural development, and further altered the behavior of zebrafish embryos/larvae.

 

https://www.sciencedirect.com/science/article/pii/S0166445X17303375

Dissociation of place preference and tolerance responses to sucrose using a dopamine antagonist in the planarian

Psychopharmacology

2017-12-02

Abstract

In rodents, sucrose has been found to elicit addictive-like behaviours like the development of tolerance and the association with cues present at the time of consumption. Furthermore, the neurochemical response to sucrose binges is equivalent to the one observed in response to the abuse of addictive substances like cocaine. The experiments reported here address the effects of sucrose on an invertebrate model, the Platyhelminth brown planarian. The animals exposed to a 10% sucrose solution in one context developed a conditioned place preference (CPP) which was subsequently extinguished in the absence of the rewarding agent. However, one exposure to sucrose per se sufficed to reinstate the CPP response, suggesting sucrose-induced CPP can be characterised as a standard Pavlovian response. The same training procedure led to the development of context-specific tolerance to the effects of sucrose. However, comparing animals treated with dopamine D1 antagonist (SCH-23390) with control animals showed that the establishment of CPP, but not the development of tolerance, is mediated by the dopamine reward system.

 

https://link.springer.com/article/10.1007/s00213-017-4801-8

Novel Kunitz-like peptides discovered in the zoanthid Palythoa caribaeorum through transcriptome sequencing

Journal of Proteome

2017-12-29

Abstract

Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, based on the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified, including allergens, neurotoxins, membrane-active and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1 to 3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong anti-locomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for bio-drug development for prospective neurodegenerative disease treatment.

Nonlinear mixed-modelling discriminates the effect of chemicals and their mixtures on zebrafish behavior

Nature

2018-01-12

Abstract

Zebrafish (Danio rerio) early-life stage behavior has the potential for high-throughput screening of neurotoxic environmental contaminants. However, zebrafish embryo and larval behavioral assessments typically utilize linear analyses of mean activity that may not capture the complexity of the behavioral response. Here we tested the hypothesis that nonlinear mixed-modelling of zebrafish embryo and larval behavior provides a better assessment of the impact of chemicals and their mixtures. We demonstrate that zebrafish embryo photomotor responses (PMRs) and larval light/dark locomotor activities can be fit by asymmetric Lorentzian and Ricker-beta functions, respectively, which estimate the magnitude of activity (e.g., maximum and total activities) and temporal aspects (e.g., duration of the responses and its excitatory periods) characterizing early life-stage zebrafish behavior. We exposed zebrafish embryos and larvae to neuroactive chemicals, including isoproterenol, serotonin, and ethanol, as well as their mixtures, to assess the feasibility of using the nonlinear mixed-modelling to assess behavioral modulation. Exposure to chemicals led to distinct effects on specific behavioral characteristics, and interactive effects on temporal characteristics of the behavioral response that were overlooked by the linear analyses of mean activity. Overall, nonlinear mixed-modelling is a more comprehensive approach for screening the impact of chemicals and chemical mixtures on zebrafish behavior.

Introduction

Zebrafish (Danio rerio) have proven to be an excellent model for ecotoxicological applications1, in part due to their tractability in laboratory settings, high fecundity, and well-understood and precisely-timed ontogeny2. The zebrafish is also an excellent model to study chemical effects on developmental programming, as well as early life-stage behavior3,4,5. For instance, as early as 30 hours post fertilization (hpf), zebrafish embryos exhibit a short photomotor response (PMR) involving a spike in movement over a 10 s period4. The PMR has high plasticity to drug exposure and has been used for high-throughput screening of neuroactive chemicals6,7. Also, after hatch (>72 hpf) the larvae exhibit distinct locomotor activity to alternating dark and light photoperiods8. Locomotor activity is supressed when larvae are held in an illuminated environment, while a switch to complete darkness evokes a spike in locomotor activity lasting around 15 min5,8. Recent studies suggest that this larval behavior may be disrupted by environmental contaminants5,8,9.

Although animal behavioral endpoints hold promise in ecotoxicology for risk assessments, they are not widely used by regulatory agencies. A major challenge in using animal behavior for risk assessment involves the wide-ranging responses that require careful assessment and selection of the most appropriate statistical analyses for biological/ecological relevance10. Animal activity is often measured by averaging the values temporally and/or spatially to make them amenable for linear analyses11,12. However, that greatly simplifies behavioral analyses because activity changes are seldom linear. This is exemplified by the nonlinear embryo PMR and larval locomotor activity profiles (i.e., asymmetric peak- and hump-shaped respectively6,8). These activity profiles reveal behavioral characteristics, including quickness, duration and maximum intensity, which are analogous to common parameters of nonlinear functions13.

Nonlinear mixed-modelling includes both fixed-effects (i.e., model parameters – phenotypical characteristics) and random-effects (i.e., within-subject effects), and allows for phenotype comparisons, while controlling for repeated measurements of activity during the behavioral trials14,15. Also, the inclusion of an interaction term in the fixed-effects component of the model allows for testing interactive effects16. Against this backdrop, our objective was to test the feasibility of using nonlinear mixed-modelling to assess the effects of chemicals either alone or as mixtures on zebrafish embryo and larval activity. Specifically, we tested the hypothesis that nonlinear mixed-modelling is a more sensitive representation of chemical effects and their interactions on early life-stage zebrafish behavior compared to linear modelling of mean activity. As a proof of concept, we carried out zebrafish embryo PMR and larval locomotor activity trials with fish exposed to model chemicals that either stimulate or suppress embryo and larvae activity. We also co-exposed embryos and larvae to a mixture of the stimulant and suppressor to demonstrate the capacity of nonlinear mixed-modelling in testing and describing mixture toxicity.

Methods

Zebrafish maintenance and embryo collection

Adult zebrafish (Tupfel long fin strain) were cultured in 10 L polypropylene tanks at 28.5 °C, pH 7.6, and 740 µS conductivity on recirculating systems (Pentair Aquatic Habitats, Apopka, Florida). The recirculating systems were housed in an animal care facility at the University of Calgary with a 14 h:10 h light:dark daily light cycle. Animals were fed with ZieglerTM adult zebrafish diet (Pentair) and live Artemia (San Francisco Bay Brand, Inc, Newark, CA, USA) in the morning and evening respectively. Zebrafish were bred and the early life-stages maintained in E3 medium17 at 28.5 °C as described previously5. The animal maintenance and all experiments were approved by the animal care committee at the University of Calgary (AC17-0079) and were in accordance with the Canadian Council on Animal Care guidelines.


Zebrafish PMR

We followed the protocol outlined in Kokel et al.6 with slight modifications. We 3D-printed a custom light emitting diode (LED)-array for use with a ZebraBox behavioral acquisition system (Viewpoint Life Sciences, Montreal, QC, Canada) that allowed for multiple configurations of LEDs (Super Bright LEDs, St. Louis, Missouri, USA) that could be activated with a remote switch. PMR trials were 30 s in duration and were carried out in total darkness, with the exception of two 1 s light pulses at 10 and 20 s6, and were recorded at 30 frames per second. Embryos were dechorionated at 24 hpf in 1 g L−1 Pronase (Sigma) and transferred (6 embryos per well) to the center 48 wells of a 96-well plate (Greiner, Sigma) with 225 µL of embryo medium per well. Only the center 48 wells were used to maximize magnification and resolution of video acquisition for activity measurements. Preliminary work identified that exposure to the β-adrenergic receptor agonist, isoproterenol, and ethanol stimulated and supressed the PMR, respectively. The treatments included 100 µM isoproterenol, 2% ethanol or a combination of both and the exposures began 30 min before each PMR trial. Final well volume was 300 uL following additions of chemical stocks. Treatments were randomly assigned by plate column, and each plate contained 2 columns (i.e., 16 wells) of control, isoproterenol, ethanol, and isoproterenol-ethanol treatments. A total of 360 embryos per treatment were used for the PMR experiments. The PMR trials began at 32 hpf with 20 min dark acclimation periods between plates. Embryo activity was quantified as Δ pixel intensity from each frame.


Zebrafish larval locomotor activity

Larvae were transferred to each well of a 96-well plate at 80 hpf along with 225 µL of embryo medium and maintained overnight at 28.5 °C. Preliminary work identified that exposure to isoproterenol and serotonin suppressed and stimulated larval locomotor activity, respectively. The next day each well received either 20 µM isoproterenol, 100 µM serotonin or a combination of both and each plate contained 24 wells/larvae per treatment (a total of 3 plates). Final well volume was 300 uL following additions of chemical stocks. Immediately following the chemical exposure, plates were transferred to the Zebrabox and behavioral trials commenced as described previously18, following the lighting regime of Emran et al.8. The Zebrabox system includes backlighting in the visible spectrum from 0% to 100% intensity. Our lighting regime included 30 min at 0% intensity (i.e., dark adaptation), 30 min at 100% intensity, and a final 30 min at 0% intensity. Larval activity was calculated as total distance travelled every 30 s as described previously5,18.

 

https://www.nature.com/articles/s41598-018-20112-x

A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target

Brain. A journal of Neurology

2018-01-24

Abstract

Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by ∼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy.

Behavioural assay

For drug screening using kcna1 morphants, 5 days post-fertilization (dpf) larval zebrafish maintained in 96-well plates were habituated for 20 min, under ambient light. This was followed by treatment with 20 μM of drugs for 20–30 min (n = 6–7 per compound), and assayed for locomotor activity, burst activity, distance moved, inactive duration and tracking in dark for 20 min in ZebraBox (ViewPoint Life Sciences). For drug screening involving PTZ induction, 5 dpf zebrafish were treated with 10 mM PTZ for 10 min, followed by 20 μM (final concentration) of drug for 20 min under ambient light, and assayed for total locomotor activity, burst activity, distance moved and inactive duration, and tracking under 100% light in ZebraBox for 20 min. All behavioural assays were repeated at least twice. Total swim activity and tracking of total distance moved as a measure of hyperactive swimming behaviour were analysed using ZebraLab V3 software (ViewPoint Life Sciences, Lyon, France).

https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awx364/4823617#109328665

NCBINCBI Logo Skip to main content Skip to navigation Resources How To About NCBI Accesskeys Sign in to NCBI PubMed US National Library of Medicine National Institutes of Health Search database Search term Clear input Advanced Help Result Filters Format: Abstract Send to Chemosphere. 2018 Jan 25;197:611-621. doi: 10.1016/j.chemosphere.2018.01.092. [Epub ahead of print] Developmental toxicity induced by PM2.5 through endoplasmic reticulum stress and autophagy pathway in zebrafish embryos.

Chemosphere

2018-02-01

Abstract

The aims of this study were to investigate the mechanism underlying the developmental toxicity of fine particulate matter (PM2.5) and provide a more thorough understanding of the toxicity of PM2.5 in an ecological environment. Zebrafish embryos at 4 h post-fertilization were exposed to PM2.5 at doses of 200, 300, 400, 500, 600 and 800 μg/mL for 120 h. The mortality, hatching rate, morphology score, body length, locomotor capacity, histological changes, antioxidant defense system, leukocyte migration, inflammation-related gene mRNA expression, endoplasmic reticulum stress (ERS) and autophagy were evaluated to study PM2.5-induced developmental toxicity and its underlying mechanisms. PM2.5 exposure significantly increased the mortality and malformations and reduced the hatching rate and body length of the zebrafish. PM2.5 significantly reduced the locomotor capacity of zebrafish larvae, increased the levels of ROS and disturbed the antioxidant defense system in zebrafish larvae. In addition, a histological examination showed that the heart, liver, intestines and muscle of the PM2.5-treated zebrafish exhibited abnormal changes and a significant increase in cellular autophagic accumulation. RT-PCR showed that the expression of genes related to inflammation (tgfβ and cox2), ERS (hspa5, chop, ire1, xbp1s, and atf6) and autophagy (lc3, beclin1 and atg3) pathways was significantly increased in the PM2.5-treated zebrafish, indicating that PM2.5 induced inflammation and promoted ERS and autophagy responses via the activation of the IRE1-XBP1 and ATF6 pathways. Together, our data indicate that PM2.5 induced a dose- and time-dependent increase in developmental toxicity to zebrafish embryos. Additionally, ERS and autophagy may play important roles in PM2.5-induced developmental toxicity.

 

Link to the publication :

 

https://www.sciencedirect.com/science/article/pii/S0045653518301097

 

 

Genetic compensation triggered by actin mutation prevents the muscle damage caused by loss of actin protein

PLOS Genetics

2018-02-08

Abstract

The lack of a mutant phenotype in homozygous mutant individuals’ due to compensatory gene expression triggered upstream of protein function has been identified as genetic compensation. Whilst this intriguing process has been recognized in zebrafish, the presence of homozygous loss of function mutations in healthy human individuals suggests that compensation may not be restricted to this model. Loss of skeletal ∞-actin results in nemaline myopathy and we have previously shown that the pathological symptoms of the disease and reduction in muscle performance are recapitulated in a zebrafish antisense morpholino knockdown model. Here we reveal that a genetic actc1b mutant exhibits mild muscle defects and is unaffected by injection of the actc1b targeting morpholino. We further show that the milder phenotype results from a compensatory transcriptional upregulation of an actin paralogue providing a novel approach to be explored for the treatment of actin myopathy. Our findings provide further evidence that genetic compensation may influence the penetrance of disease-causing mutations.


Author summary


Many healthy individuals carry loss of function mutations in essential genes that would normally be deleterious for survival. Intriguingly, it may be the presence of the genomic lesion itself in these individuals that triggers the compensatory pathways. It is not known how widespread this phenomenon is in vertebrate populations and how genetic compensation is activated. We have shown that knockdown of actin causes nemaline myopathy as indicated by the formation of nemaline bodies within the skeletal muscle and reduced muscle function which, remarkably, we did not observe in an actin genetic mutant. We have identified that protection from the disease phenotype results from transcriptional upregulation of an actin paralogue restoring actin protein in the skeletal muscle. This study demonstrates that genetic compensation may be more prevalent than previously anticipated and highlights phenotypic differences resulting from genetic mutations versus antisense knockdown approaches. Furthermore, we suggest that activating compensatory pathways may be exploited as a potential novel therapeutic approach for human disorders caused by loss of function mutations.

 

Link to the publication :

http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007212

Ezh2 promotes clock function and hematopoiesis independent of histone methyltransferase activity in zebrafish

Nucleic Acids Research

2018-02-13

Abstract

EZH2 is a subunit of polycomb repressive complex 2 (PRC2) that silences gene transcription via H3K27me3 and was shown to be essential for mammalian liver circadian regulation and hematopoiesis through gene silencing. Much less, however, is known about how Ezh2 acts in live zebrafish. Here, we show that zebrafish ezh2 is regulated directly by the circadian clock via both E-box and RORE motif, while core circadian clock genes per1a, per1b, cry1aa and cry1ab are down-regulated in ezh2 null mutant and ezh2 morphant zebrafish, and either knockdown or overexpression of ezh2 alters locomotor rhythms, indicating that Ezh2 is required for zebrafish circadian regulation. In contrast to its canonical silencing function, zebrafish Ezh2 up-regulates these key circadian clock genes independent of histone methyltransferase activity by directly binding to key circadian clock proteins. Similarly, Ezh2 contributes to hematopoiesis by enhancing expression of hematopoietic genes such as cmyb and lck. Together, our findings demonstrate for the first time that Ezh2 acts in both circadian regulation and hematopoiesis independent of silencing PRC2.

Link to the publication :

https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gky101/4852815

Lethal and sub-lethal effects of cyproconazole on freshwater organisms: a case study with Chironomus riparius and Dugesia tigrina

Environmental Science and Pollution Research

2018-02-18

Abstract

The fungicide cyproconazole (CPZ) inhibits the biosynthesis of ergosterol, an essential sterol component in fungal cell membrane and can also affect non-target organisms by its inhibitory effects on P450 monooxygenases. The predicted environmental concentration of CPZ is up to 49.05 μg/L and 145.89 μg/kg in surface waters and sediments, respectively, and information about CPZ toxicity towards non-target aquatic organisms is still limited. This study aimed to address the lack of ecotoxicological data for CPZ, and thus, an evaluation of the lethal and sub-lethal effects of CPZ was performed using two freshwater invertebrates (the midge Chironomus riparius and the planarian Dugesia tigrina). The estimated CPZ 48 h LC50 (95% CI) was 17.46 mg/L for C. riparius and 47.38 mg/L for D. tigrina. The emergence time (EmT50) of C. riparius was delayed by CPZ exposure from 0.76 mg/L. On the other hand, planarians showed higher tolerance to CPZ exposure. Sub-lethal effects of CPZ on planarians included reductions in locomotion (1.8 mg/L), delayed photoreceptors regeneration (from 0.45 mg/L), and feeding inhibition (5.6 mg/L). Our results confirm the moderate toxicity of CPZ towards aquatic invertebrates but sub-lethal effects observed also suggest potential chronic effects of CPZ with consequences for population dynamics.

Effects of UV filter 4-methylbenzylidene camphor during early development of Solea senegalensis Kaup, 1858

Science of The Total Environment

2018-02-20

Highlights

4MBC induced mortality, malformations and reduced growth in S. senegalensis larvae.

Swimming behaviour was affected by 4MBC at low concentrations.

4MBC caused alterations in AChE and LDH biochemical markers.

Assessment of behaviour is recommended in ecotoxicological studies with sole larvae.


Abstract

The inclusion of organic UV filters in personal care products (PCPs) has increased in recent years. 4-Methylbenzylidene camphor (4MBC) is one of the most used UV filters, and thus it is commonly found in aquatic ecosystems, with proved negative effects on aquatic organisms. Effects on early life stages of marine vertebrates are largely unknown. Therefore, the main goal of this work was to evaluate 4MBC effects on Senegalese sole (Solea Senegalensis Kaup, 1858) larvae at different levels of biological organization. S. senegalensis were exposed to increasing concentrations of 4MBC from egg stage until 96 h. Mortality, growth, malformations, behaviour and biochemical responses, including enzymatic biomarkers were studied. The exposure to 4MBC until 96 h post-fertilization (hpf) induced mortality and malformations in a dose-response manner. Besides, reduced growth with increasing concentrations was observed. The exposure to 4MBC also caused alterations on behaviour, including overall lower swimming time during light and dark periods. Biomarkers alterations caused by 4MBC included imbalance of neurotransmission related endpoints (increased acetylcholinesterase activity) and decreased activity of enzymes related to anaerobic metabolism (lower cellular lactate dehydrogenase activity) at the lower concentrations tested. Furthermore, our results suggest that 4MBC do not induce oxidative stress in S. senegalensis larvae, since catalase and lipid peroxidation levels were not significantly altered by 4MBC.

S. senegalensis revealed to be a good model species for vertebrate animal testing in the marine environment. Sub-lethal concentrations of 4MBC induced toxic effects at all organizational levels. Swimming behaviour was a sensitive endpoint and showed that exposure to 4MBC causes impairment on response to light stimulus which is possibly linked with the observed imbalances on cholinesterase activity in larvae. Conservation concerns along distribution range of S. senegalensis should consider that increasing levels of UV filters in marine environment might have impact on the ecology of the species.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0048969718305023

Advancement of Passive Sampling Applications for Assessing Contaminant Transport, Bioaccumulation, and Toxicity

Oregon State University

2018-02-20

Abstract

 

In the environment, it is the unbound fraction of chemical (C free ) which is able to diffuse across environmental interfaces and biological membranes. It is therefore C free  which drives many important biological-environmental processes including contaminant transport, bioaccumulation and toxicity. Passive sampling devices (PSDs) offer a simplified and more accurate approach for measuring C free  compared to traditional methods. The chapters of this dissertation extend the applications of passive sampling for answering questions related to the transport, bioaccumulation, and toxicity of contaminants, specifically polycyclic aromatic hydrocarbons (PAHs). In Chapter 1, using PSDs, we measured the diffusive flux of PAHs between sediment, water, and air at the Portland Harbor Superfund site (PHSS). Data indicated that modern (atmospheric) sources of 2- and 3-ring PAHs were more significant than legacy (sediment) sources. Additionally, the data pointed toward PHSS sediments as potential atmospheric sources of 4-ring and larger PAHs through diffusion. This result may have significant health risk
implications for those living near the PHSS and other contaminated sites. Ultimately, data generated by this study was used to make a regulatory decision at the McCormick and Baxter Superfund site, highlighting the growing acceptance and applicability of passive sampling devices. Transport of contaminants may lead to exposure and bioaccumulation in humans and organisms. When organisms are consumed by humans, measuring bioaccumulation of contaminants in those organisms is essential for assessing human health risk. This is especially
true for subsistence consumers who have elevated ingestion rates including Native American tribes. Traditional predictive methods for bioaccumulation in benthic organisms are often inaccurate because of reliance on poorly characterized and understood site specific sediment characteristics. Passive sampling devices directly measure C free  and therefore inherently account for these site specific differences. In Chapter 2, sediment PSDs were used to build a model for predicting PAH concentrations in traditionally harvested clams on Native American tribal land in
the Puget Sound region of the Salish Sea. The model was able to predict PAH concentrations in butter clams (Saxidomus giganteus) within a factor of 1.9 ± 0.2. This model will provide a more accurate and simplified method to monitor PAH concentrations in clams without having to remove clams from the environment. Additionally, data from this study highlighted spatial differences in carcinogenic risk associated with the consumption of clams and was used to inform local communities. Bioaccumulation of PAHs may result in health effects if the PAHs are toxic. In the environment PAHs exist as mixtures and it is therefore essential to consider the toxicity of relevant environmental PAH mixtures. There is growing evidence for developmental effects (morphological and neurological) from PAH exposure. In Chapter 4, data from passive sampling devices deployed in surface water at the PHSS was used to construct a surrogate mixture of the 10 most abundant PAHs (Supermix10). Using the zebrafish model, we assessed the developmental toxicity of Supermix10 (SM10), its toxic (Supermix3) and non-toxic (Supermix7) sub-fractions, and the 10 individual PAHs. Data indicated that the general additivity model may be sufficient for explaining the overall developmental effects caused by these PAH mixtures. However, we showed that individual PAH toxic endpoints may not be predictive of the toxic endpoints in PAH mixtures. Finally, SM10 caused behavioral effects in adult fish following exposure during development at concentrations below those which caused overt morphological effects. Ultimately the work in this dissertation advances the application of passive sampling technologies toward a better understanding of PAH transport, bioaccumulation, and toxicity.

 

https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/0z7092360

Development of a high-throughput in vivo screening platform for particulate matter exposures

Environmental Pollution

2018-02-21

Highlights

Standardized methods for PM exposure established in developmental zebrafish model.

PM associated bioactivity included mortality, morphological and behavioral responses.

Concentration dependent cyp1a/GFP expression was observed for PM exposures.

Zebrafish is a sensitive in vivo model of PM toxicity.

High-throughput screening of PM will expand knowledge of PM-health effects.


Abstract

Particulate matter (PM) exposure is a public health burden with poorly understood health effect mechanisms and lacking an efficient model to compare the vast diversity of PM exposures. Zebrafish (Danio rerio) are amenable to high-throughput screening (HTS), but few studies have investigated PM toxicity in zebrafish, despite the multitude of advantages. To develop standardized exposure procedures, the urban PM standard reference material (SRM) 1649b was used to systematically determine sample preparation methods, design experimental controls, determine concentration ranges and evaluation procedures. Embryos (n = 32/treatment) were dechorionated and placed into 96-well plates containing SRM1649b (0–200 μg/mL) at 6 h post fertilization (hpf). Developmental toxicity was assessed at 24 and 120 hpf by evaluating morphological changes, embryonic/larval photomotor behavior, and mortality. Differences from blank medium and particle controls were observed for all biological responses measured. Differences due to SRM1649b concentration and preparation method were also observed. Exposure to SRM1649b from DMSO extraction was associated with changes in morphology and mortality and hypoactivity in photomotor responses compared to the DMSO control for the whole particle suspension (76, 68%) and soluble fraction (59, 54%) during the embryonic and larval stages, respectively. Changes in behavioral responses were not observed following exposure to the insoluble fraction of SRM1649b from DMSO extraction. The toxicity bias from PM preparation provided further impetus to select a single HTS exposure method. Based on the biological activity results, the soluble fraction of SRM1649b from DMSO extraction was selected and shown to have concentration dependent cyp1a/GFP expression. This rapid, sensitive and consistently scalable model is a potentially cost-effective vertebrate approach to study the toxicology of PM from diverse locations, and provides a path to identifying the toxic material(s) in these samples, and discover the mechanisms of toxicity.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749117344469

 

Response of zebrafish larvae to mild electrical stimuli: a 96-well setup for behavioural screening

Journal of Neuroscience Methods

2018-03-06

Abstract

Background

Zebrafish larvae have a high potential as model system to replace rodents, especially in screening and drug discovery applications. However, an experimental setup to deliver mild electrical stimuli with simultaneous high throughput behavioural tracking has not yet been described.


New method

A new tool was designed, making the delivery of electrical stimuli in a 96-well plate format possible. Using custom made electrode clips that can be slid over the walls of a square 96-well plate, 80 larvae could be tested simultaneously and behavioural responses recorded.

Results

As proof of principle, two applications were tested: 1) The behavioural response after a single stimulus and the effect of buprenorphine on this response. 2) Habituation of locomotor activity to multiple stimuli and the involvement of the NMDA receptor. Reduced locomotor activity was observed after a single 5 V stimulus, however not with lower intensity stimuli. Pre-treatment with the analgesic buprenorphine prevented this response. Specificity of buprenorphine was confirmed using the antagonist naloxone. Habituation of locomotor activity was seen in response to multiple stimuli, depending on the inter stimulus interval. Treatment with the NMDA receptor antagonist memantine disrupted behavioural habituation.


Comparison with existing methods

The equipment and setup described here are the first of its kind using a 96-well plate format, thereby increasing the potential throughput in screening applications using zebrafish larvae.

Conclusion

The combination of the described electrode clips for stimulus delivery and behavioural tracking allows for the use of zebrafish larvae in a new array of medium to high throughput applications.

Keywords

electrical stimulation; buprenorphine; naloxone; memantine; NMDA; zebrafish larvae

 

https://www.sciencedirect.com/science/article/pii/S016502701830061X

Movement maintains forebrain neurogenesis via peripheral neural feedback in larval zebrafish

Developmental Biology and Stem Cells, Neuroscience

2018-03-12

Abstract

The postembryonic brain exhibits experience-dependent development, in which sensory experience guides normal brain growth. This neuroplasticity is thought to occur primarily through structural and functional changes in pre-existing neurons. Whether neurogenesis also mediates the effects of experience on brain growth is unclear. Here, we characterized the importance of motor experience on postembryonic neurogenesis in larval zebrafish. We found that movement maintains an expanded pool of forebrain neural precursors by promoting progenitor self-renewal over the production of neurons. Physical cues associated with swimming (bodily movement) increase neurogenesis and these cues appear to be conveyed by dorsal root ganglia (DRG) in the zebrafish body: DRG-deficient larvae exhibit attenuated neurogenic responses to movement and targeted photoactivation of DRG in immobilized larvae expands the pallial pool of proliferative cells. Our results demonstrate the importance of movement in neurogenic brain growth and reveal a fundamental sensorimotor association that may couple early motor and brain development.

 

https://elifesciences.org/articles/31045

 

Tris (2-butoxyethyl) phosphate affects motor behavior and axonal growth in zebrafish (Danio rerio) larvae

Aquatic Toxicology

2018-03-13

Abstract

Tris (2-butoxyethyl) phosphate (TBOEP) is an environmental contaminant that poses serious risks to aquatic organisms and their associated ecosystem. Recently, the reproductive and developmental toxicology of TBOEP has been reported. However, fewer studies have assessed the neurotoxic effects in zebrafish (Danio rerio) larvae. In this study, zebrafish embryos were subjected to waterborne exposure of TBOEP at 0, 50, 500, 1500 and 2500 μg/L from 2 to 144-h post-fertilization (hpf). Behavioral measurements showed that TBOEP exposure reduced embryonic spontaneous movement and decreased swimming speed of larvae in response to dark stimulation. In accordance with these motor effects, TBOEP treatment reduced neuron-specific GFP expression in transgenic Tg (HuC-GFP) zebrafish larvae and inhibited the growth of secondary motoneurons, as well as decreased expression of marker genes related to central nervous system development in TBOEP treated group. Furthermore, increased concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as reduction of SOD activity were detected in TBOEP exposure group. The present results showed that the alteration in motor neuron and oxidative stress could together lead to the motor behavior alterations induced by TBOEP.


Keywords

TBOEP; Zebrafish larvae; Axonal growth; Neurobehavioral toxicity; Oxidative stress

 

https://www.sciencedirect.com/science/article/pii/S0166445X18302522

Involvement of the GluN2C subunit in the behavioral syndrome induced by non-competitive NMDA antagonists

European Neuropsychopharmacology

2018-03-14

Abstract

 

Non-competitive NMDA receptor (NMDA-R) antagonists like ketamine, dizocilpine (MK-801) and phencyclidine (PCP) are used as pharmacological models of schizophrenia and they are also being studied as antidepressant drugs [1]. In rodents, NMDA-R antagonists induce a behavioral syndrome including hyperlocomotion, ataxia signs and stereotypies, as well as other behavioral responses like disorganization in the locomotor pattern and a decrease in the exploratory activity [2]. Previous studies proposed that PCP activates thalamo-cortical circuits after the blockade of NMDA-R in reticular thalamic GABAergic neurons [3]. Since the GluN2C subunit of NMDA-R is densely expressed in thalamus and cerebellum [4], our purpose was to study the involvement of the GluN2C subunit in the behavioral syndrome induced by ketamine, MK-801 and PCP under the working hypothesis that those effects would partly depend on the blockade of thalamo-cortical NMDA-R containing the GluN2C receptor subunit.

Male adult (25-35g) wild-type (WT) and GluN2C receptor subunit knockout (KOGluN2C) mice backcrossed onto a C57BL/6J genetic background were used. Ketamine hydrochloride (Ketolar®, Pfizer), dizocilpine maleate (Sigma-Aldrich) and phencyclidine hydrochloride (Sigma-Aldrich) were dissolved in saline (PCP solution was pH adjusted to 6–6.5 with sodium bicarbonate). Ketamine (10 mg/kg, i.p.), MK-801 (0.10 and 0.25 mg/kg, i.p.) and PCP (5 mg/kg, s.c.) were injected immediately, 30 min and 15 min before behavioral testing, respectively. Mice were placed in a dimly lighted open field cage where activity was recorded with a video camera during 30 min or 2 h. Distance moved (cm) was calculated by the videotrack View Point software (France). Behavioral signs such as number of rearings, number of falls, circling and hind-limb abduction were observed and scored by an experimenter blind to mice genotype and treatment. Statistical analyses were carried out using two-way ANOVA followed by Newman-Keuls post-hoc comparisons. Data are expressed as mean ± SEM and the level of significance was set at p<0,05.

The administration of ketamine and MK-801 significantly enhanced locomotor activity and decreased exploratory behavior (number of rearings) similarly in both genotypes (p<0.01). However, KOGluN2C mice treated with PCP showed increased hyperlocomotion and number of rearings compared to WT mice (p<0.01 and p<0.05, respectively). Moreover, the number of falls induced by all three non-competitive NMDA-R antagonists was markedly and significantly reduced in mice lacking the GluN2C subunit (p<0.01).

Overall, these results show that the GluN2C subunit appears to be strongly involved in the stereotyped behavior induced by non-competitive NMDA-R antagonists. KOGluN2C mice showed less motor impairment, as suggested by the decrease in the number of falls and the increased exploratory behavior, which may account for the genotype differences in the locomotor activity observed in PCP-treated mice. Further, the dramatic difference in the number of falls between WT and KOGluN2C mice suggests the involvement of the cerebellum, a key brain structure for the control of motor coordination.

 

http://www.europeanneuropsychopharmacology.com/article/S0924-977X(17)32093-X/abstract

Functional changes in the dorsolateral striatum in loss of control over reward seeking

European Neuropsychopharmacology

2018-03-14

Abstract

 

Substance addiction inflicts an enormous burden on patients with formidable costs to society. Loss of control over substance use is a hallmark of addiction. It marks the stage where substance use has progressed from casual to compulsive and persists despite negative consequences. It is thought that this progression arises after lengthy and excessive substance use, as a result of neurobiological dysfunctions. One of the neural mechanisms that has been proposed to contribute to loss of control use is exaggerated involvement of the dorsolateral striatum (DLS), which mediates habitual behaviour [1,2]. This study examines how changes in DLS functioning contribute to loss of control over reward seeking. First, we assessed the effect of long-term exposure to alcohol on synaptic plasticity in a cortical projection onto neurons in the DLS. To map cortical inputs towards the DLS, rats were bilaterally injected with a retrograde rabies virus (Rabies-SadDG-GFP) in the DLS. Using immunohistochemistry, we found GFP expression in different cortical areas, including the primary and secondary motor cortex. Next, voluntary home-cage alcohol consumption was monitored in outbred Lister-Hooded rats. We used a two-month intermittent alcohol access paradigm and found substantial individual variation in alcohol intake, consistent with our previous findings. Based on a rank score of their voluntary alcohol intake, low and high consuming animals were then selected and bilaterally injected with an adeno-associated Channelrhodopsin virus (AAV5-CamKII-Chr2-eYFP) targeted at the identified motor cortices that project onto neurons in the DLS. Whole-cell patch clamp recordings were performed to measure optically induced excitatory post-synaptic currents in neurons in the DLS. Our preliminary results indicate increased facilitation of paired-pulse responses in the DLS in rats that show a high voluntary alcohol consumption. This increase is caused by reduced amplitude of the first post-synaptic response, indicative of a reduced presynaptic release probability. In parallel, we assessed whether activation of the DLS is sufficient to drive habitual behaviour and loss of control over sucrose seeking. To investigate habitual behavior, changes in sensitivity for outcome devaluation were measured after pre-feeding animals with either sucrose or chow before operant training. Loss of control over sucrose seeking was examined in a task in which we measure continued responding during a conditioned cue that predicts probabilistic footshock punishment. To enhance neuronal activity of the DLS, rats received bilateral injections of an adeno-associated DREADD virus (AAV5-hSyn-hMD3q-mCherry). Systemic clozapine N-oxide (CNO) injections did not change the sensitivity to outcome devaluation. Furthermore, we show that the presentation of the footshock-predictive cue profoundly suppresses responding for sucrose, but this is unaffected by CNO injections. In sum, our data indicate that long-term ingestion of large quantities of alcohol modulates short-term synaptic plasticity in glutamatergic cortical inputs into the DLS. However, chemogenetic activation of the DLS does not seem sufficient to evoke habitual or compulsive behaviour.

 

http://www.europeanneuropsychopharmacology.com/article/S0924-977X(17)32090-4/abstract

Effect of titanium dioxide nanoparticles on the bioavailability and neurotoxicity of cypermethrin in zebrafish larvae

Aquatic Toxicology

2018-03-31

Abstract

In aquatic environment, the presence of nanoparticles (NPs) has been reported to modify the bioavailability and toxicity of the organic toxicants. Nevertheless, the combined toxicity of NPs and the pesticides that were used world-widely still remains unclear. Cypermethrin (CYP), a synthetic pyrethroid insecticide, is commonly used for controlling agricultural and indoor pests. Therefore, the effects of titanium dioxide NPs (nTiO2) on CYP bioconcentration and its effects on the neuronal development in zebrafish were investigated in our study. Zebrafish embryos (2- hour-post-fertilization, hpf) were exposed to CYP (0, 0.4, 2 and 10 μg/L) alone or co-exposed with nTiO2 (1 mg/L) until 120-hpf. nTiO2 is taken up by zebrafish larvae and also it can adsorb CYP. The zebrafish body burdens of CYP was observed and CYP uptake was increased by nTiO2, indicating that the nTiO2 could accelerate the bioaccumulation of CYP in larvae. Co-exposure of nTiO2 and CYP induced the generation of reactive oxygen species. Exposure to CYP alone significantly decreased the mRNA expression of genes, including glial fibrillary acidic protein (gfap), α1-tubulin, myelin basic protein (mbp) and growth associated protein (gap-43). Besides, reductions of serotonin, dopamine and GABA concentrations were observed in zebrafish and the larval locomotion was significantly decreased in response to the lower level of the neurotransmitters. Moreover, co-exposure of nTiO2 and CYP caused further significantly decreased in the locomotion activity, and enhanced the down-regulation of the mRNA expression of specific genes and the neurotransmitters levels. The results demonstrated that nTiO2 increased CYP accumulation and enhanced CYP-induced developmental neurotoxicity in zebrafish.

Keywords

Cypermethrin; nTiO2; Neurotoxicity; Zebrafish; Co-exposure

 

https://www.sciencedirect.com/science/article/pii/S0166445X18302832

CRISPR/Cas9-induced shank3b mutant zebrafish display autism-like behaviors

Molecular Autism

2018-04-02

Abstract

Background

Human genetic and genomic studies have supported a strong causal role of SHANK3 deficiency in autism spectrum disorder (ASD). However, the molecular mechanism underlying SHANK3 deficiency resulting in ASD is not fully understood. Recently, the zebrafish has become an attractive organism to model ASD because of its high efficiency of genetic manipulation and robust behavioral phenotypes. The orthologous gene to human SHANK3 is duplicated in the zebrafish genome and has two homologs, shank3a and shank3b. Previous studies have reported shank3 morphants in zebrafish using the morpholino method. Here, we report the generation and characterization of shank3b mutant zebrafish in larval and adult stages using the CRISPR/Cas9 genome editing technique.

Methods

CRISPR/Cas9 was applied to generate a shank3b loss-of-function mutation (shank3b −/− ) in zebrafish. A series of morphological measurements, behavioral tests, and molecular analyses were performed to systematically characterize the behavioral and molecular changes in shank3b mutant zebrafish.

Results

shank3b−/− zebrafish exhibited abnormal morphology in early development. They showed reduced locomotor activity both as larvae and adults, reduced social interaction and time spent near conspecifics, and significant repetitive swimming behaviors. Additionally, the levels of both postsynaptic homer1 and presynaptic synaptophysin were significantly reduced in the adult brain of shank3b-deficient zebrafish.

Conclusions

We generated the first inheritable shank3b mutant zebrafish model using CRISPR/Cas9 gene editing approach. shank3b−/− zebrafish displayed robust autism-like behaviors and altered levels of the synaptic proteins homer1 and synaptophysin. The versatility of zebrafish as a model for studying neurodevelopment and conducting drug screening will likely have a significant contribution to future studies of human SHANK3 function and ASD.

Keywords

shank3 - CRISPR/Cas9 - Zebrafish - ASD - Social behavior - Animal model

 

https://molecularautism.biomedcentral.com/articles/10.1186/s13229-018-0204-x

Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish

Annals of Clinical and Translational Neurology

2018-04-06

Abstract


Objective: DEPDC5 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling.

Methods: Zebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy-related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression.

Results: Here we report a zebrafish model of Depdc5 loss-of-function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy-associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein.

Interpretation: This study demonstrates that Depdc5 knockdown leads to early-onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy-associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1-dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5-inherited forms of focal epilepsy.

 

https://onlinelibrary.wiley.com/doi/full/10.1002/acn3.542

Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish

Toxicology and Applied Pharmacology

2018-04-06

Abstract

Superfund sites often consist of complex mixtures of polycyclic aromatic hydrocarbons (PAHs). It is widely recognized that PAHs pose risks to human and environmental health, but the risks posed by exposure to PAH mixtures are unclear. We constructed an environmentally relevant PAH mixture with the top 10 most prevalent PAHs (SM10) from a Superfund site derived from environmental passive sampling data. Using the zebrafish model, we measured body burden at 48 hours post fertilization (hpf) and evaluated the developmental and neurotoxicity of SM10 and the 10 individual constituents at 24 hours post fertilization (hpf) and 5 days post fertilization (dpf). Zebrafish embryos were exposed from 6 to 120 hpf to (1) the SM10 mixture, (2) a variety of individual PAHs: pyrene, fluoranthene, retene, benzo[a]anthracene, chrysene, naphthalene, acenaphthene, phenanthrene, fluorene, and 2-methylnaphthalene. We demonstrated that SM10 and only 3 of the individual PAHs were developmentally toxic. Subsequently, we constructed and exposed developing zebrafish to two sub-mixtures: SM3 (comprised of 3 of the developmentally toxicity PAHs) and SM7 (7 non-developmentally toxic PAHs). We found that the SM3 toxicity profile was similar to SM10, and SM7 unexpectedly elicited developmental toxicity unlike that seen with its individual components. The results demonstrated that the overall developmental toxicity in the mixtures could be explained using the general concentration addition model. To determine if exposures activated the AHR pathway, spatial expression of CYP1A was evaluated in the 10 individual PAHs and the 3 mixtures at 5 dpf. Results showed activation of AHR in the liver and vasculature for the mixtures and some individual PAHs. Embryos exposed to SM10 during development and raised in chemical-free water into adulthood exhibited decreased learning and responses to startle stimulus indicating that developmental SM10 exposures affect neurobehavior. Collectively, these results exemplify the utility of zebrafish to investigate the developmental and neurotoxicity of complex mixtures.

Keywords

Passive sampling; Polycyclic aromatic hydrocarbons; Behavior; Aryl hydrocarbon receptor; Cytochrome P4501A; Biomarker

 

https://www.sciencedirect.com/science/article/pii/S0041008X18301212

Zebrafish-based identification of the antiseizure nucleoside inosine from the marine diatom Skeletonema marinoi

PLOS one

2018-04-24

Abstract

With the goal of identifying neuroactive secondary metabolites from microalgae, a microscale in vivo zebrafish bioassay for antiseizure activity was used to evaluate bioactivities of the diatom Skeletonema marinoi, which was recently revealed as being a promising source of drug-like small molecules. A freeze-dried culture of S. marinoi was extracted by solvents with increasing polarities (hexane, dichloromethane, methanol and water) and these extracts were screened for anticonvulsant activity using a larval zebrafish epilepsy model with seizures induced by the GABAA antagonist pentylenetetrazole. The methanolic extract of S. marinoi exhibited significant anticonvulsant activity and was chosen for bioassay-guided fractionation, which associated the bioactivity with minor constituents. The key anticonvulsant constituent was identified as the nucleoside inosine, a well-known adenosine receptor agonist with previously reported antiseizure activities in mice and rat epilepsy models, but not reported to date as a bioactive constituent of microalgae. In addition, a UHPLC-HRMS metabolite profiling was used for dereplication of the other constituents of S. marinoi. Structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution spectrometry. These results highlight the potential of zebrafish-based screening and bioassay-guided fractionation to identify neuroactive marine natural products.

 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196195

Mutation of IPO13 causes recessive ocular coloboma, microphthalmia, and cataract

Experimental & Molecular Medicine

2018-04-27

Abstract

Ocular coloboma is a developmental structural defect of the eye that often occurs as complex ocular anomalies.
However, its genetic etiology remains largely unexplored. Here we report the identification of mutation (c.331C>T, p.R111C) in the IPO13 gene in a consanguineous family with ocular coloboma, microphthalmia, and cataract by a combination of whole-exome sequencing and homozygosity mapping. IPO13 encodes an importin-B family protein and has been proven to be associated with the pathogenesis of coloboma and microphthalmia. We found that Ipo13 was expressed in the cornea, sclera, lens, and retina in mice. Additionally, the mRNA expression level of Ipo13 decreased significantly in the patient compared with its expression in a healthy individual. Morpholinooligonucleotide-induced knockdown of ipo13 in zebrafish caused dose-dependent microphthalmia and coloboma, which is highly similar to the ocular phenotypes in the patient. Moreover, both visual motor response and optokinetic response were impaired severely. Notably, these ocular phenotypes in ipo13-deficient zebrafish could be rescued remarkably by full-length ipo13 mRNA, suggesting that the phenotypes observed in zebrafish were due to insufficient ipo13 function. Altogether, our findings demonstrate, for the first time, a new role of IPO13 in eye morphogenesis and that loss of function of IPO13 could lead to ocular coloboma, microphthalmia, and cataract in humans and zebrafish.

 

https://www.nature.com/articles/s12276-018-0079-0

High-throughput behavioral assay to investigate seizure sensitivity in zebrafish implicates ZFHX3 in epilepsy

Journal of Neurogenetics

2018-05-02

Abstract

Epilepsy, which affects ∼1% of the population, is caused by abnormal synchronous neural activity in the central nervous system (CNS). While there is a significant genetic contribution to epilepsy, the underlying causes for the majority of genetic cases remain unknown. The NIH Undiagnosed Diseases Project (UDP) utilized exome sequencing to identify genetic variants in patients affected by various conditions with undefined etiology, including epilepsy. Confirming the functional relevance of the candidate genes identified by exome sequencing in a timely manner is crucial to translating exome data into clinically useful information. To this end, we developed a high throughput version of a seizure-sensitivity assay in zebrafish (Danio rerio) to rapidly evaluate candidate genes found by exome sequencing. We developed open access software, Studying Epilepsy In Zebrafish using R (SEIZR), to efficiently analyze the data. SEIZR was validated by disrupting function of a known epilepsy gene, prickle 1. Next, using SEIZR, we analyzed a candidate gene from the UDP screen (Zinc Finger Homeobox 3, ZFHX3), and showed that reduced ZFHX3 function in zebrafish results in a significant hyperactive response to the convulsant drug pentylenetetrazol (PTZ). We find that ZFHX3 shows strong expression in the CNS during neurogenesis including in the pallium, thalamus, tegmentum, reticular formation, and medulla oblongata – all regions which have roles in motor control and coordination. Our findings in the zebrafish confirm human ZFHX3 is a strong candidate for further neurological studies. We offer SEIZR to other researchers as a tool to rapidly and efficiently analyze large behavioral data sets.


Keywords

Epilepsy, central nervous system, ZFHX3, tracking data analysis, neurogenesis, development

 

https://www.tandfonline.com/doi/abs/10.1080/01677063.2018.1445247

Sodium tanshinone IIA sulfonate promotes endothelial integrity via regulating VE-cadherin dynamics and RhoA/ROCK-mediated cellular contractility and prevents atorvastatin-induced intracerebral hemorrhage in zebrafish

Toxicology and Applied Pharmacology

2018-05-04

Abstract

Impaired vascular integrity leads to serious cerebral vascular diseases such as intracerebral hemorrhage (ICH). In addition, high-dose statin therapy is suggested to cause increased ICH risk due to unclear effects of general inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) on the vascular system. Here we evaluated the protective effects of sodium tanshinone IIA sulfonate (STS), which has high efficacy and safety in clinical studies of ischemic stroke, by using atorvastatin (Ator) induced ICH zebrafish embryos and human umbilical vein endothelial cells (HUVECs). By using double transgenic Tg(fli1a:EGFP)y1 & Tg(gata1a:dsRed)sd2 zebrafish, we demonstrated that STS effectively reduced the occurrence and area of hemorrhage induced by Ator in zebrafish and restored impairment in motor function. We further demonstrated that Ator-induced disruption in VE-cadherin (VEC)-containing cell-cell adherens junctions (AJs) in HUVECs by enhancing Src-induced VEC internalization and RhoA/ROCK-mediated cellular contraction. STS inhibited Ator-induced Src activation and subsequent VEC internalization and actin depolymerization near cell borders, reducing lesions between neighboring cells and increasing barrier functions. STS also inhibited the Ator-induced RhoA/ROCK-mediated cellular contraction by regulating downstream LIMK/cofilin and MYPT1/MLC phosphatase signaling. These results showed that STS significantly promoted the stability of cell junctions and vascular integrity. Moreover, we observed that regulations of both Src and RhoA/ROCK are required for the maintenance of vascular integrity, and Src inhibitor (PP2) or ROCK inhibitors (fasudil and H1152) alone could not reduce the occurrence Ator-induced ICH. Taken together, we investigated the underlying mechanisms of Ator-induced endothelial instability, and provided scientific evidences of STS as potential ICH therapeutics by promoting vascular integrity.

Keywords

Sodium tanshinone IIA sulfonate; Salvia miltiorrhiza; Hemorrhagic stroke; Atorvastatin; ROCK

 

https://www.sciencedirect.com/science/article/pii/S0041008X18301911

Essential oil of Siparuna guianensis as an alternative tool for improved lepidopteran control and resistance management practices

Scientific Reports

2018-05-08

Abstract

Although the cultivation of transgenic plants expressing toxins of Bacillus thuringiensis (Bt) represents a successful pest management strategy, the rapid evolution of resistance to Bt plants in several lepidopteran pests has threatened the sustainability of this practice. By exhibiting a favorable safety profile and allowing integration with pest management initiatives, plant essential oils have become relevant pest control alternatives. Here, we assessed the potential of essential oils extracted from a Neotropical plant, Siparuna guianensis Aublet, for improving the control and resistance management of key lepidopteran pests (i.e., Spodoptera frugiperda and Anticarsia gemmatalis). The essential oil exhibited high toxicity against both lepidopteran pest species (including an S. frugiperda strain resistant to Cry1A.105 and Cry2Ab Bt toxins). This high insecticidal activity was associated with necrotic and apoptotic effects revealed by in vitro assays with lepidopteran (but not human) cell lines. Furthermore, deficits in reproduction (e.g., egg-laying deterrence and decreased egg viability), larval development (e.g., feeding inhibition) and locomotion (e.g., individual and grouped larvae walking activities) were recorded for lepidopterans sublethally exposed to the essential oil. Thus, by similarly and efficiently controlling lepidopteran strains susceptible and resistant to Bt toxins, the S. guianensis essential oil represents a promising management tool against key lepidopteran pests.

 

https://www.nature.com/articles/s41598-018-25721-0

Effects of effluent wastewater in developing zebrafish (Danio rerio)

Uppsala Universitet

2018-05-22

Abstract


Traditional wastewater treatment is known not to be specifically designed to eliminate the new generation of chemical residues that ends up in the sewage system. Polluted wastewater effluent therefore reaches the aquatic environment possibly causing adverse effects in aquatic wildlife. The effects of effluent water from five Swedish sewage treatment plants sampled on 6 occasions 2017, were studied in developing zebrafish (Danio rerio). The study included morphological, physiological and behavioural endpoints. Overall there were few effects where deviations from control animals could be seen in the exposed zebrafish. The overall outcome of this assessment was that the wastewater effluent had no consistent effects on the early development of zebrafish. The consequences of continuous low-level exposure during the whole life-cycle of wild fish are presently unknown and further studies are needed to evaluate potential risks.

 

http://uu.diva-portal.org/smash/get/diva2:1197925/FULLTEXT01.pdf

Phenelzine, a cell adhesion molecule L1 mimetic small organic compound, promotes functional recovery and axonal regrowth in spinal cord-injured zebrafish

Pharmacology Biochemistry and Behavior

2018-05-24

Abstract

Injury to the spinal cord initiates a cascade of cellular and molecular events that contribute to the tissue environment that is non-permissive for cell survival and axonal regrowth/sprouting in the adult mammalian central nervous system. The endogenous repair response is impaired in this generally inhibitory environment. Previous studies indicate that homophilic interactions of the neural cell adhesion molecule L1 (L1CAM) promote recovery after spinal cord injury and ameliorate neurodegenerative processes in experimental rodent and zebrafish models. In light of reports that phenelzine, a small organic compound that mimics L1, stimulates neuronal survival, neuronal migration, neurite outgrowth, and Schwann cell proliferation in vitro in a L1-dependent manner, we examined the restorative potential of phenelzine in a zebrafish model of spinal cord injury. Addition of phenelzine into the aquarium water immediately after spinal cord injury accelerated locomotor recovery and promoted axonal regrowth and remyelination in larval and adult zebrafish. Phenelzine treatment up-regulated the expression and proteolysis of L1.1 (a homolog of the mammalian recognition molecule L1) and phosphorylation of Erk in the spinal cord caudal to lesion site. By combining the results of the present study with those of other studies, we propose that phenelzine bears hopes for therapy of nervous system injuries.

 

Abbreviations

CNS, central nervous system; dpf, days post fertilization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MBP, myelin basic protein; MS-222, ethyl 3-aminobenzoate methanesulfonate; PS, phenelzine sulfate; PBS, phosphate-buffered saline, pH 7.3; SCI, spinal cord injury

Keywords

L1; Phenelzine; Spinal cord injury; Remyelination; Axonal regrowth; Zebrafish

 

https://www.sciencedirect.com/science/article/pii/S0091305717307062

Predation and behavioral changes in the neotropical lacewing Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) exposed to lambda-cyhalothrin

Ecotoxicology

2018-05-24

Abstract

Pyrethroid insecticides are widely recommended against various defoliating pest species, but usually lack efficacy against sucking pests such as aphids, which are preferred prey of lacewing larvae. Interaction of pyrethroids and lacewings are likely to occur in fields infested by both defoliating and sap-sucking pests and should provide a complementary control. Therefore, our study aimed to estimate dose-mortality curves and behavioral changes by Chrysoperla externa exposed to lambda-cyhalothrin. We tested the susceptibility of two populations from different locations and insecticide history exposure through topical application. Based on the LD50-calculated, the population exhibiting the greater LD50 was exposed to resistance enhancement (Sel) by treating larvae once for seven successive generations. The population with lower LD50 was kept without selection (Nsel). Subsequently, walking, predation and oviposition behavioral after exposure to dried insecticide residues were investigated. After seven generations with insecticide selection, the resistance rations between Sel and Nsel populations were 5.85- and 9.37-fold higher for larvae and 3.38- and 2.75-fold higher for adults, respectively. Selected females caged in partially treated arenas laid similar eggs number on both treated and untreated surfaces, while Nsel females laid fewer eggs on treated surfaces. Insecticide repellency was not observed in either population, although irritability was observed for Nsel larvae. Selected larvae confined on fully and partially treated surfaces walked further, for a longer time, and with greater speeds compared to Nsel larvae. Furthermore, Sel and Nsel larvae had reduced predation rates when confined on treated surfaces, and Nsel larvae consumed less prey than Sel larvae. Results indicate changes in susceptibility, behavior, and predation rate of C. externa following exposure to lambda-cyhalothrin.

 

Keywords


Non-target insects Pyrethroid Physiology selectivity Insecticide resistance Cotton

 

https://link.springer.com/article/10.1007/s10646-018-1949-x

Toxicity of acaricides to and the behavioural response of Steneotarsonemus concavuscutum (Acari: Tarsonemidae)

Crop Protection

2018-05-25

Abstract

Steneotarsonemus concavuscutum Lofego and Gondim Jr. causes chlorosis, necrosis, deformation, resinosis and death of coconut fruit (Cocos nucifera L.). These types of damage are commonly interpreted to result from Aceria guerreronis Keifer. The control of mites in coconut fruits is carried out primarily by acaricide applications. However, no records exist on the effectiveness of acaricides for S. concavuscutum control. Therefore, this study aimed to evaluate the toxicity of acaricides registered for the control of A. guerreronis on S. concavuscutum, in addition to analysing the walking behaviour of tarsonemid mites when exposed to the products. The behaviour of mites exposed to acaricides was studied with a video-tracking system in choice and no-choice arenas treated with acaricides. The LC50 estimate for abamectin and fenpyroximate was 1.1 mg/L and 2757 mg/L, respectively. Azadirachtin, spirodiclofen and hexythiazox caused mortality of up to 25%. Only exposure to abamectin reduced the number of mites that reached the perianth and remained live, but no difference was observed in the number of eggs found at this site. No acaricide showed an ovicidal or sterilizing effect on the females. The total distance walked and the walking velocity were higher when S. concavuscutum was exposed to azadirachtin and lower when exposed to abamectin. All acaricides irritated S. concavuscutum; however, repellence was observed in only 5% of the tested population for abamectin and azadirachtin. Among the products recommended for the control of A. guerreronis, only abamectin showed potential in controlling S. concavuscutum.

 

Keywords

Mite; Pest; Coconut; Toxicology; Behaviour; Management

 

https://www.sciencedirect.com/science/article/pii/S0261219418301303

Influence of pH on the uptake and toxicity of β-blockers in embryos of zebrafish, Danio rerio

Aquatic Toxicology

2018-05-28

Abstract

ß-blockers are weak bases with acidity constants related to their secondary amine group. At environmental pH they are protonated with tendency to shift to their neutral species at more alkaline pH. Here we studied the influence of pH from 5.5 to 8.6 on the toxicity of the four ß-blockers atenolol, metoprolol, labetalol and propranolol in zebrafish embryos, relating toxicity not only in a conventional way to external aqueous concentrations but also to measured internal concentrations.

Besides lethality we evaluated changes in swimming activity and heartbeat, using the Locomotor Response (LMR) method and the Vertebrate Automated Screening Technology (VAST) for high throughput imaging.

Effects of metoprolol, labetalol and propranolol were detected on phenotype, heart rate and swimming activity. External effect concentrations decreased with increasing neutral fraction for all three pharmaceuticals, attributed by an enhanced uptake of the neutral species in comparison to the corresponding charged form. The LC50 of metoprolol decreased by a factor of 35 from 1.91 mM at almost complete cationic state at pH 7.0 to 0.054 mM with 8% neutral fraction at pH 8.6. For propranolol the LC50 of 2.42 mM at pH 5.5 was even 100 fold higher than the LC50 of 0.023 mM at pH 8.0 where 3% were neutral fraction. No effects were detected in the zebrafish embryo exposed to atenolol.

The internal concentrations for metoprolol and propranolol were quantified at non-toxic concentrations and at LC10. Apparent bioconcentration factors (BCF) ranged from 1.96 at pH 7.0 to 32.0 at pH 8.6 for metoprolol and from 1.86 at pH 5.5 to 169 at pH 8.0 for propranolol. The BCFs served to model the internal effect concentrations from the measured external effect concentrations.

Internal effect concentrations of metoprolol and propranolol were in a similar range for all pH-values and for all endpoints. Interestingly the internal effect concentrations were in the internal concentration range of baseline toxicity, which suggests that the effects of the ß-blockers are rather unspecific, even for sublethal effects on heart rate. In summary, our data confirm that the pH-dependent toxicity related to external concentrations can be explained by toxicokinetic effects and that the internal effect concentrations are pH-independent.

Keywords

β-blocker; Internal effect concentration; pH; Zebrafish

 

https://www.sciencedirect.com/science/article/pii/S0166445X18301152

Sex Differences in the Effects of Parp Inhibition on Microglial Phenotypes Following Neonatal Stroke

Brain, Behavior, and Immunity

2018-05-28

Abstract

Neonatal acute ischemic stroke is a cause of neonatal brain injury that occurs more frequently in males, resulting in associated neurobehavioral disorders. The bases for these sex differences are poorly understood but might include the number, morphology and activation of microglia in the developing brain when subjected to stroke. Interestingly, poly (ADP-ribose) polymerase (PARP) inhibition preferentially protects males against neonatal ischemia. This study aims to examine the effects of PJ34, a PARP inhibitor, on microglial phenotypes at 3 and 8 days and on neurobehavioral disorders in adulthood for both male and female P9 mice subjected to permanent middle cerebral artery occlusion (pMCAo). PJ34 significantly reduced the lesion size by 78% and reduced the density of CX3CR1gfp-labeled microglial cells by 46% when examined 3 days after pMCAo in male but not in female mice. Eight days after pMCAo, the number of Iba1+/Cox-2+ cells did not differ between male and female mice in the cortical peri-infarct region. In the amygdala, Iba1+/Cox-2+ (M1-like) cell numbers were significantly decreased in PJ34-treated males but not in females. Conversely, Iba1+/Arg-1+ (M2-like) and Arg-1+/Cox-2+ (Mtransitional) cell numbers were significantly increased in PJ34-treated females. Regarding neurobehavioral disorders during adulthood, pMCAo induced a motor coordination deficit and a spatial learning deficit in female mice only. PJ34 prevented MBP fibers, motor coordination and learning disorders during adulthood in female mice. Our data show significant sex differences in the effects of PARP inhibition on microglia phenotypes following neonatal ischemia, associated with improved behavior and myelination during adulthood in females only. Our findings suggest that modulating microglial phenotypes may play key roles in behavior disorders and white matter injury following neonatal stroke.

Keywords

Amygdala; cortex; microglial phenotype; neonatal stroke; neurobehavioral deficits; poly (ADP-ribose) polymerase inhibitor

 

https://www.sciencedirect.com/science/article/pii/S0889159118301983

Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement

PLOS One

2018-05-29

Abstract

In occidental societies, high fat and high sugar diets often coincide with episodes of stress. The association is likely to modify brain energy control. Brain insulin signalling is rarely studied in stressed individuals consuming high fat diets. Furthermore the effects of cinnamon supplement are not known in these conditions. Therefore, we exposed rats, over a 12-week period, to a control (C) or a high fat/high fructose (HF/HFr) diet that induces peripheral insulin resistance. A cinnamon supplement (C+CN and HF/HFr +CN) was added or not. After diet exposure, one group of rats was exposed to a 30-min restraint followed by a 10-min open-field test, their combination featuring a moderate stressor, the other rats staying unstressed in their home cages. The insulin signalling in hippocampus and frontal cortex was studied through the mRNA expression of the following genes: insulin receptor (Ir), insulin receptor substrate (Irs1), glucose transporters (Glut1 and Glut3), glycogen synthase (Gys1) and their modulators, Akt1 and Pten. In C rats, stress enhanced the expression of Ir, Irs1, Glut1, Gys1 and Akt1 mRNA. In C+CN rats, stress induced an increase in Pten but a decrease in Gys1 mRNA expression. In HF/HFr rats, stress was associated with an increase in Pten mRNA expression. In HF/HFr+CN rats, stress increased Pten mRNA expression but also decreased Gys1 mRNA expression. This suggests that a single moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement.

 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197094

Testing of therapies in a novel nebulin nemaline myopathy model demonstrate a lack of efficacy

Acta Neuropathologica Communications

2018-05-30

Abstract

Nemaline myopathies are heterogeneous congenital muscle disorders causing skeletal muscle weakness and, in some cases, death soon after birth. Mutations in nebulin, encoding a large sarcomeric protein required for thin filament function, are responsible for approximately 50% of nemaline myopathy cases. Despite the severity of the disease there is no effective treatment for nemaline myopathy with limited research to develop potential therapies. Several supplements, including L-tyrosine, have been suggested to be beneficial and consequently self-administered by nemaline myopathy patients without any knowledge of their efficacy. We have characterized a zebrafish model for nemaline myopathy caused by a mutation in nebulin. These fish form electron-dense nemaline bodies and display reduced muscle function akin to the phenotypes observed in nemaline myopathy patients. We have utilized our zebrafish model to test and evaluate four treatments currently self-administered by nemaline myopathy patients to determine their ability to increase skeletal muscle function. Analysis of muscle pathology and locomotion following treatment with L-tyrosine, L-carnitine, taurine, or creatine revealed no significant improvement in skeletal muscle function emphasizing the urgency to develop effective therapies for nemaline myopathy.

Keywords


NebulinNemaline myopathyZebrafishTreatment

 

https://actaneurocomms.biomedcentral.com/articles/10.1186/s40478-018-0546-9

Improvement of BDNF signalling by P42 peptide in Huntington's disease

Human Molecular Genetics

2018-06-01

Abstract

Huntington’s disease (HD) is caused by a mutation in the Huntingtin (Htt) protein. We previously reported that the 23aa peptide of Htt protein, P42, is preventing HD pathological phenotypes, such as aggregation, reduction of motor performances and neurodegeneration. A systemic treatment with P42 during the pre-symptomatic phase of the disease showed therapeutic potential in R6/2 mice. We here tested P42 effects when administered during the post-symptomatic phase. The P42 treatment alleviated deficits in motor performances, even when symptoms have already started.

Because changes in the level and activity of brain-derived neurotrophic factor (BDNF) have been shown to play a central role in HD, we analysed the influence of P42 on BDNF deficit and associated phenotypes.

Our data suggest that P42 is involved in the spatio-temporal control of bdnf and trkB mRNA and their protein levels. Related to this enhancement of BDNF-TrkB signalling, R6/2 mice treated with P42, exhibit reduced anxiety, better learning and memory performances, and better long-term potentiation (LTP) response. Finally we identified a direct influence of P42 peptide on neuronal plasticity and activity.

These results suggest that P42 offers an efficient therapeutic potential not only by preventing aggregation of mutant Htt at early stages of the disease, but also by favouring some physiological functions of normal Htt, as P42 is naturally part of it, at the different stages of the disease. This makes P42 peptide potentially suitable not only to prevent, but also to treat HD.

 

https://doi.org/10.1093/hmg/ddy207

High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics

Perioperative Medicine

2018-06-11

Abstract

Background: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds.

Methods: The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent.

Results: Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-d-aspartate and neuronal nicotinic receptors.

Conclusions: Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.

 

http://anesthesiology.pubs.asahq.org/article.aspx?articleid=2685007

Behavioral Profiling of Zebrafish (Danio rerio) Larvae Following Teratogen Exposure

Teratogenicity Testing

2018-06-13

Abstract

Apart from morphological, biochemical, and genetic alterations induced by teratogen compounds, there is an increase interest in characterizing behavioral alterations. Behavior is a sensitive parameter that can provide information regarding developmental disruptions noninvasively, as it is the result of brain processes. Behavioral disturbances interfere with animals’ capacity to cope with the environment, having an impact on the organism’s life. Hereby, it is proposed behavioral assays consisting on recording larvae in multiwell plates and video analysis with a proper software, allowing for teratogen screening of behavior. How to evaluate locomotor, anxiety-like and avoidance-like behaviors, and the integrity of sensory-motor functions and learning are discussed in this chapter.

Keywords


Teratogen - Zebrafish larvae - Behavior - Activity - Thigmotaxis - Avoidance behavior - Dark–light challenge - Startle response

 

https://link.springer.com/protocol/10.1007/978-1-4939-7883-0_22

Comparative behavioral toxicology with two common larval fish models: Exploring relationships among modes of action and locomotor responses

Science of The Total Environment

2018-06-18

Abstract

Behavioral responses inform toxicology studies by rapidly and sensitively detecting molecular initiation events that propagate to physiological changes in individuals. These behavioral responses can be unique to chemical specific mechanisms and modes of action (MOA) and thus present diagnostic utility. In an initial effort to explore the use of larval fish behavioral response patterns in screening environmental contaminants for toxicity and to identify behavioral responses associated with common chemical specific MOAs, we employed the two most common fish models, the zebrafish and the fathead minnow, to define toxicant induced swimming activity alterations during interchanging photoperiods. Though the fathead minnow (Pimephales promelas) is a common model for aquatic toxicology research and regulatory toxicology practice, this model has received little attention in behavioral studies compared to the zebrafish, a common biomedical model. We specifically compared behavioral responses among 7 different chemicals (1-heptanol, phenol, R-(-)-carvone, citalopram, diazinon, pentylenetetrazole (PTZ), and xylazine) that were selected and classified based on anticipated MOA (nonpolar narcosis, polar narcosis, electrophile, specific mechanism) according to traditional approaches to examine whether these comparative responses differ among chemicals with various structure-based predicted toxicity. Following standardized experimental guidelines, zebrafish embryos and fathead minnow larvae were exposed for 96 h to each compound then were observed using digital behavioral analysis. Behavioral observations included photomotor responses, distance traveled, and stimulatory, refractory and cruising locomotor activity. Though fathead minnow larvae displayed greater behavioral sensitivity to 1-heptanol, phenol and citalopram, zebrafish were more sensitive to diazinon and R-(-)-carvone. Both fish models were equally sensitive to xylazine and PTZ. Further, the pharmaceuticals citalopram and xylazine significantly affected behavior at therapeutic hazard values, and each of the seven chemicals elicited unique behavioral response profiles. Larval fish behaviors appear useful as early tier diagnostics to identify mechanisms and pathways associated with diverse biological activities for chemicals lacking mechanistic data.

Keywords

High throughput screening; Behavior; Alternative toxicity testing; Chemical bioactivity; Comparative toxicology

 

https://www.sciencedirect.com/science/article/pii/S004896971832059X

Translational control of depression-like behavior via phosphorylation of eukaryotic translation initiation factor 4E

Nature Communications

2018-06-25

Abstract

Translation of mRNA into protein has a fundamental role in neurodevelopment, plasticity, and memory formation; however, its contribution in the pathophysiology of depressive disorders is not fully understood. We investigated the involvement of MNK1/2 (MAPK-interacting serine/threonine-protein kinase 1 and 2) and their target, eIF4E (eukaryotic initiation factor 4E), in depression-like behavior in mice. Mice carrying a mutation in eIF4E for the MNK1/2 phosphorylation site (Ser209Ala, Eif4e ki/ki), the Mnk1/2 double knockout mice (Mnk1/2−/−), or mice treated with the MNK1/2 inhibitor, cercosporamide, displayed anxiety- and depression-like behaviors, impaired serotonin-induced excitatory synaptic activity in the prefrontal cortex, and diminished firing of the dorsal raphe neurons. In Eif4e ki/ki mice, brain IκBα, was decreased, while the NF-κB target, TNFα was elevated. TNFα inhibition in Eif4e ki/ki mice rescued, whereas TNFα administration to wild-type mice mimicked the depression-like behaviors and 5-HT synaptic deficits. We conclude that eIF4E phosphorylation modulates depression-like behavior through regulation of inflammatory responses.

 

https://www.nature.com/articles/s41467-018-04883-5

Biodegradable Si3N4 bioceramic sintered with Sr, Mg and Si for spinal fusion: Surface characterization and biological evaluation

Applied Materials Today

2018-06-27

Abstract

Silicon nitride (Si3N4) is an industrial ceramic used in spinal fusion and maxillofacial reconstruction because of its excellent mechanical properties and good biocompatibility. This study compares the surface properties, apatite formation ability, bacterial infection, cell-biomaterial interactions, and in vivo toxicity (zebrafish) of newly developed Si3N4 bioceramics (sintered with bioactive sintering additives SrO, MgO and SiO2) with two standard biomaterials; titanium (Ti) and traditional Si3N4 bioceramics (sintered with standard sintering additives Al2O3 and Y2O3). In general, Si3N4 bioceramics (both the newly developed and the traditional) displayed less in vitro bacterial affinity than Ti, which may arise from differences in the surface properties between these two types of material. The newly developed Si3N4 bioceramics developed lower biofilm coverage and thinner biofilm, compared to traditional Si3N4 bioceramics. The effects of ionic dissolution products (leach) on proliferation and differentiation of MC3T3-E1 cell were also investigated. Ionic dissolution products containing moderate amount of Sr, Mg and Si ions (approximately 4.72 mg/L, 3.26 mg/L and 3.67 mg/L, respectively) stimulated osteoblast proliferation during the first 2 days in culture. Interestingly, ionic dissolution products from the traditional Si3N4 bioceramics that contained small amount of Si and Y ions achieved the greatest stimulatory effect for alkaline phosphatase activity after 7 days culture. The toxicity of ionic dissolution products was investigated in a putative developmental biology model: zebrafish (Danio rerio). No toxicity, or developmental abnormalities, was observed in zebrafish embryos exposed to ionic dissolution products, for up to 144 h post fertilization. These newly developed Si3N4 bioceramics with bioactive sintering additives show great potential as orthopedic implants, for applications such as spinal fusion cages. Future work will focus on evaluation of the newly developed Si3N4 bioceramics using a large animal model.

 

Keywords

Si3N4 bioceramic - Spinal fusion - Biocompatibility - Bioactive ions - Zebrafish

 

https://www.sciencedirect.com/science/article/pii/S2352940718302099

Is the 3,4-methylendioxypyrovalerone/mephedrone combination responsible for enhanced stimulant effects? A rat study with investigation of the effect/concentration relationships

Psychopharmacology

2018-07-03

Abstract


Rationale

The use of synthetic cathinones as recreational drugs frequently sold in combination has been increasing exponentially. However, the consequences of combining cathinones on the resulting stimulant effects and the pharmacokinetics have been poorly investigated.

Objective and methods

To study 3,4-methylenedioxypyrovalerone (MDPV; 3 mg/kg) and mephedrone (4-MMC; 30 mg/kg)-induced effects on rat locomotor activity and pharmacokinetics, administered alone or in combination by the intragastric route. The pharmacokinetic parameters were determined using non-compartmental analysis and the relationships between the locomotor activity and drug concentrations using sigmoidal Emax modeling.

Results

Locomotor activity significantly increased during the first hour post-administration with the MDPV/4-MMC combination in comparison to MDPV (p < 0.001) and 4-MMC (p < 0.01) alone. The pharmacokinetic profile of MDPV, but not 4-MMC, was significantly modified with the combination resulting in decreases in Cmax (16.4 ± 5.5 versus 62.2 ± 14.2 μg/L, p < 0.05) and AUC0 → ∞ (708 ± 91 versus 3316 ± 682 μg/L/min, p < 0.01) and increases in V/F (582.6 ± 136.8 versus 115.9 ± 42.7 L/kg, p < 0.05) and Cl/F (4.6 ± 0.7 versus 1.2 ± 0.4 L/kg/min, p < 0.01) in comparison to MDPV alone. The sigmoidal Emax model fitted the observed data well; MDPV being markedly more potent than 4-MMC (EC50, 0.043 versus 0.7 μmol/L). The enhancing factor representing the MDPV contribution to the alteration in the relationships between locomotor activity and 4-MMC concentrations was 0.3.

Conclusion

An MDPV/4-MMC combination results in enhanced stimulant effects in the rat, despite significant reduction in MDPV bioavailability. Enhanced effects could be explained by increased MDPV distribution and/or possible complementation at the brain dopaminergic targets. However, the exact consequences of the MDPV/4-MMC combination in humans remain to be clarified.

Keywords


MDPV 4-MMC Locomotor activity Pharmacokinetics Effect/concentration relationships

 

https://link.springer.com/article/10.1007/s00213-018-4962-0

Chromosome-level reference genome of the Siamese fighting fish Betta splendens, a model species for the study of aggression

Giga Science

2018-07-11

Abstract


Background: Siamese fighting fish Betta splendens (NCBI Taxonomy ID: 158456) are notorious for their aggressiveness and accordingly have been widely used to study aggression. However, the lack of a reference genome has so far limited the understanding of the genetic basis of aggression in this species. Here we present the first reference genome assembly of the Siamese fighting fish.

Findings: We first sequenced and de novo assembled a 465.24 Mb genome for the B. splendens variety Giant, with a weighted average (N50) scaffold size of 949.03 Kb and an N50 contig size of 19.01 Kb, covering 99.93% of the estimated genome size. To obtain a chromosome-level genome assembly, we constructed one Hi-C library and sequenced 75.24 Gb reads using the BGISEQ-500 platform. We anchored approximately 93% of the scaffold sequences into 21 chromosomes and evaluated the quality of our assembly using the high contact frequency heatmap and BUSCO. We also performed comparative chromosome analyses between Oryzias latipes and B. splendens, revealing a chromosome conservation evolution in B. splendens. We predicted a total of 23,981 genes assisted by RNA-seq data generated from brain, liver, muscle and heart tissues of Giant, and annotated 15% repetitive sequences in the genome. Additionally, we resequenced other five B. splendens varieties and detected ~3.4M single-nucleotide variations (SNVs) and 27,305 indels.

Conclusions: We provide the first chromosome-level genome for the Siamese fighting fish. The genome will lay a valuable foundation for future research on aggression in B. splendens.

Keywords: Betta splendens; fish genome; aggression; Hi-C; chromosomal genome assembly; resequencing

 

https://doi.org/10.1093/gigascience/giy087

Auriculasin from Flemingia philippinensis roots shows good therapeutic indexes on hyperactive behavior in zebrafish

Biochemical and Biophysical Research Communications

2018-07-17

Abstract

Previously, period1b−/− zebrafish mutants were used to establish an attention deficit hyperactivity disorder (ADHD) model, in which hyperactive behavior was found to be a typical characteristic of ADHD due to down-regulated dopamine levels. Here, we used five prenylated isoflavones from Flemingia philippinensis roots to study their therapeutic effects on hyperactivity behavior in period1b−/− zebrafish. Results of locomotor activity assay showed that auriculasin, one of the prenylated isoflavones, significantly reduced the hyperactivity behavior in period1b−/− zebrafish. Hormone measurement results showed that auriculasin increased melatonin and dopamine content. Results of quantitative real-time polymerase chain reaction showed that auriculasin down-regulated the expression of mao but up-regulated the expression of th and per1b. Thus, auriculasin demonstrated a potential biological effect on dopamine activity to inhibit hyperactivity behavior in the ADHD zebrafish model by regulating circadian clock gene per1b.

Keywords
Isoflavones ; ADHD ; Hyperactive behavior ; Zebrafish ; Circadian clock ; per1b

 

https://www.sciencedirect.com/science/article/pii/S0006291X18315286

Biodegradable Si3N4 bioceramic sintered with Sr, Mg and Si for spinal fusion: Surface characterization and biological evaluation

Applied materialstoday

2018-07-27

Abstract

Silicon nitride (Si3N4) is an industrial ceramic used in spinal fusion and maxillofacial reconstruction because of its excellent mechanical properties and good biocompatibility. This study compares the surface properties, apatite formation ability, bacterial infection, cell-biomaterial interactions, and in vivo toxicity (zebrafish) of newly developed Si3N4 bioceramics (sintered with bioactive sintering additives SrO, MgO and SiO2) with two standard biomaterials; titanium (Ti) and traditional Si3N4 bioceramics (sintered with standard sintering additives Al2O3 and Y2O3). In general, Si3N4 bioceramics (both the newly developed and the traditional) displayed less in vitro bacterial affinity than Ti, which may arise from differences in the surface properties between these two types of material. The newly developed Si3N4 bioceramics developed lower biofilm coverage and thinner biofilm, compared to traditional Si3N4 bioceramics. The effects of ionic dissolution products (leach) on proliferation and differentiation of MC3T3-E1 cell were also investigated. Ionic dissolution products containing moderate amount of Sr, Mg and Si ions (approximately 4.72 mg/L, 3.26 mg/L and 3.67 mg/L, respectively) stimulated osteoblast proliferation during the first 2 days in culture. Interestingly, ionic dissolution products from the traditional Si3N4 bioceramics that contained small amount of Si and Y ions achieved the greatest stimulatory effect for alkaline phosphatase activity after 7 days culture. The toxicity of ionic dissolution products was investigated in a putative developmental biology model: zebrafish (Danio rerio). No toxicity, or developmental abnormalities, was observed in zebrafish embryos exposed to ionic dissolution products, for up to 144 h post fertilization. These newly developed Si3N4 bioceramics with bioactive sintering additives show great potential as orthopedic implants, for applications such as spinal fusion cages. Future work will focus on evaluation of the newly developed Si3N4 bioceramics using a large animal model.

 

https://www.sciencedirect.com/science/article/pii/S2352940718302099

Influence of diltiazem on fathead minnows across dissolved oxygen gradients

SETAC Environmental Toxicology and Chemistry

2018-07-28

ABSTRACT

Water resources in many arid to semi‐arid regions are stressed by population growth and drought. Growing populations and climatic changes are influencing contaminant and water chemistry dynamics in urban inland waters where flows can be dominated by, or even dependent on, wastewater effluent discharge. In these watersheds, interacting stressors such as dissolved oxygen (DO) and environmental contaminants (e.g., pharmaceuticals) have the potential to affect fish physiology and populations. Recent field observations from our group identified the calcium channel blocker diltiazem in fish plasma exceeding human therapeutic doses (e.g., Cmin) in aquatic systems impaired due to nonattainment of DO water quality standards (WQS) and criteria. Thus, our study objectives examined: 1) standard acute and chronic effects of DO and diltiazem to fish, 2) influences of DO, at criteria levels deemed protective of aquatic life, on diltiazem toxicity to fish, and 3) whether sublethal effects occur at diltiazem water concentrations predicted to cause a human therapeutic level in fish plasma (therapeutic hazard value, THV). DO x diltiazem co‐exposures significantly decreased survival at typical stream, lake, and reservoir WQS of 5.0 and 3.0 mg DO/L. DO and diltiazem growth effects were observed at 2x and 10x their LC50 values (1.7 and 28.2 mg/L, respectively). Larval fathead minnow swimming behavior following all DO and diltiazem exposures generally decreased and significantly reduced light:dark bursting distance traveled, number of movements, and duration at concentrations as low as the THV. Individual and population level consequences of such responses are not yet understood; however, these observations suggest that assessments with pharmaceuticals and other contaminants may underestimate the effects in fish across DO levels considered protective of aquatic life.

 

https://setac.onlinelibrary.wiley.com/doi/abs/10.1002/etc.4242

Swimming activity in zebrafish larvae exposed to veterinary antiparasitic pharmaceuticals

Environmental Toxicology and Pharmacology

2018-08-25

Abstract

Veterinary antiparasitic pharmaceuticals have been detected in surface waters and several of these pharmaceuticals act on the nervous system on the target organisms implying that neurological effects also might be of concern in non-target animals such as fish. We tested if exposure to antiparasitic pharmaceuticals affect swimming activity in 6 days old zebrafish larvae. The results revealed that most pharmaceuticals did not cause any effects in swimming activity. However, larvae exposed to 0.58 mg/L doramectin displayed reduced swimming activity even though they were classified as normal, having no morphological abnormalities.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S1382668918302874

The Locus Coeruleus Modulates Intravenous General Anesthesia of Zebrafish via a Cooperative Mechanism

Cell Press

2018-09-01

Highlights

• Intravenous anesthetics induce general anesthesia in larval zebrafish
• Impairment of the locus coeruleus (LC)-norepinephrine system affects anesthesia
• Anesthetics suppress LC neuronal activities via pre- and postsynaptic mechanisms
• Larval zebrafish is an ideal model for investigation of general anesthesia

Summary

How general anesthesia causes loss of consciousness has been a mystery for decades. It is generally thought that arousal-related brain nuclei, including the locus coeruleus (LC), are involved. Here, by monitoring locomotion behaviors and neural activities, we developed a larval zebrafish model for studying general anesthesia induced by propofol and etomidate, two commonly used intravenous anesthetics. Local lesion of LC neurons via two-photon laser-based ablation or genetic depletion of norepinephrine (NE; a neuromodulator released by LC neurons) via CRISPR/Cas9-based mutation of dopamine-β-hydroxylase (dbh) accelerates induction into and retards emergence from general anesthesia. Mechanistically, in vivo whole-cell recording revealed that both anesthetics suppress LC neurons’ activity through a cooperative mechanism, inhibiting presynaptic excitatory inputs and inducing GABAA receptor-mediated hyperpolarization of these neurons. Thus, our study indicates that the LC-NE system plays a modulatory role in both induction of and emergence from intravenous general anesthesia.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S2211124718313329

Ecotoxicological effects, water quality standards and risk assessment for the anti-diabetic metformin

Environmental Pollution

2018-09-08

Abstract

Metformin (MET) is among the most consumed pharmaceuticals worldwide. This compound has been frequently detected in fresh surface water. However, ecotoxicological information for MET is still too limited, particularly regarding chronic and behavioral data. This study aimed to help filling these knowledge gaps, by carrying out both acute and chronic studies with four different test organisms from three different trophic levels. We assessed different endpoints, including the swimming behavior of Danio reriolarvae. We also derived both short-term and long-term environmental quality standards (EQS) for the protection of freshwater pelagic biota towards MET adverse effects. A risk quotient (RQ) was calculated for MET in fresh surface water, considering a worst-case scenario. Daphnia similis was by far the most sensitive species evaluated. An EC10 of 4.4 mg L−1 was obtained from the reproduction test with D. similis. A long-term EQS of 88 μg L−1 was derived and a RQ of 0.38 was obtained. An ecological risk is not expected for the chronic exposure of pelagic freshwater species to MET, considering the endpoints and the standard bioassays usually recommended in standard protocols. However, endocrine disruptive effects and potential interactive effects of MET with other co-occurring contaminants cannot be ruled out. To the best of our knowledge, this study presents the first data related with MET effects on population endpoints of D. similis and Hydra attenuata, as well as on the locomotor activity of D. rerio.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749118316294

Automated morphological feature assessment for zebrafish embryo developmental toxicity screens

Toxicological Sciences

2018-10-01

Abstract


Detection of developmental phenotypes in zebrafish embryos typically involves a
visual assessment and scoring of morphological features by an individual
researcher. Subjective scoring could impact results and be of particular concern
when phenotypic effect patterns are also used as a diagnostic tool to classify
compounds. Here we introduce a quantitative morphometric approach based on
image analysis of zebrafish embryos. A software called FishInspector was developed
to detect morphological features from images collected using an automated system
to position zebrafish embryos. The analysis was verified and compared with visual
assessments of three participating laboratories using three known developmental
toxicants (methotrexate, dexamethasone and topiramate) and two negative
compounds (loratadine and glibenclamide). The quantitative approach exhibited
higher sensitivity and made it possible to compare patterns of effects with the
potential to establish a grouping and classification of developmental toxicants. Our
approach improves the robustness of phenotype scoring and reliability of assay
performance and, hence, is anticipated to improve the predictivity of developmental
toxicity screening using the zebrafish embryo.

 

Link to the publication :

http://scholar.google.fr/scholar_url?url=https://academic.oup.com/toxsci/advance-article-pdf/doi/10.1093/toxsci/kfy250/25949135/kfy250.pdf&hl=fr&sa=X&d=693064754340786427&scisig=AAGBfm1hNXS2Dp3SEr7wAAzh0W_DnrNFOg&nossl=1&oi=scholaralrt

 

Sub-lethal UV radiation during early life stages alters the behaviour, heart rate and oxidative stress parameters in zebrafish (Danio rerio)

Ecotoxicology and Environmental Safety

2018-10-01

Abstract

Environmental UV radiation in sufficient doses, as a possible consequence of climate change, is potent enough to affect living organisms with different outcomes, depending on the exposure life stage. The aim of this project was to evaluate the potentially toxic effects of exposure to sub-lethal and environmentally relevant doses of UVA (9.4, 18. 7, 37.7 J/cm2) and UVB radiation (0.013, 0.025, 0.076 J/cm2) on the development and behaviour in early life stages (4.5–5.5 h post fertilization, hpf) of the zebrafish (Danio rerio). The used doses were all below the median lethal dose (LD50) and caused no significant difference in survival, deformities, or hatching between exposed and control groups. Compared to controls, there were transient UVA and UVB exposure effects on heart rate, with dose dependent reductions at 50 hpf, and at 60 hpf for UVA only. The UVB exposure caused an increasing trend in reactive oxygen species (ROS) formation at the two highest doses, even though only significant at 120 hpf for the second highest dose. Both UVA and UVB caused an increasing trend in lipid peroxidation (LPO) at the highest doses tested at 72 hpf. Furthermore, UVA exposure led to significant reductions in larval movement following exposure to the two highest doses of UVA, i.e., reduction in the time spent active and the total distance moved compared to control at 100 hpf, while no effect on the swimming speed was observed. The lowest dose of UVA had no effect on behaviour. In contrast, the highest dose of UVB led to a possible increase in the time spent active and a slower average swimming speed although these effects were not significant (p = 0.07). The obtained results show that UV doses below LD50 levels are able to cause changes in the behaviour and physiological parameters of zebrafish larvae, as well as oxidative stress in the form of ROS formation and LPO. Further testing is necessary to assess how this type of radiation and the effects observed could affect fish population dynamics.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651318309436

Motor Neuron Abnormalities Correlate with Impaired Movement in Zebrafish that Express Mutant Superoxide Dismutase 1

Zebrafish

2018-10-09

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p < 0.002) and traveled a significantly shorter distance during behavioral testing (p < 0.001) when compared with WT SOD1 and noninjected larvae. Furthermore, there was a positive correlation between distance traveled and motor axon length (R2 = 0.357, p < 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS.

 

Link to the publication :

https://www.liebertpub.com/doi/abs/10.1089/zeb.2018.1588

Expression of pathogenic SCN9A mutations in the zebrafish: A model to study small fiber neuropathy

2018-10-11

Highlights

• Set up of two read-outs reflecting Small-fiber neuropathy in zebrafish
• Expression of mutant SCN9A provides a model for Small-fiber neuropathy in zebrafish.
• Development of a temperature-controlled water compartment

Abstract

Small fiber neuropathy (SFN) patients experience a spectrum of sensory abnormalities, including attenuated responses to non-noxious temperatures in combination with a decreased density of the small-nerve fibers. Gain-of-function mutations in the voltage-gated sodium channels SCN9A, SCN10A and SCN11A have been identified as an underlying genetic cause in a subpopulation of patients with SFN. Based on clinical-diagnostic tests for SFN, we have set up a panel of two read-outs reflecting SFN in zebrafish, being nerve density and behavioral responses. Nerve density was studied using a transgenic line in which the sensory neurons are GFP-labelled. For the behavioral experiments, a temperature-controlled water compartment was developed. This device allowed quantification of the behavioral response to temperature changes. By using these read-outs we demonstrated that zebrafish embryos transiently overexpressing the pathogenic human SCN9A p.(I228M) or p.(G856D) mutations both have a significantly decreased density of the small-nerve fibers. Additionally, larvae overexpressing the p.(I228M) mutation displayed a significant increase in activity induced by temperature change. As these features closely resemble the clinical hallmarks of SFN, our data suggest that transient overexpression of mutant human mRNA provides a model for SFN in zebrafish. This disease model may provide a basis for testing the pathogenicity of novel genetic variants identified in SFN patients. Furthermore, this model could be used for studying SFN pathophysiology in an in vivo model and for testing therapeutic interventions.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0014488618305727

Novel neurotoxic peptides from Protopalythoa variabilis virtually interact with voltage-gated sodium channel and display anti-epilepsy and neuroprotective activities in zebrafish

Archieves of Toxicology

2018-10-17

Abstract

We previously reported a novel toxic peptide identified from the anthozoan Protopalythoa variabilis transcriptome which is homologous to a novel structural type of sodium channel toxin isolated from a parental species (Palythoa caribaeorum). The peptide was named, according to its homologous, as Pp V-shape α-helical peptide (PpVα) in the present study. Through molecular docking and dynamics simulation, linear and hairpin folded PpVα peptides were shown to be potential voltage-gated sodium channel blockers. Nowadays, sodium channel blockers have been the mainstream of the pharmacological management of epileptic seizures. Also, sodium channel blockers could promote neuronal survival by reducing sodium influx and reducing the likelihood of calcium importation resulting in suppressing microglial activation and protecting dopaminergic neurons from degeneration. The folded PpVα peptide could decrease pentylenetetrazol (PTZ)-induced c-fos and npas4a expression level leading to reverse PTZ-induced locomotor hyperactivity in zebrafish model. In vitro, the folded PpVα peptide protected PC12 cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via activating heme oxygenase-1 (HO-1) and attenuating inducible nitric oxide synthase (iNOS) expression. In vivo, PpVα peptide suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish and, importantly, prevented the 6-OHDA-induced excessive ROS generation and subsequent dopaminergic neurons loss. This study indicates that the single S–S bond folded PpVα peptide arises as a new structural template to develop sodium channel blockers and provides an insight on the peptide discovery from cnidarian transcriptome to potentially manage epilepsy and neurodegenerative disorders.

 

Link to the publication :

https://link.springer.com/article/10.1007/s00204-018-2334-5

A Zebrafish Behavior Assay for Assessing Anti- Epileptic Drug Efficacy

NeuroQuantology

2018-10-17

ABSTRACT


The availability of zebrafish larvae model of epileptic seizure provides opportunities to identify novel anticonvulsants
for treatment of people with epilepsy. However, the major parameters of zebrafish behavior assay for assessing antiepileptic drug efficacy existed disparity. In this study, we chose the high, medium, slow-speed moved distances and the total distances moved for seizure-like activity quantification in individual wells of a 48-well plate in the dark phase.
Results also showed that the high-speed moved distances were given a more reasonable and sensitive dose-response
curve than the total distances in zebrafish larvae model exposed to 10 mM pentylenetetrazole (PTZ). Moreover, this
seizure-like swimming pattern was alleviated by the addition of either phenytoin (PHT) or valproate sodium (VPA),
two commonly prescribed anti-epileptic drugs (AEDs). They exhibited concentration-dependent inhibition of both
locomotor activity and PTZ-induced c-fos transcription, confirming their anticonvulsant characteristics. Whereas
carbamazepine (CBZ) promoted zebrafish larvae movement by 26% at 30 μM and 116.7% at 100 μM, and sharply
suppressed zebrafish larvae motility about 94.8% at 300 μM. C-fos expression significantly decreased at 30 μM and
300 μM CBZ, which was found to oppose various aspects of the PTZ-induced changes in activity. These results indicated
that this zebrafish larvae model could be useful for studying drug efficacy, predicting drug targets and aiding our
understanding of disease etiology.

 

 

 

Knockdown of chondroitin-4-sulfotransferase-1, but not of dermatan-4-sulfotransferase-1, accelerates regeneration of zebrafish after spinal cord injury

The Faseb Journal

2018-10-19

Glycosaminoglycans such as chondroitin sulfate (CS) and dermatan sulfate (DS) are long chains of repeating disaccharide units, covalently linked to core proteins to form proteoglycans. Proteoglycans can be cell membrane–bound or are part of the extracellular matrix. They are important in a wide range of biologic processes, including development, synaptic plasticity, and regeneration after injury, as well as modulation of growth factor signaling, cell migration, survival, and proliferation. Synthesis of CS and DS in the Golgi apparatus is mediated by sulfotransferases that modify sugar chains through transfer of sulfate groups to specific positions on the sugar moieties. To clarify the functions of CS and DS during nervous system regeneration, we studied the effect of chondroitin 4-O-sulfotransferase-1/carbohydrate sulfotransferase-11 (C4ST-1/Chst-11) and dermatan 4-O-sulfotransferase-1/Chst-14 (D4ST-1/Chst-14) down-regulation on spinal cord regeneration in larval and adult zebrafish. In our study, knockdown of C4ST1/Chst-11 accelerated regeneration after spinal cord injury in larval and adult zebrafish and knockdown of D4ST1/Chst-14 did not alter regenerative capacity. From these and previous observations, we drew the conclusion that different CS and DS expression patterns can be growth permitting, growth inhibiting, or neutral for regrowing or sprouting axons, depending on the tissue environment of a particular animal species.—Sahu, S., Li, R., Loers, G., Schachner, M. Knockdown of chondroitin-4-sulfotransferase-1, but not of dermatan-4-sulfotransferase-1, accelerates regeneration of zebrafish after spinal cord injury.

Link to the publication :

https://www.fasebj.org/doi/abs/10.1096/fj.201800852RR

Effects of 17α‑ethinylestradiol on caudal fin regeneration in zebrafish larvae

Science of the total environment

2018-10-21

Highlights

• Effects of EE2 on the regenerative capacity of larval zebrafish were assessed.
• EE2 exposure inhibited fin regeneration and changed locomotor behavior.
• EE2 exposure down-regulated the transcriptions of regeneration-related genes.
• EE2 exposure changed the immune competence in the amputated fish.

Abstract

The ability to restore tissue function and morphology after injury is a key advantage of many fish for a greater chance of survival. The tissue regeneration process is regulated by multiple pathways, and it can therefore be hypothesized that environmental contaminants targeting components of these signaling pathways, may disrupt the fish's capability to repair or regenerate. This could lead to higher mortality and eventually even to a decline in populations. In this study, the effects of 17α‑ethinylestradiol (EE2), a synthetic estrogen, were assessed on the regenerative capacity of larval zebrafish. Zebrafish aged 2 hour post fertilization (hpf) were exposed to 1, 10, or 100 ng/L EE2, and the caudal fins were amputated at 72 hpf. It was found that EE2 exposure significantly inhibited fin regeneration and changed locomotor behavior. The transcription levels for most of the genes involved in the signaling networks regulating the fin regeneration, such as axin2, fgfr1, bmp2b and igf2b, were down-regulated in the amputated fish in response to EE2 exposure, which was in contrast to their increased patterns in the vehicle-exposed control fish. Additionally, the mRNA levels of several immune-related genes, such as il-, il-6, il-10 and nf-κb2, were significantly decreased after EE2 exposure, accompanied by a lower density of neutrophils migrated into the wound site. In conclusion, the present study indicated for the first time that estrogenic endocrine disrupting chemicals (EEDCs) could inhibit the regenerative capacity of zebrafish, and this effect was speculated to be mediated through the alteration in regeneration-related signaling pathways and immune competence. This work expands our knowledge of the potential effects of EEDCs on injured aquatic organisms, and highlights the ecotoxicological significance of relationships between regenerative process and endocrine system. This study also implies the potential application of fin regeneration assay for assessing immunotoxicity in ecotoxicological risk assessment.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0048969718341718

Developmental protein kinase C hyper-activation results in microcephaly and behavioral abnormalities in zebrafish

Transl Psychiatry

2018-10-23

Abstract

Susceptible genetic polymorphisms and altered expression levels of protein kinase C (PKC)-encoding genes suggest overactivation of PKC in autism spectrum disorder (ASD) development. To delineate the pathological role of PKC, we pharmacologically stimulated its activity during the early development of zebrafish. Results demonstrated that PKC hyper-activation perturbs zebrafish development and induces a long-lasting head size deficit. The anatomical and cellular analysis revealed reduced neural precursor proliferation and newborn neuron formation. β-Catenin that is essential for brain growth is dramatically degraded. Stabilization of β-catenin by gsk3β inhibition partially restores the head size deficit. In addition, the neuropathogenic effect of developmental PKC hyper-activation was further supported by the alterations in the behavioral domain including motor abnormalities, heightened stress reactivity and impaired habituation learning. Taken together, by causally connecting early-life PKC hyper-activation to these neuropathological traits and the impaired neurogenesis, these results suggest that PKC could be a critical pathway in ASD pathogenesis.

 

Link to the publication :

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199330/

Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy

ACS Chemical Neuroscience

2018-10-23

Abstract

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3′-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.

High-performance counter-current chromatography isolation and initial neuroactivity characterization of furanocoumarin derivatives from Peucedanum alsaticum L (Apiaceae)

Phytomedicine

2018-10-26

Abstract

Background

Medicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity.

Purpose

To accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesteraseELISAs and zebrafish epilepsy models.

Research methods and procedure

In this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABAAantagonistpentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior.

Results

The solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ± 1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ± 2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ± 1.25%). The DCM extract of P. alsaticum fruits (0.62–1.75 µg/ml) and bergamottin (2–10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10–16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A.

Conclusion

Our findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0944711318305518

Drug repositioning in epilepsy reveals novel antiseizure candidates

Annals of clinical and transnational neurology

2018-10-26

Abstract


Objective: Epilepsy treatment falls short in ~30% of cases. A better understanding
of epilepsy pathophysiology can guide rational drug development in
this difficult to treat condition. We tested a low-cost, drug-repositioning strategy
to identify candidate epilepsy drugs that are already FDA-approved and
might be immediately tested in epilepsy patients who require new therapies.
Methods: Biopsies of spiking and nonspiking hippocampal brain tissue from
six patients with unilateral mesial temporal lobe epilepsy were analyzed by
RNA-Seq. These profiles were correlated with transcriptomes from cell lines
treated with FDA-approved drugs, identifying compounds which were tested
for therapeutic efficacy in a zebrafish seizure assay. Results: In spiking versus
nonspiking biopsies, RNA-Seq identified 689 differentially expressed genes, 148
of which were previously cited in articles mentioning seizures or epilepsy. Differentially
expressed genes were highly enriched for protein–protein interactions
and formed three clusters with associated GO-terms including myelination,
protein ubiquitination, and neuronal migration. Among the 184 compounds, a
zebrafish seizure model tested the therapeutic efficacy of doxycycline, metformin,
nifedipine, and pyrantel tartrate, with metformin, nifedipine, and
pyrantel tartrate all showing efficacy. Interpretation: This proof-of-principle
analysis suggests our powerful, rapid, cost-effective approach can likely be
applied to other hard-to-treat diseases.

Developmental neurotoxicity and immunotoxicity induced by graphene oxide in zebrafish embryos

Environmental Toxicology

2018-12-13

Abstract

Graphene oxide (GO) has emerged as the worldwide promising candidate for biomedical application, such as for drug delivery, bio‐sensing and anti‐cancer therapy. This study was focused on the zebrafish and RAW264.7 cell line as in vivo and in vitro models to assess the potential developmental neurotoxicity and immunotoxicity of GO. No obvious acute developmental toxicity was observed upon treatments with 0.01, 0.1, and 1 μg/mL GO for five consecutive days. However, decreased hatching rate, increased malformation rate, heart beat rate and hypoactivity of locomotor behavior were detected when exposed to 10 μg/mL GO. Also, RT‐PCR analysis revealed that expressions of genes related to the nervous system were up‐regulated. The potential risk of GO for developmental neurotoxicity may be ascribed to the high level of oxidative stress induced by high concentration of GO. Most importantly, the mRNA levels of immune response associated genes, such as interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor‐α (TNFα), interferon‐γ (IFN‐γ) were significantly increased under environmental concentration exposure. The activation of pro‐inflammatory immune response was also observed in macrophage cell line. Taken together, our results demonstrated that immunotoxicity is a sensitive indicator for assessment of bio‐compatibility of GO.

 

Link to the publication :

https://onlinelibrary.wiley.com/doi/abs/10.1002/tox.22695

Bioassay-guided isolation of anti-seizure principles from Semen Pharbitidis using a zebrafish pentylenetetrazol seizure model

Journal of Ethnopharmacology

2018-12-17

Abstract

Ethnopharmacological relevance

Semen Pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae) is a well-known traditional Chinese medicinal plant used for treating helminthiasis and epilepsy in China.

Aim of the study

This study aims to identify the anti-seizure components from Semen Pharbitidis.

Methods

A bioassay-guided isolation of anti-seizure compounds from Semen Pharbitidis was performed using a zebrafishpentylenetetrazol seizure model. The structures of active compounds were elucidated by high resolution mass spectrometry. The fragments of active compounds were tested for anti-seizure activity as well.

Results

The bioassay-guided isolation of ethanol extract of Semen Pharbitidis led to a group of resin glucosides, namely pharbitin. One of the fragments of pharbitin, 2-methylbutyric acid, also showed anti-seizure activity.

Conclusions

We provided further experimental scientific evidence to support the traditional use of Semen Pharbitidis for the treatment of epilepsy. Pharbitin was identified to be the main anti-seizure component in Semen Pharbitidis.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0378874118320609

RGD inhibition of itgb1 ameliorates laminin-a2 deficient zebrafish fibre pathology

Human Molecular Genetics

2018-12-19

Abstract

Deficiency of muscle basement membrane (MBM) component laminin-α2, leads to muscular dystrophy congenital type 1a MDC1a, a currently untreatable myopathy. Laminin-α2 has two main binding partners within the MBM, dystroglycan and integrin. Integrins co-ordinate both cell adhesion and signalling, however there is little mechanistic insight into integrin’s function at the MBM. In order to study integrin’s role in basement membrane development and how this relates to the MBM’s capacity to handle force, an itgβ1.b-/- zebrafish line was created. Histological examination revealed increased extracellular matrix (ECM) deposition at the MBM in the itgβ1.b-/- fish, when compared to controls. Surprisingly, both laminin and collagen proteins were found to be increased in expression at the MBM of the itgβ1.b-/-larvae, when compared to controls. This increase in extracellular matrix components resulted in a decrease in myotomal elasticity as determined by novel passive force analyses. To determine if it was possible to control ECM deposition at the MBM by manipulating integrin activity, RGD peptide, a potent inhibitor of integrin-β1, was injected into in a zebrafish model of MDC1a. As postulated an increase in laminin and collagen was observed in the lama2-/- mutant MBM. Importantly, there was also an improvement in fibre stability at the MBM, judged by a reduction in fibre pathology. These results therefore show that, blocking ITGβ1 signalling increases ECM deposition at the MBM, a process that could be potentially exploited for treatment of MDC1a.

 

Link to the publication :

https://academic.oup.com/hmg/advance-article-abstract/doi/10.1093/hmg/ddy426/5253587

Multigenerational consequences of early-life cannabinoid exposure in zebrafish

Toxicology and Applied Pharmacology

2018-12-26

Highlights

• CBD caused adverse developmental outcomes at much lower concentrations than THC.
• THC and CBD exposure led to expression changes in developmentally imperative genes.
• Sub-lethal concentrations of THC and CBD affected larval and adult zebrafish behavior.
• THC and CBD decreased zebrafish offspring in the F0 but not the F1 generation.

Abstract

While Δ9-tetrahydrocannabinol (THC) has been widely studied in the realm of developmental and reproductive toxicology, few studies have investigated potential toxicities from a second widely used cannabis constituent, cannabidiol (CBD). CBD is popularized for its therapeutic potential for reducing seizure frequencies in epilepsy. This study investigated developmental origins of health and disease (DOHaD) via multigenerational gene expression patterns, behavior phenotypes, and reproductive fitness of a subsequent F1 following an F0 developmental exposure of zebrafish (Danio rerio) to THC (0.024, 0.12, 0.6 mg/L; 0.08, 0.4, 2 μM) or CBD (0.006, 0.03, 0.15 mg/L; 0.02, 0.1, 0.5 μM). Embryonic exposure at these concentrations did not cause notable morphological abnormalities in either F0 or F1 generations. However, during key developmental stages (14, 24, 48, 72, and 96 h post fertilization) THC and CBD caused differential expression of c-fos,brain-derived neurotrophic factor (bdnf), and deleted-in-azoospermia like (dazl), while in F1 larvae only CBD differentially expressed dazl. Larval photomotor behavior was reduced (F0) or increased (F1) by THC exposure, while CBD had no effect on F0 larvae, but decreased activity in the unexposed F1 larvae. These results support our hypothesis of cannabinoid-related developmental neurotoxicity. As adults, F0 fecundity was reduced, but it was not in F1 adults. Conversely, in the adult open field test there were no significant effects in F0 fish, but a significant reduction in the time in periphery was seen in F1 fish from the highest THC exposure group. The results highlight the need to consider long-term ramifications of early-life exposure to cannabinoids.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0041008X18305805

Post-retrieval re-learning strengthens hippocampal memories via destabilization and reconsolidation

The Journal of neuroscience

2018-12-26

Abstract

Memory reconsolidation is hypothesised to be a mechanism by which memories can be updated with new information. Such updating has previously been shown to weaken memory expression or change the nature of the memory. Here we demonstrate that retrieval-induced memory destabilization also allows that memory to be strengthened by additional learning. We show that for rodent contextual fear memories, this retrieval-conditioning effect is observed only when conditioning occurs within a specific temporal window opened by retrieval. Moreover, it necessitates hippocampal protein degradation at the proteasome and engages hippocampal Zif268 protein expression, both of which are established mechanisms of memory destabilization-reconsolidation. We also demonstrate a conceptually analogous pattern of results in human visual paired-associate learning. Retrieval-relearning strengthens memory performance, again only when relearning occurs within the temporal window of memory reconsolidation. These findings link retrieval-mediated learning in humans to the reconsolidation literature, and have potential implications both for the understanding of endogenous memory gains and strategies to boost weakly-learned memories.

Evaluation of neuroactive effects of ethanol extract of Schisandra chinensis, Schisandrin, and Schisandrin B and determination of underlying mechanisms by zebrafish behavioral profiling

Chinese Journal of Natural Medicines

2018-12-27

Abstract

Schisandra chinensis, a traditional Chinese medicine (TCM), has been used to treat sleep disorders. Zebrafish sleep/wake behavioral profiling provides a high-throughput platform to screen chemicals, but has never been used to study extracts and components from TCM. In the present study, the ethanol extract of Schisandra chinensis and its two main lignin components, schisandrin and schisandrin B, were studied in zebrafish. We found that the ethanol extract had bidirectional improvement in rest and activity in zebrafish. Schisandrin and schisandrin B were both sedative and active components. We predicted that schisandrin was related to serotonin pathway and the enthanol extract of Schisandra chinensis was related to seoronin and domapine pathways using a database of zebrafish behaviors. These predictions were confirmed in experiments using Caenorhabditis elegans. In conclusion, zebrafish behavior profiling could be used as a high-throughput platform to screen neuroactive effects and predict molecular pathways of extracts and components from TCM.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S187553641830133X

Evaluation of neuroactive effects of ethanol extract of Schisandra chinensis, Schisandrin, and Schisandrin B and determination of underlying mechanisms by zebrafish behavioral profiling

Chinese Journal of Chinese neurosciences

2018-12-27

Abstract

Schisandra chinensis, a traditional Chinese medicine (TCM), has been used to treat sleep disorders. Zebrafish sleep/wake behavioral profiling provides a high-throughput platform to screen chemicals, but has never been used to study extracts and components from TCM. In the present study, the ethanol extract of Schisandra chinensis and its two main lignin components, schisandrin and schisandrin B, were studied in zebrafish. We found that the ethanol extract had bidirectional improvement in rest and activity in zebrafish. Schisandrin and schisandrin B were both sedative and active components. We predicted that schisandrin was related to serotonin pathway and the enthanol extract of Schisandra chinensis was related to seoronin and domapine pathways using a database of zebrafish behaviors. These predictions were confirmed in experiments using Caenorhabditis elegans. In conclusion, zebrafish behavior profiling could be used as a high-throughput platform to screen neuroactive effects and predict molecular pathways of extracts and components from TCM.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S187553641830133X

The mineralocorticoid receptor is essential for stress axis regulation in zebrafish larvae

Nature

2018-12-27

Abstract

The mineralocorticoid receptor (MR) in mammals mediates the effects of aldosterone in regulating fluid balance and potassium homeostasis. While MR signalling is essential for survival in mammals, there is no evidence that MR has any physiological role in ray-finned fish. Teleosts lack aldosterone and emerging evidence suggest that cortisol mediates ion and fluid regulation by activating glucocorticoid receptor (GR) signalling. Consequently, a physiological role for MR signalling, despite its conserved and ancient origin, is still lacking. We tested the hypothesis that a key physiological role for MR signalling in fish is the regulation of stress axis activation and function. Using either MR or GR knockout zebrafish, our results reveal distinct and complementary role for these receptors in stress axis function. GR−/− mutants were hypercortisolemic and failed to elicit a cortisol stress response, while MR−/− mutants showed a delayed, but sustained cortisol response post-stressor. Both these receptors are involved in stress-related behaviour, as the loss of either receptors abolished the glucocorticoid-mediated larval hyperactivity to a light stimulus. Overall, the results underscore a key physiological role for MR signalling in ray-finned fishes, and we propose that the regulation of the highly conserved stress axis as the original function of this receptor.

 

Link to the publication :

https://www.nature.com/articles/s41598-018-36681-w

Ferredoxin 1b deficiency leads to testis disorganization, impaired spermatogenesis and feminization in zebrafish

Endocrinology

2019-01-19

Abstract

The roles of steroids in zebrafish sex differentiation, gonadal development and function of the adult gonad are poorly understood. Herein, we have employed a ferredoxin 1b (fdx1b) mutant zebrafish to explore such processes. Fdx1b is an essential electron-providing cofactor to mitochondrial steroidogenic enzymes, which are crucial for glucocorticoid and androgen production in vertebrates. Fdx1b-/- zebrafish mutants develop into viable adults, in which concentrations of androgens and the glucocorticoid, cortisol, are significantly reduced. Adult fdx1b-/- mutant zebrafish display predominantly female secondary sex characteristics but may possess either ovaries or testes, confirming that androgen signaling is dispensable for testicular differentiation in this species, as previously demonstrated in androgen receptor mutant zebrafish. Adult male fdx1b-/- mutant zebrafish do not exhibit characteristic breeding behaviors, and sperm production is reduced, resulting in infertility in standard breeding scenarios. However, eggs collected from wild-type females can be fertilized by the sperm of fdx1b-/- mutant males by IVF. The testes of fdx1b-/- mutant males are disorganized and lack defined seminiferous tubule structure. Expression of several pro-male and spermatogenic genes is decreased in the testes of fdx1b-/-mutant males, including pro-male transcription factor SRY-box 9a (sox9a) and spermatogenic genes insulin-like growth factor 3 (igf3) and insulin-like 3 (insl3). This study establishes an androgen- and cortisol-deficient fdx1bzebrafish mutant as a model for understanding the impacts of steroid deficiency on sex development and reproductive function. This model will be particularly useful for further investigation of the roles of steroids in spermatogenesis, gonadal development and regulation of reproductive behavior, thus enabling further elucidation of the physiological consequences of endocrine disruption in vertebrates.

 

Link to the publication : https://academic.oup.com/endo/advance-article-abstract/doi/10.1210/en.2019-00068/5535648

Embryonic development, locomotor behavior, biochemical, and epigenetic effects of the pharmaceutical drugs paracetamol and ciprofloxacin in larvae and embryos of Danio rerio when exposed to environmental realistic levels of both drugs

Environmental Toxicology

2019-01-19

Abstract

For several years, the scientific community has been concerned about the presence of pharmaceuticals in the wild, since these compounds may have unpredictable deleterious effects on living organisms. Two examples of widely used pharmaceuticals that are present in the environment are paracetamol and ciprofloxacin. Despite their common presence in the aquatic environment due to their poor removal by sewage treatment plants, knowledge concerning their putative toxic effects is still scarce. This work aimed to characterize the effects of paracetamol (0.005, 0.025, 0.125, 0.625, and 3.125 mg/L) and ciprofloxacin (0.005, 0.013, 0.031, 0.078, 0.195, and 0.488 μg/L) in zebrafish embryos and larvae, exposed to environmentally relevant levels, close to the real concentrations of these pharmaceuticals in surface waters and effluents. The adopted toxic end points were developmental, a behavioral parameter (total swimming time), and a biomarker‐based approach (quantification of the activities of catalase, glutathione‐S‐transferase, cholinesterases, glutathione peroxidase, and lipid peroxidation levels) combined with epigenetic analysis (immunohistochemical detection of 5‐methylcytidine). Exposure to paracetamol had effects on all of the adopted toxic end points; however, ciprofloxacin only caused effects on behavioral tests and alterations in biomarkers. It is possible to ascertain the occurrence of oxidative stress following exposure to both drugs, which was more evident regarding paracetamol, an effect that may be related to the observed epigenetic modifications.

 

Link to the publication : https://onlinelibrary.wiley.com/doi/abs/10.1002/tox.22819

A zebrafish model to study small-fiber neuropathy reveals a potential role for GDAP1

Mitochondrion

2019-01-22

Highlights

• Depletion of mitochondrial gdap1 affects temperature sensitivity and nerve growth in zebrafish embryos.
• In contrast, knockdown of the mitochondrial fusion protein opa1 did not affect one of these read-outs.
• We propose that genes involved in mitochondrial dynamics should be screened for mutations in patients with SFN.

Abstract

Mutations in genes involved in mitochondrial dynamics (fusion and fission) have been implicated in many peripheral neuropathies. We hypothesized that defects in these genes could result in a phenotype resembling features of small-fiber neuropathy (SFN). This was investigated in zebrafish by knocking down two genes involved in mitochondrial dynamics gdap1 (possibly fission and motility) and opa1 (fusion) using established morpholinos. Our read-outs were nerve density in the caudal fin and a behavioral response to temperature changes, both based on comparable hallmarks of SFN in patients. Knockdown of gdap1 resulted in zebrafish embryos with a reduced density of sensory neurites compared to control morpholino-injected embryos. Furthermore, these embryos demonstrated a decreased temperature-related activity. In contrast, a knockdown of opa1 did not affect the density of sensory neurites nor the temperature-related activity. However, only the opa1morphants had an effect on mitochondrial network morphology. As we were not able to visualize the mitochondria in the neurons, it could well be that changes in the mitochondrial network remained undetected. Our data indicate that GDAP1 knockdown affects sensory neurite development, however, it is unclear if a problem in mitochondrial fission and network formation is the pathophysiological mechanism. Although we did not observe an effect of inhibiting mitochondrial fusion during development, we still propose that genes involved in mitochondrial dynamics should be screened for mutations in patients with SFN.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S1567724918300606

Spared cognitive and behavioral functions prior to epilepsy onset in a rat model of subcortical band heteropia

Brain Research

2019-01-24

Highlights

• Subcortical band heterotopia (SBH) does not impair motor functions and somatosensory processing in pre-epileptic rats.
• Learning and memory are normal in rats with SBH before seizure onset.
• Our data suggest that the SBH presence is not sufficient to impair behavioral functions.

ABSTRACT


Subcortical band heterotopia (SBH), also known as doublecortex syndrome, is a malformation of cortical development resulting from mutations in the doublecortin gene (DCX). It is characterized by a lack of migration of cortical neurons that accumulate in the white matter forming a heterotopic band. Patients with SBH may present mild to moderate intellectual disability as well as epilepsy. The SBH condition can be modeled in rats by in utero knockdown (KD) of Dcx. The affected cells form an SBH reminiscent of that observed in human patients and the animals develop a chronic epileptic condition in adulthood. Here, we investigated if the presence of an SBH is sufficient to induce cognitive impairment in juvenile Dcx-KD rats, before the onset of epilepsy. Using a wide range of behavioral tests, we found that the presence of SBH did not appear to affect motor control or somatosensory processing. In addition, cognitive abilities such as learning, short-term and long-term memory, were normal in pre-epileptic Dcx-KD rats. We suggest that the SBH presence is not sufficient to impair these behavioral functions.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0006899319300526

Varying the exposure period and duration of neuroactive pharmaceuticals and their metabolites modulates effects on the visual motor response in zebrafish (Danio rerio) larvae

Neurotoxicology and Teratology

2019-01-31

Highlights

• 7 out of 10 antidepressants altered swimming behavior in zebrafish larvae.
• 2 out of 3 metabolites tested elicited similar effects as parent compounds.
• 2 of 15 compounds altered behavior in repeated, late, and early exposure scenarios.

Abstract

Pharmaceuticals and personal care products are emerging contaminants that are increasingly detected in surface waters around the world. Despite the rise in environmental detections, measured concentrations are still typically low, raising the importance of environmental risk assessments that focus on ecologically relevant sublethal endpoints, such as altered behavior. Neuroactive pharmaceuticals, like mental health medications, pain killers, etc., may be particularly potent in this regard as they are specifically designed to cause behavioral changes without causing physiologic impairment in mammalian systems. We screened 15 different popular neuroactive pharmaceuticals, ranging from antidepressants (including 3 major antidepressant metabolites), anxiety medications, and pain killers, under three different exposure scenarios (repeated, late acute and early transient exposure) to look for behavioral effects in larval zebrafish using the visual motor response (VMR). Drugs were screened at 0, 1, 10, and 100 μg/L in the repeated exposure scenario, and at 0 and 100 μg/L in the late acute and early transient exposure scenarios. Eight of the 15 compounds tested, specifically the antidepressants amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, sertraline, nor-sertraline, venlafaxine, and the antipsychotic drug haloperidol decreased swimming activity by 25% to 40% under repeated exposure conditions. Five of the compounds (amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, and sertraline) also significantly decreased activity by 17% to 31% in the late acute exposure paradigm. Three compounds (fluoxetine, paroxetine and venlafaxine) significantly altered swimming activity with early transient exposure, however creating a hyperactive response and increasing activity from 24% to 28%, while haloperidol significantly decreased activity by 31%. This paper is, to our knowledge, the first to screen so many neuroactive pharmaceuticals, including major metabolites, in parallel under multiple exposure conditions. We show that antidepressants most consistently alter VMR swimming activity. Additionally, we show that major antidepressant metabolites can potentially alter behavior as much as their parent compounds. Furthermore, we show that the magnitude and direction of behavioral effect is dependent on the exposure duration and period, indicating that a more diverse experimental approach might be needed to more accurately assess the risk these compounds pose to the environment.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0892036218301466

Toxicity and neurotoxicity profiling of contaminated sediments from Gulf of Bothnia (Sweden): a multi-endpoint assay with Zebrafish embryos

Environmental Sciences Europe

2019-02-04

Abstract

Background

The toxicological characterization of sediments is an essential task to monitor the quality of aquatic environments. Many hazardous pollutants may accumulate in sediments and pose a risk to the aquatic community. The present study provides an attempt to integrate a diagnostic whole mixture assessment workflow based on a slightly modified Danio rerio embryo acute toxicity test with chemical characterization. Danio rerio embryos were directly exposed to sieved sediment (≤ 63 μm) for 96 h. Sediment samples were collected from three polluted sites (Kramfors, Sundsvall and Örnsköldsvik) in the Gulf of Bothnia (Sweden) which are characterized by a long history of pulp and paper industry impact. Effect data were supported by chemical analyses of 237 organic pollutants and 30 trace elements.

Results

The results show that malformations and neurotoxic compounds are the main drivers of differentiation in chemical and effects analyses, respectively. Specific spinal cord malformations and delayed hatching were observed only in sediments from Kramfors while light hyperactivity was seen only after exposure to sediments from Sundsvall.

Conclusions

Our experiments demonstrate that specific chemical profiles lead to specific effect patterns in Danio rerio embryos. In fact, behavioral endpoints could help detect the exposure to neurotoxins, and the observation of body malformations seems to be a potential tool for the identification of site-specific pollutants as polychlorinated biphenyl (PCBs), brominated flame retardants (BFRs) and several pesticides. Overall, results show the suitability of Danio rerio embryos for the fast screening of sediment samples.

Role of Olfactorily Responsive Neurons in the Right Dorsal Habenula - Ventral Interpeduncular Nucleus Pathway in Food- Seeking Behaviors of Larval Zebrafish

Neuroscience

2019-02-05

Highlights

• Olfactorily responsive neurons preferentially locate in the right (R) but not left dorsal habenula (dHb).
• This right-to-left Hb asymmetry converts to a ventral-to-dorsal pattern in the downstream interpeduncular nucleus (IPN).
• Loss-of-function of either the R-dHb or ventral IPN (V-IPN) impairs food-seeking associated increase of swimming activity.
• Asymmetrical olfactory responses in the R-dHb to V-IPN pathway are important for food-seeking behavior of larval zebrafish.

Abstract

The habenula (Hb) plays important roles in emotion-related behaviors. Besides receiving inputs from the limbic system and basal ganglia, Hb also gets inputs from multiple sensory modalities. Sensory responses of Hb neurons in zebrafish are asymmetrical: the left and right dorsal Hb (dHb) preferentially respond to visual and olfactory stimuli, respectively, implying different functions of the left and right dHb. While visual responses of the left dHb (L-dHb) has been implicated in light-preference behavior, the significance of olfactory responses of the right dHb (R-dHb) remains under-examined. It was reported that the R-dHb can gate innate attraction to a bile salt. However, considering a broad range of odors that R-dHb respond to, it is of interest to examine the role of R-dHb in other olfactory behaviors, especially food seeking, which is essential for animals' survival. Here, using in vivo whole-cell recording and calcium imaging, we first characterized food extract-evoked responses of Hb neurons. Responsive neurons preferentially locate in the R- but not L-dHb and exhibit either ON- (~ 87%) or OFF-type responses (~ 13%). Interestingly, this right-to-left asymmetry of olfactory responses converts into a ventral-to-dorsal pattern in the interpeduncular nucleus (IPN), a main downstream target of Hb. Combining behavior assay, we further found that genetic dysfunction or lesion of the R-dHb and its corresponding downstream ventral IPN (V-IPN) impairs the food seeking-associated increase of swimming activity. Thus, our study indicates that the asymmetrical olfactory response in the R-dHb to V-IPN pathway plays an important role in food-seeking behavior of zebrafish larvae.

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0306452219300843

2,4-Dichlorophenoxyacetic acid herbicide effects on zebrafish larvae: development, neurotransmission and behavior as sensitive endpoints

Environmental Science and Pollution Research

2019-02-05

Abstract

Assessment of pesticides toxicity using zebrafish early life stages is relevant for aquatic systems safety. This study aimed to evaluate the short-term effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on zebrafish (Danio rerio) embryos from 3 h post fertilization to 96 hpf. A set of 2,4-D concentrations ranging from 0.32 to 80 mg/L were tested and median lethal concentration (LC50) at 96-h was calculated as 2.86 mg/L. A sub-teratogenic concentrations range from 0.02 to 0.8 mg/L was then used to assess effects at ontogenic, biochemical, and behavioral levels. The main developmental defects were tail deformities and pericardial edema at concentrations equal or above 0.32 mg/L. Cholinesterase activity (at 96 hpf) and larvae swimming behavior (at 120 hpf) were affected even at the lowest tested dose (0.02 mg/L). The behavior analysis was a sensitive endpoint, with a decrease in the swimming distance of exposed larvae during light period. The effect of 2,4-D in ChE was translated by an inhibition of the enzyme activity in all treated groups. These findings demonstrate that 2,4-D can alter the cholinergic system by affecting ChE activity which may be involved in the locomotion reduction of exposed larvae and emphasize the potential of neurotransmission and behavioral endpoints as early warning signs of herbicides contamination in aquatic ecosystems.

 

Link to the publication : https://link.springer.com/article/10.1007/s11356-019-04488-5

Synergistic effects of Pb and repeated heat pulse on developmental neurotoxicity in zebrafish

Ecotoxicology and Environmental Safety

2019-02-06

Highlights

• Repeated heat pulse increases Pb-induced developmental toxicity in zebrafish.
• Synergistic effects of repeated heat pulse on Pb-induced abnormal neurodevelopment.
• Repeated heat pulse enhances Pb-induced suppression on zebrafish locomotor capacity.
• Caspase 3 regulated apoptosis is involved in this synergistic process.

Abstract

Pollutant discharges to the aquatic environment often contain multiple environmental stressors, affecting aquatic organisms. To mimic the discharges from nuclear and industry facilities, the combined effects of two independent types of stressors, heavy metal Pb and repeated heat pulse were addressed in this study. We investigated the developmental toxicity of combined treatment, especially its toxic effects on zebrafish neurodevelopment. The normal embryos at 4 hpf were exposed to 0.2 mM of Pb dissolved in the bathing medium with different temperatures (30, 32, and 34 °C) and then maintained in an incubator at 28 °C. After performing above treatment once every 24 h for 6 days, we found that combined treatment significantly affected neural development, including loss of dopaminergic (DA) neurons and brain vasculature, disruption of locomotor activity and neurodevelopmental genes expression in a temperature-dependent manner as compared to the Pb alone exposure group, indicating that repeated heat pulse enhances these negative impacts induced by Pb. In contrast, no apparent toxicity was observed in repeated heat pulse alone groups, suggesting that Pb treatment reduces thermal tolerance in zebrafish, which emphasized the importance to evaluate synergistic effects of Pb and repeated heat pulse. Moreover, repeated heat pulse aggravated Pb-induced apoptosis in the zebrafish brain. Further study of the underlying mechanism suggested that Caspase 3 regulated apoptosis was involved in this process. Taken together, our findings shed light on the full understanding of toxic effects of discharges from industrial applications on living organisms and its environmental impact.

Subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize in zebrafish sensory hair cells

Plos One Genetics

2019-02-06

Abstract

Mutations in transmembrane inner ear (TMIE) cause deafness in humans; previous studies suggest involvement in the mechano-electrical transduction (MET) complex in sensory hair cells, but TMIE’s precise role is unclear. In tmie zebrafish mutants, we observed that GFP-tagged Tmc1 and Tmc2b, which are subunits of the MET channel, fail to target to the hair bundle. In contrast, overexpression of Tmie strongly enhances the targeting of Tmc1-GFP and Tmc2b-GFP to stereocilia. To identify the motifs of Tmie underlying the regulation of the Tmcs, we systematically deleted or replaced peptide segments. We then assessed localization and functional rescue of each mutated/chimeric form of Tmie in tmie mutants. We determined that the first putative helix was dispensable and identified a novel critical region of Tmie, the extracellular region and transmembrane domain, which is required for both mechanosensitivity and Tmc2b-GFP expression in bundles. Collectively, our results suggest that Tmie’s role in sensory hair cells is to target and stabilize Tmc channel subunits to the site of MET.

 

Link to the publication :

 

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007635

Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish

Nature Communications

2019-02-12

Abstract

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.

Introduction

Aminoacyl tRNA synthetases (ARSs) play a key role in protein translation as they catalyze the attachment of specific amino acids to their cognate transfer RNA (tRNA) molecules1,2. The nuclear encoded ARS gene loci are subdivided into 17 cytoplasmic, 17 mitochondrial, and three bi-functional ARSs3,4. The canonical aminoacylation and proofreading functions of ARSs are highly conserved across species. In addition, during evolution many ARSs acquired additional domains with unique structural characteristics that are not essential for tRNA charging but account for non-canonical functions5,6. These alternative functions are critical for cellular homeostasis and include among others: regulation of signal transduction and cell migration, angiogenesis and tumorigenesis, inflammatory responses, and control of cell death5. This functional diversity may in part account for the association between mutations in ARS genes and a broad range of human disorders, including neurological disorders, cancer, and auto-immune diseases2.

Both monoallelic and biallelic pathogenic variants in ARS genes, encoding dominant and recessive disease traits, respectively, have been increasingly reported in patients with various disorders that often have predominantly neurological features. Dominant heterozygous mutations in ARS genes have been identified in patients with Charcot-Marie-Tooth disease and related peripheral neuropathies, including AARS7, GARS8, HARS9,10, MARS11,12, WARS13, and YARS12,14. Recessive mutations have been identified in complex disorders often involving the central nervous system such as hypomyelination with brainstem and spinal cord involvement (DARS)15, leukodystrophy (RARS)16, congenital visual impairment and progressive microcephaly (KARS)17, developmental delay with progressive microcephaly and intractable seizures (QARS)18,19 and early onset epileptic encephalopathy with myelination defect (AARS)20. Interestingly, some ARS genes have been associated with both dominant and recessive disease traits including mutations in AARS7,20KARS21, and YARS14,22.

In this study, we report five newly diagnosed families with biallelic variants in valyl-tRNA synthetase (VARS), including seven novel VARS variants. In addition, we present an in-depth description of two families previously reported in a large study on brain malformations in mainly consanguineous families wherein VARS was reported as a candidate disease gene23. In vitro studies with patient-derived cell lines, including enzymatic assays, and yeast complementation assays show that recessive VARS mutations most likely lead to a loss-of-protein function, i.e. loss of function (LoF) alleles. A vars knockout (KO) zebrafish model further demonstrates that deficiency of vars results in microcephaly and epileptiform activity, replicating key characteristics of the human disease.

Results

Biallelic VARS variants cause developmental encephalopathy

In total, ten patients from seven families with biallelic VARS variants were identified (Fig. 1a)23. All families were included through international collaborations or via the program GeneMatcher24. All patients had global developmental delay (DD), which was already present in the first months of life in most patients, and prior to seizure onset or unrelated to epilepsy in five patients. All patients at a sufficient age for IQ testing had severe or profound intellectual disability (ID) and were nonverbal. Only two of the nine patients who had reached the walking age were able to walk independently, though both acquired this skill only at later age.

 

Link to the publication :

https://www.nature.com/articles/s41467-018-07953-w

Normalization of large-scale behavioural data collected from zebrafish

Plos One

2019-02-15

Abstract

Many contemporary neuroscience experiments utilize high-throughput approaches to simultaneously collect behavioural data from many animals. The resulting data are often complex in structure and are subjected to systematic biases, which require new approaches for analysis and normalization. This study addressed the normalization need by establishing an approach based on linear-regression modeling. The model was established using a dataset of visual motor response (VMR) obtained from several strains of wild-type (WT) zebrafish collected at multiple stages of development. The VMR is a locomotor response triggered by drastic light change, and is commonly measured repeatedly from multiple larvae arrayed in 96-well plates. This assay is subjected to several systematic variations. For example, the light emitted by the machine varies slightly from well to well. In addition to the light-intensity variation, biological replication also created batch-batch variation. These systematic variations may result in differences in the VMR and must be normalized. Our normalization approach explicitly modeled the effect of these systematic variations on VMR. It also normalized the activity profiles of different conditions to a common baseline. Our approach is versatile, as it can incorporate different normalization needs as separate factors. The versatility was demonstrated by an integrated normalization of three factors: light-intensity variation, batch-batch variation and baseline. After normalization, new biological insights were revealed from the data. For example, we found larvae of TL strain at 6 days post-fertilization (dpf) responded to light onset much stronger than the 9-dpf larvae, whereas previous analysis without normalization shows that their responses were relatively comparable. By removing systematic variations, our model-based normalization can facilitate downstream statistical comparisons and aid detecting true biological differences in high-throughput studies of neurobehaviour.

Linarin improves the dyskinesia recovery in Alzheimer's disease zebrafish by inhibiting the acetylcholinesterase activity

Life Sciences

2019-02-22

Abstract

Background

Due to complex pathogenesis of Alzheimer's disease (AD), currently there is no effective disease-modifying treatment. Acetylcholinesterase (AChE) has introduced itself as an important target for AD therapy. Linarin as the representative active ingredient of flavonoid glycoside in Flos chrysanthemi indici has been found to have anti-acetylcholinesterase effect.

Aims

The present study intended to explore the potential effect of linarin for treatment of AD.

Main methods

In this study, molecular docking simulation was used to evaluate whether linarin could dock with AChE and decipher the mechanism of linarin as an AChE inhibitor. After molecular docking simulation, AlCl3-induced Alzheimer's disease zebrafish model was established. Effects of linarin on treating AD zebrafish dyskinesia and AChE inhibition were compared with donepezil (DPZ) which was used as a positive control drug.

 

Link to the publication :

 

https://www.sciencedirect.com/science/article/abs/pii/S0024320519301365

A long noncoding RNA cluster-based genomic locus maintains proper development and visual function

Nucleic Acids Research

2019-05-10

Abstract

Long noncoding RNAs (lncRNAs) represent a group of regulatory RNAs that play critical roles in numerous cellular events, but their functional importance in development remains largely unexplored. Here, we discovered a series of previously unidentified gene clusters harboring conserved lncRNAs at the nonimprinting regions in brain (CNIBs). Among the seven identified CNIBs, human CNIB1 locus is located at Chr 9q33.3 and conserved from Danio rerio to Homo sapiens. Chr 9q33.3-9q34.11 microdeletion has previously been linked to human nail-patella syndrome (NPS) which is frequently accompanied by developmental and visual deficiencies. By generating CNIB1 deletion alleles in zebrafish, we demonstrated the requirement of CNIB1 for proper growth and development, and visual activities. Furthermore, we found that the role of CNIB1on visual activity is mediated through a regulator of ocular development-lmx1bb. Collectively, our study shows that CNIB1 lncRNAs are important for zebrafish development and provides an lncRNA cluster-mediated pathophysiological mechanism for human Chr 9q33.3-9q34.11 microdeletion syndrome.

 

Link to the publication :

https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkz444/5498619

Zebrafish neurobehavioral phenomics applied as the behavioral warning methods for fingerprinting endocrine disrupting effect by lead exposure at environmentally relevant level

Chemosphere

2019-05-20

Highlights

 

• Pb of environmentally relevant concentration are of potential reproductive toxicity.
• Pb disturbed expression of genes involved in testicular steroidogenesis and apoptosis.
• Pb exerted biphasic effects on courtship behavior and endocrine regulation in male.

 

Abstract

Environmental lead (Pb) exposure is a great hazard to the public health. Although environmentally relevant Pb poisoning is preventable, insidious Pb contaminants are still a major threat to human health. Herein, we reported that exposure to Pb at environmentally relevant concentration level (1 μg/L, 10 μg/L and 100 μg/L), disturbed the courtship behavior of adult male zebrafish and further altered the transcriptional patterns of key genes involved in testicular steroidogenesis (igf3, amh, piwil1, lhcgr, fshr, cyp11c1star, cyp19a1a, cyp19a1b) and apoptosis (bax, cytoC, caspase 9, caspase 3, puma). Both the behavioral and the transcriptional profiles share a similar biphasic dose response, with stimulatory effects after low-level exposure and inhibitory effects after high-level exposure. This results revealed the endocrine disrupting effects of Pb even at an environmentally relevant level within the concentration range of ambient water quality criteria (AWQC) and the reliability of locomotion fingerprint as the indicator for detecting the risk induced by Pb pollution. Current research, for the first time, employed the ZebraTower system as the biological early warning system (BEWS) to find that Pb exerted biphasic effects on the courtship behavior and endocrine regulation of male adult zebrafish. Methodologically, we firstly propose an efficient solution to monitor and assess the risk of Pb exposure by combining the (BEWS) and data analyzing methods such as zebrafish phenomics, which would make a contribution to the detection and prevention of environmentally relevant Pb poisoning.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653519310586

Neurodevelopmental toxicity assessments of alkyl phenanthrene and Dechlorane Plus co-exposure in zebrafish

Ecotoxicology and Environmental Safety

2019-05-30

Highlights

 

• Neurotoxicity of 3-MP and DP co-exposure in zebrafish was firstly studied.
• Co-exposure with 3-MP and DP had synergistic effects on neurobehavioral deficits.
• Behavioral anomalies were due to altered neuronal connectivity and muscle function.

 

Abstract

Alkyl phenanthrene (A-Phen) and Dechlorane Plus (DP) are ubiquitous environmental pollutants that widely co-exist in the environment. It has been established that both A-Phen and DP elicit neurotoxicity, but the potential interactive toxicity of these contaminants is not well-known. To determine whether a mixture of A-Phen and DP would exhibit interactive effects on neurodevelopment, we co-exposed 3-methylphenanthrene (3-MP), a representative of A-Phen, with DP. Our results illustrated that exposure to 5 or 20 μg/L 3-MP alone or in combination with 60 μg/L DP caused neurobehavioral anomalies in zebrafish. In accordance with the behavioral deficits, 3-MP alone or co-exposed with DP significantly decreased axonal growth of secondary motoneurons, altered intracellular Ca2+ homeostasis and induced cell apoptosis in the muscle of zebrafish. Additionally, 3-MP alone or co-exposed with DP significantly increased reactive oxygen species (ROS) and the mRNA levels of apoptosis-related genes. These findings indicate that 3-MP alone or co-exposed with DP induces neurobehavioral deficits through the combined effects on neuronal connectivity and muscle function. Chemical analysis revealed significant increases in 3-MP and DP bioaccumulation in zebrafish co-exposed with 3-MP and DP. Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of the synergistic effects observed in combined chemical exposure.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651319306074

Impact of co-exposure to titanium dioxide nanoparticles and Pb on zebrafish embryos

Chemosphere

2019-06-03

Highlights

 

• n-TiO2-Pb complexes were formed in co-exposure suspension.
• Bioconcentration and bioavailability of Pb was increased by n-TiO2 in zebrafish embryos.
• Neurotoxicity of Pb was enhanced in the presence of n-TiO2.

 

Abstract

Complex interactions have been established between nanoparticles (NPs) and heavy metals in real environments. Herein we used zebrafish embryos to investigate the influence of titanium dioxide NPs (n-TiO2) on the uptake, bioconcentration, and depuration, and toxicity of Pb. The formation of n-TiO2–Pb complexes was confirmed in an exposure suspension. An increase in Pb bioconcentration was observed in zebrafish embryos upon co-exposure to n-TiO2 and Pb; moreover, n-TiO2–Pb complexes could be found in the embryos, indicating the bioavailability of NPs. However, there was no difference in the depuration rates of Pb in the presence of n-TiO2. Metallothionein (MT) content was significantly increased upon exposure to Pb alone, and the content significantly increased even further upon co-exposure. A downregulation in the expression levels of the neurodevelopment-related genes gfapsyn2α, and elavl3 was observed in the embryos, and we also noted a reduction in the swimming speed of and the total distance traveled by the larvae. To summarize, our results indicate that n-TiO2 can act as an effective carrier of Pb to enhance its uptake, bioavailability, and toxicity in zebrafish embryos.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S0045653519312299

 

Long‑term acclimation to near‑future ocean acidification has negligible effects on energetic attributes in a juvenile coral reef fsh

Oecologia

2019-06-04

Abstract

Increased levels of dissolved carbon dioxide (CO2) drive ocean acidification and have been predicted to increase the energy use of marine fishes via physiological and behavioural mechanisms. This notion is based on a theoretical framework suggesting that detrimental effects on energy use are caused by plasma acid–base disruption in response to hypercapnic acidosis, potentially in combination with a malfunction of the gamma aminobutyric acid type A (GABAA) receptors in the brain. However, the existing empirical evidence testing these effects primarily stems from studies that exposed fish to elevated CO2 for a few days and measured a small number of traits. We investigated a range of energetic traits in juvenile spiny chromis damselfish (Acanthochromis polyacanthus) over 3 months of acclimation to projected end-of-century CO2 levels (~ 1000 µatm). Somatic growth and otolith size and shape were unaffected by the CO2 treatment across 3 months of development in comparison with control fish (~ 420 µatm). Swimming activity during behavioural assays was initially higher in the elevated CO2 group, but this effect dissipated within ~ 25 min following handling. The transient higher activity of fish under elevated CO2 was not associated with a detectable difference in the rate of oxygen uptake nor was it mediated by GABAA neurotransmitter interference because treatment with a GABAA antagonist (gabazine) did not abolish the CO2 treatment effect. These findings contrast with several short-term studies by suggesting that end-of-century levels of CO2 may have negligible direct effects on the energetics of at least some species of fish.

 

Link to the publication :

https://link.springer.com/article/10.1007/s00442-019-04430-z

Sleep deprivation caused a memory defects and emotional changes in a rotenone-based zebrafish model of Parkinson’s disease

Behavioural Brain Research

2019-06-10

Highlights

• Sleep deprivation worsened the memory impairment of rotenone treated zebrafish.

• DA metabolic process was significantly increases after sleep deprivation.
• The expression of dopamine D2 receptors were decreased in SD + Rotenone group.

 

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disorder in the world. Apart from motor deficits, PD reduces patient’s quality of life through sleep disturbances, cognitive impairment and emotional disorders. However, it’s unclear whether bad life habits such as stay up late exacerbate the patient’s cognition and emotional disorders. Thus we investigated the consequences of sleep deprivation (SD) on memory and emotions using a rotenone-based zebrafish model of PD. Behavioral assays, using locomotor activity assay, showed that rotenone treated zebrafish exhibited PD-like symptoms, whereas sleep deprivation didn’t exacerbate the progression of them. The object discrimination task exhibited that the short-term cognitive deficits of rotenone group are more serious than the sham group after SD. Light-dark box test showed that rotenone treated fish are more dysphoric than the sham fish after SD. Dopamine and DOPAC significantly reduced in rotenone treated fish compared with the sham fish. However, this DOPAC reduction recovered after SD. The expression of D2 and D3 in rotenone treated zebrafish elevated compared with sham group and SD group. However, the rotenone treated zebrafish manifested a decrease level of D2 and D3 after SD. D1 did not show any significantly changes among the four groups. Our findings suggest that zebrafish treated with rotenone may have a more severe damage of memory and emotional function after SD, which may be related to the changes in the DA systems.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0166432819301949

Combined transcriptomic and proteomic analysis reveals a diversity of venom-related and toxin-like peptides expressed in the mat anemone Zoanthus natalensis (Cnidaria, Hexacorallia)

Archives of Toxicology

2019-06-15

Abstract

Venoms from marine animals have been recognized as a new emerging source of peptide-based therapeutics. Several peptide toxins from sea anemone have been investigated as therapeutic leads or pharmacological tools. Venom complexity should be further highlighted using combined strategies of large-scale sequencing and data analysis which integrated transcriptomics and proteomics to elucidate new proteins or peptides to be compared among species. In this work, transcriptomic and proteomic analyses were combined to identify six groups of expressed peptide toxins in Zoanthus natalensis. These include neurotoxin, hemostatic and hemorrhagic toxin, protease inhibitor, mixed function enzymes, venom auxiliary proteins, allergen peptides, and peptides related to the innate immunity. Molecular docking analysis indicated that one expressed Zoanthus Kunitz-like peptide, ZoaKuz1, could be a voltage-gated potassium channels blocker and, hence, it was selected for functional studies. Functional bioassays revealed that ZoaKuz1 has an intrinsic neuroprotective activity in zebrafish model of Parkinson’s disease. Since pharmacological blockade of KV channels is known to induce neuroprotective effects, ZoaKuz1 holds the potential to be developed in a therapeutic tool to control neural dysfunction by slowing or even halting neurodegeneration mediated by ion-channel hyperactivity.

 

Link to the publication : 

https://link.springer.com/article/10.1007/s00204-019-02456-z

Effects of antidepressants with different modes of action on early life stages of fish and amphibians

Environmental Pollution

2019-08-06

Highlights

 

• Lethal effects were not observed at environmentally relevant concentrations.
• Behaviour was effected only at higher tested concentrations.
• mRNA expression was impacted at environmentally relevant concentrations.

 

Abstract

Drugs are excreted from the human body as both original substances and as metabolites and enter aquatic environment through waste water. The aim of this study was to widen the current knowledge considering the effects of waterborne antidepressants with different modes of action—amitriptyline, venlafaxine, sertraline—on embryos of non-target aquatic biota—fish (represented by Danio rerio) and amphibians (represented by Xenopus tropicalis). The tested concentrations were 0.3; 3; 30; 300 and 3000 μg/L in case of amitriptyline and venlafaxine and 0.1; 1; 10; 100 and 1000 μg/L for sertraline. Test on zebrafish embryos was carried out until 144 h post fertilization, while test on Xenopus embryos was terminated after 48 h. Lethal and sublethal effects as well as swimming alterations were observed at higher tested concentrations that are not present in the environment. In contrast, mRNA expression of genes related to heart, eye, brain and bone development (nkx2.5, otx 2, bmp4 and pax 6) seems to be impacted also at environmentally relevant concentrations. In a wider context, this study reveals several indications on the ability of antidepressants to affect non target animals occupying environments which may be contaminated by such compounds.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0269749119304257

Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks

Cell

2019-08-08

Highlights

 

• Identification of rare inherited variants associated with ASD and 16 new ASD risk genes
• Inherited risk reveals both new biological pathways and shared PPI with known genes
• We develop and validate a machine learning algorithm (ARC) to remove WGS artifacts
• NR3C2 mutations define a novel syndromic form of ASD, which we model in zebrafish

 

Summary

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0092867419307809

Coexposure to environmental concentrations of cis-bifenthrin and graphene oxide: Adverse effects on the nervous system during metamorphic development of Xenopus laevis

Journal of Hazardous Materials

2019-08-12

Highlights

 

• The adverse effects of coexposure of Xenopus laevis to cis-BF and GO were studied.
• GO increased the bioconcentration of cis-BF and the fraction of 1S-enantiomer.
• Coexposure to cis-BF and GO resulted in activation of neurotransmitters.
• cis-BF and GO coexposure led to metamorphic development delay and neurotoxicity.

 

Abstract

Despite the great concerns associated with the combined biological effects of nanoparticles and insecticides, the current understanding of the corresponding ecological risks remains limited. Xenopus laevis (X. laevis) tadpoles were exposed to various concentrations of typical pyrethroid (cis-bifenthrin; cis-BF), either alone or in combination with graphene oxide (GO), for 21 days. The presence of GO resulted in increased bioconcentration of cis-BF and a higher 1S-enantiomer fraction. Exposure to cis-BF and GO caused further reduction in pre-metamorphic developmental rates and activated dopaminergic, noradrenergic, and serotonergic neurotransmitter systems. Reduced tadpole activity and levels of genomic DNA methylation at cytosine nucleotides (5hmC) were observed in the coexposure groups. These results indicate that GO enhance the bioconcentration of cis-BF and promote the conversion of its 1R-enantiomer to the 1S form, which lead to disruption of neurotransmitter systems as well as interference in metamorphic development.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0304389419309495

Behavioral effects of citalopram, tramadol, and binary mixture in zebrafish (Danio rerio) larvae

Chemosphere

2019-08-13

Highlights

 

• Citalopram and tramadol binary mixture effects studied in zebrafish (Danio rerio).
• Embryo-larvae were exposed from fertilization until six days post-fertilization.
• Heart rate, spontaneous tail coiling, and death/malformation incidence unaffected.
• Anxiolytic effects in citalopram (EC50: 471 μg L−1) and tramadol (EC50: 411 μg L−1).
• The mixture was less anxiolytic (EC50: 713 μg L−1), indicating interaction effects.

 

Abstract

Pharmaceuticals are emerging as environmentally problematic compounds. As they are often not appropriately removed by sewage treatment plants, pharmaceutical compounds end up in surface water environments worldwide at concentrations in the ng to μg L−1 range. There is a need to further explore single compound and mixture effects using e.g. in vivo test model systems. We have investigated, for the first time, behavioral effects in larval zebrafish (Danio rerio) exposed to a binary mixture of an antidepressant drug (citalopram) and a synthetic opioid (tramadol). Citalopram and tramadol have a similar mode of action (serotonin reuptake inhibition) and are known to produce drug-drug interactional effects resulting in serotonin syndrome (SS) in humans. Zebrafish embryo-larvae were exposed to citalopram, tramadol and 1:1 binary mixture from fertilization until 144 h post-fertilization. No effects on heart rate, spontaneous tail coiling, or death/malformations were observed in any treatment at tested concentrations. Behavior (hypoactivity in dark periods) was on the other hand affected, with lowest observed effect concentrations (LOECs) of 373 μg L−1 for citalopram, 320 μg L−1 for tramadol, and 473 μg L−1 for the 1:1 mixture. Behavioral EC50 was calculated to be 471 μg L−1 for citalopram, 411 μg L−1 for tramadol, and 713 μg L−1 for the 1:1 mixture. The results of this study conclude that tramadol and citalopram produce hypoactivity in 144 hpf zebrafish larvae. Further, a 1:1 binary mixture of the two caused the same response, albeit at a higher concentration, possibly due to SS.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0045653519318119

Involvement of peroxisome proliferator-activated receptor γ in anticonvulsant activity of α-asaronol against pentylenetetrazole-induced seizures in zebrafish

Neuropharmacology

2019-09-02

Highlights

 

• α-asaronol, a metabolic product of α-asarone, attenuated seizures in zebrafish.
• RNA-seq revealed the involvement of PPAR γ in anticonvulsant effects of α-asaronol.
• Blocking of PPAR γ reversed the anti-seizures actions of α-asaronol.
• Docking simulation implied the physical interaction between α-asaronol and PPAR γ.

 

Abstract

In mammals, peroxisome proliferators activated receptors (PPARs), the nuclear hormone receptors, have been reported to be involved in seizure control. Selective agonists and antagonists of PPARs raise seizure thresholds and suppress seizures, respectively. In this study, we evaluated the anticonvulsant effects of α-asaronol, a metabolic product of α-asarone, on pentylenetetrazole (PTZ)-induced seizures in zebrafish and investigated the underlying mechanisms. As a result, α-asaronol ameliorated seizures with increase of seizure latency, as well as decrease of seizure-like behavior, c-fos expression, and abnormal neuronal discharge in a concentration dependent manner. By comparing gene expression profiles of zebrafish undergoing seizures and α-asaronol pretreated zebrafish, we found that α-asaronol attenuate seizures through increase of PPAR γ expression, while PPAR γ antagonist GW9662 inhibit the anti-seizures actions of α-asaronol. Moreover, molecular docking simulation implied the physical interaction between α-asaronol and PPAR γ. The overall results indicated that the anticonvulsant effects of α-asaronol are regulated through PPAR γ-mediated pathway, which shed light on development of α-asaronol as a potential antiepileptic drug. In addition, it is for first time to report that PPAR γ is associated with seizures in zebrafish, supporting previous evidence that zebrafish is a suitable alternative for studying seizures.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0028390819303193

Correspondence Between Behavioral, Physiological, and Anatomical Measurements of Visual Function in Inhibitory Neuron–Ablated Zebrafish

Visual Neuroscience

2019-10-05

Abstract

PurposeTo compare the effects of reduced inhibitory neuron function in the retina across behavioral, physiological, and anatomical levels.

 

Link to the publication : 

https://iovs.arvojournals.org/article.aspx?articleid=2755676

Biochemical and behavioral responses of zebrafish embryos to magnetic graphene/nickel nanocomposites

Ecotoxicology and Environmental Safety

2019-10-10

Highlights

• No acute or developmental toxicity of graphene/Ni nanocomposites (G/Ni) was observed.

• G/Ni sublethal toxicity to zebrafish embryos depends on their shape and size.
• Locomotor behavior was the most sensitive parameter evaluated.
• Predicted environmental concentrations (PEC) of these compounds should be calculated.
• Relevance of findings to be determined in light of the PEC values.

Abstract

Graphene nanocomposites are emerging carbon-based materials with interesting electrical, mechanical, optical and magnetic properties, relevant for applications in different fields. Despite this increased use, the impact of graphene nanocomposites residues in the environment has not been properly studied. Thus, the goal of this work was to assess the toxicity of two nickel/graphene nanocomposites (G/Ni1 and G/Ni2) differing in size and shape to Danio rerio embryos. Their toxicity was evaluated using apical (mortality, development and hatching), biochemical [cholinesterase (ChE), glutathione-S-transferase (GST), and catalase (CAT) activities] and behavioral (locomotor activity) endpoints. At the tested concentrations, neither of the nanocomposites presented lethal or developmental effects. Nevertheless, both nanocomposites induced behavioral effects, reducing swimming distances. This effect was, however detected at lower concentrations in the G/Ni1 nanocomposite. At biochemical level, only G/Ni1 nanocomposite showed to interfere with the measured parameters, increasing the activities of ChE, CAT and GST. Differences in the effects induced by the two nanocomposites seem to be related not only with their size, but also with the shape and the ability to continuously release nickel ions to aqueous medium. This work highlights the importance of studying graphene nanocomposites effects to aquatic organisms even when acute toxicity is not expected. The relevance of the effects found in this work need to be further analyzed in light of the consequences to the long-term fitness of the organisms and in light of the environmental concentrations expected for this type of compounds.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0147651319310917

ABCC9-related Intellectual disability Myopathy Syndrome is a KATP channelopathy with loss-of-function mutations in ABCC9

Nature Communications

2019-11-01

Mutations in genes encoding KATP channel subunits have been reported for pancreatic disorders and Cantú syndrome. Here, we report a syndrome in six patients from two families with a consistent phenotype of mild intellectual disability, similar facies, myopathy, and cerebral white matter hyperintensities, with cardiac systolic dysfunction present in the two oldest patients. Patients are homozygous for a splice-site mutation in ABCC9 (c.1320 + 1G> A), which encodes the sulfonylurea receptor 2 (SUR2) subunit of KATP channels. This mutation results in an in-frame deletion of exon 8, which results in non-functional KATP channels in recombinant assays. SUR2 loss-of-function causes fatigability and cardiac dysfunction in mice, and reduced activity, cardiac dysfunction and ventricular enlargement in zebrafish. We term this channelopathy resulting from loss-of-function of SUR2-containing KATP channels ABCC9-related Intellectual disability Myopathy Syndrome (AIMS). The phenotype differs from Cantú syndrome, which is caused by gain-of-function ABCC9 mutations, reflecting the opposing consequences of KATP loss- versus gain-of-function.

 

Link to the publication :

https://digitalcommons.wustl.edu/cgi/viewcontent.cgi?article=9291&context=open_access_pubs

Flumazenil-insensitive benzodiazepine binding sites in GABAA receptors contribute to benzodiazepine-induced immobility in zebrafish larvae

Life Sciences

2019-11-04

Abstract

Aims

Benzodiazepines (BZDs) produce various pharmacological actions by binding to and allosterically regulating GABAA receptors. Several in vitro studies have demonstrated diazepam, the prototypic BZD, produces a high-dose action that cannot be countered with the classical BZD-binding site antagonist flumazenil. Here, we investigate the existence and behavioral relevance of non-classical BZD binding sites in zebrafish larvae.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0024320519309609

Zebrafish (Danio rerio) embryo-larvae locomotor activity data analysis: Evaluating anxiolytic effects of the antidepressant compound citalopram

Journal Data in Brief

2019-11-15

Abstract

Newly fertilized zebrafish (Danio rerio) embryos were exposed to increasing concentrations of the selective serotonin reuptake inhibitor (SSRI) citalopram from fertilization until six days post-fertilization (dpf). Locomotor activity data were acquired at six dpf using an automated ZebraBox® infrared tracking system. Individual (n = 32) locomotor activity was recorded during 75 minutes in total during alternating illumination conditions (0% light, i.e. dark periods, and 100% light, i.e. light periods). The first 15 minutes of the test consisted of a dark period, i.e an acclimatization phase. Afterward, six alternating light and dark periods were conducted. Individual zebrafish embryo-larvae locomotion was tracked and aggregated in ten-second bins. The dataset, containing nine locomotor-related quantified endpoints (factors), was parsed, analyzed, and visualized using R software. The dataset and its associated custom R script may be used to further explore locomotor activity outcomes (e.g. anxiolytic or anxiogenic properties) following exposure to citalopram or other neuroactive chemicals.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S2352340919311679

Developmental exposure to lead at environmentally relevant concentrations impaired neurobehavior and NMDAR-dependent BDNF signaling in zebrafish larvae

Environmental Pollution

2019-11-21

Highlights

 

• The potential ecological risks of Pb could be underestimated in the real environment.
• Environmental levels of Pb induced neurobehavioral changes in zebrafish larvae.
• The neurotoxicity of Pb could be related with NMDAR-dependent BDNF signaling.

 

Abstract

Lead (Pb) is one of the predominant heavy metals in e-waste recycling arears and recognized as a notorious environmental neurotoxic substance. However, whether Pb at environmentally relevant concentrations could cause neurobehavioral alteration and even what kind of signaling pathway Pb exposure would disrupt in zebrafish were not fully uncovered. In the present study, 6 h postfertilization (hpf) zebrafish embryos were exposed to Pb at the concentrations of 0, 5, 10, and 20 μg/L until 144 hpf. Then the neurobehavioral indicators including locomotor, turnings and social behaviors, and the expressions of selected genes concerning brain-derived neurotrophic factor (BDNF) signaling were investigated. The results showed that significant changes were obtained under 20 μg/L Pb exposure. The hypoactivity of zebrafish larvae in locomotor and turning behaviors was induced during the dark period, while hyperactivity was observed in a two-fish social assay during the light period. The significantly downregulation of genes encoding BDNF, its receptor TrkB, and N-methyl-D-aspartate glutamate receptor (NMDAR) suggested the involvement of NMDAR-dependent BDNF signaling pathway. Overall, our study demonstrated that developmental exposure to Pb at environmentally relevant concentrations caused obvious neurobehavioral impairment of zebrafish larvae by disrupting the NMDAR-dependent BDNF signaling, which could exert profound ecological consequences in the real environment.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749119339235

Pyridox(am)ine 5′-phosphate oxidase (PNPO) deficiency in zebrafish results in fatal seizures and metabolic aberrations

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

2019-11-21

Highlights

 

• PNPO deficient zebrafish (Danio rerio) and HEK293 cell line were generated using CRISPR/Cas9 gene editing technology.
• PNPO deficiency resulted in strong shortening of the survival and in development of fatal spontaneous seizures.
• PLP treatment increased survival and normalized PLP, pyridoxal, 4-pyridoxic acid, GABA, glutamate and glycine levels.
• PLP treatment did not prevent accumulation of pyridoxamine, pyridoxamine 5’-phosphate and several essential amino acids.
• Accumulation of pyridoxamine 5’-phosphate may play a role in the residual clinical phenotype of PNPO deficiency.

 

Abstract

Pyridox(am)ine 5′-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5′-phosphate (PNP) and pyridoxamine 5′-phosphate (PMP) to pyridoxal 5′-phosphate (PLP), the active form of vitamin B6. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo−/−) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo−/− zebrafish develop seizures resulting in only 38% of pnpo−/− zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo−/− zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S0925443919303308

The α1-adrenoceptor inhibitor ρ-TIA facilitates net hunting in piscivorous Conus tulipa

Nature - Scientific Report

2019-11-28

Abstract

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of “nirvana cabal” peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in Cgeographus and Ctulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in Ctulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

 

Link to the publication :

https://www.nature.com/articles/s41598-019-54186-y

Towards less hazardous industrial compounds: coupling quantum mechanical computations, biomarker responses and behavioral profiles identify bioactivity of SN2 electrophiles in alternative vertebrate models

Chemical research in toxicology

2019-12-02

Abstract

Abstract Image

Sustainable molecular design of less hazardous chemicals promises to reduce risks to public health and the environment. Computational chemistry modeling coupled with alternative toxicology models (e.g., larval fish) present unique high-throughput opportunities to understand structural characteristics eliciting adverse outcomes. Numerous environmental contaminants with reactive properties can elicit oxidative stress, an important toxicological response associated with diverse adverse outcomes (i.e., cancer, diabetes, neurodegenerative disorders, etc.). We examined a common chemical mechanism (bimolecular nucleophilic substitution (SN2)) associated with oxidative stress using property-based computational modeling coupled with acute (mortality) and sublethal (glutathione, photomotor behavior) responses in the zebrafish (Danio rerio) and the fathead minnow (Pimephales promelas) models to identify whether relationships exist among biological responses and molecular attributes of industrial chemicals. Following standardized methods, embryonic zebrafish and larval fathead minnows were exposed separately to eight different SN2 compounds for 96 h. Acute and sublethal responses were compared to computationally derived in silico chemical descriptors. Specifically, frontier molecular orbital energies were significantly related to acute LC50 values and photomotor response (PMR) no observed effect concentrations (NOECs) in both fathead minnow and zebrafish. This reactivity index, LC50 values, and PMR NOECs were also significantly related to whole body glutathione (GSH) levels, suggesting that acute and chronic toxicity results from protein adduct formation for SN2 electrophiles. Shared refractory locomotor response patterns among study compounds and two alternative vertebrate models appear informative of electrophilic properties associated with oxidative stress for SN2 chemicals. Electrophilic parameters derived from frontier molecular orbitals were predictive of experimental in vivo acute and sublethal toxicity. These observations provide important implications for identifying and designing less hazardous industrial chemicals with reduced potential to elicit oxidative stress through bimolecular nucleophilic substitution.

 

Link to the publication : 

https://pubs.acs.org/doi/abs/10.1021/acs.chemrestox.9b00290

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Journal of Visualized Experiments

2019-12-05

Abstract

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

 

Link to the publication :

https://www.jove.com/video/58837/behavioral-and-physiological-analysis-in-a-zebrafish-model-of-epilepsy?status=a60843k

Bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate affects lipid metabolism in zebrafish larvae via DNA methylation modification

Environmental Science and Technology

2019-12-05

Abstract

Abstract Image

Bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) is a ubiquitous environmental contaminant, but its toxicity is not fully understood. Accordingly, we investigated the effects of TBPH and its metabolite, mono-(2-ethyhexyl)tetrabromophthalate (TBMEHP), on lipid metabolism using a zebrafish model. The molecular docking study revealed that TBPH and TBMEHP bind to zebrafish peroxisome proliferator-activated receptor γ (PPARγ), with binding energies similar to rosiglitazone, a PPARγ agonist. Zebrafish embryos 0.75 hpf were exposed to TBPH (0.2–2000 nM) or TBMEHP (0.2–2000 nM) until 72 hpf, and their effects on PPARγ-mediated lipid metabolism were evaluated. Significant regional DNA demethylation of the PPARγ promoter was observed in the larvae at 72 hpf. Demethylation of the PPARγ promoter accompanied by upregulation of tet1 and tet2 transcription caused upregulation of PPARγ transcription and certain downstream genes involved in lipid lipolysis, transport, and metabolism. The triglyceride and total cholesterol concentrations in the larvae were significantly reduced following exposure to TBPH or TBMEHP. Furthermore, significant increases in the whole ATP content and locomotor activity in the 120 hpf larvae were observed. The overall results suggest that both TBPH and TBMEHP affect methylation of the PPARγ promoter, subsequently influencing larvae lipid metabolism via the PPARγ signaling pathway and disrupting energy homeostasis.

 

Link to the publication : 

https://pubs.acs.org/doi/abs/10.1021/acs.est.9b05796

Municipal wastewater effluent exposure disrupts early development, larval behavior, and stress response in zebrafish

Environmental Pollution

2019-12-13

Highlights

 

• Municipal wastewater effluent (MWWE) contains pharmaceutically-active compounds.
• MWWE delays somitogenesis and hatching rates in embryonic zebrafish.
• Short-term exposures to MWWE alters zebrafish embryo and larval behaviors.
• MWWE dulls the cortisol and lactate stress response in juvenile zebrafish.
• Zebrafish at various lifestages are a sensitive model for assessing MWWE toxicity.

 

Abstract

While wastewater treatment standards have been progressively increasing, emerging contaminants such as pharmaceuticals can nonetheless pass through treatment and end up in our watersheds. Pharmaceuticals in the parts-per-billion range can impact fish behavior, survival, and recruitment in the wild. However, the ecological risk posed by whole municipal wastewater effluents (MWWE), a complex mixture, is not clear. This knowledge gap is particularly evident for early lifestages (ELS) of fish, and because effluent discharge events are typically short, the effects of short-term MWWE exposures to ELS fish are particularly important from an environmental perspective. Here we tested the effects of rapid 30-min exposures, and short-term 24- and 72-h exposures to MWWE on development, behaviors, and stress response in zebrafish (Danio rerio) embryos, larvae, and juveniles. We obtained 24-h composite samples of tertiary-treated MWWE that contained a mixture of chemicals with affinities for serotonergic, adrenergic, dopaminergic, and ion-channel receptors. Embryos exposed to 5%, 10%, and 50% MWWE experienced developmental delays in somitogenesis and hatching rate, although there was no effect on survival. Embryonic photomotor responses were affected following 30-min and 24-h exposures to 10% and 50% MWWE, and larval visual motor responses were reduced from 24-h exposure to 10% MWWE. Exposure to 10% MWWE dulled the juvenile cortisol and lactate response following an acute air-exposure. Compromised behavioral and stress performances demonstrate the capacity of MWWE to impact phenotypes critical to the survival of fish in the environment. Taken together, we found that zebrafish were sensitive to toxic effects of MWWE at multiple life-stages.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749119350638

Assessment of the ecotoxicity of the pharmaceuticals bisoprolol, sotalol, and ranitidine using standard and behavioral endpoints

Environmental Science and Pollution Research

2019-12-18

Abstract

The pharmaceuticals bisoprolol (BIS), sotalol (SOT), and ranitidine (RAN) are among the most consumed pharmaceuticals worldwide and are frequently detected in different aquatic ecosystems. However, very few ecotoxicity data are available in the literature for them. To help fill these data gaps, toxicity tests with the algae Raphidocelis subcapitata, the macrophyte Lemna minor, the cnidarian Hydra attenuata, the crustacean Daphnia similis, and the fish Danio rerio were performed for assessing the ecotoxicity of these pharmaceuticals. Standard, as well as non-standard endpoint, was evaluated, including the locomotor behavior of D. rerio larvae. Results obtained for SOT and RAN showed that acute adverse effects are not expected to occur on aquatic organisms at the concentrations at which these pharmaceuticals are usually found in fresh surface waters. On the other hand, BIS was classified as hazardous to the environment in the acute III category. Locomotor behavior of D. rerio larvae was not affected by BIS and RAN. A disturbance on the total swimming distance at the dark cycle was observed only for larvae exposed to the highest test concentration of 500 mg L−1 of SOT. D. similis reproduction was affected by BIS with an EC10 of 3.6 (0.1–34.0) mg L−1. A risk quotient (RQ) of 0.04 was calculated for BIS in fresh surface water, considering a worst-case scenario. To the best of our knowledge, this study presents the first chronic toxicity data with BIS on non-target organisms.

 

Link to the publication :

https://link.springer.com/article/10.1007/s11356-019-07322-0

DMSO effects larval zebrafish (Danio rerio) behavior, with additive and interaction effects when combined with positive controls

Science of the Total Environment

2019-12-19

Highlights

 

• Solvents are frequently used during zebrafish toxicity testing but their effects are unknown.
• DMSO affected behavior at a concentration of ≥0.55%
• Different zebrafish strains showed different basal activity, but the same behavioral response to DMSO.
• DMSO had an additive and interaction effects on behavior when co-exposed with positive controls.

 

Abstract

Embryonic and larval zebrafish (Danio rerio) behavior is commonly used to identify neurotoxic compounds. Here, we investigated whether sub-lethal exposures to the common solvents dimethyl sulfoxide (DMSO, 0.01–1%) and methanol (MeOH, 0.01–1%), or the anti-fungal agent methylene blue (MB, 0.0001–0.0005%), can influence larval behavior in a simple light/dark paradigm conducted in 96-well plates. In addition, we tested whether the media volume within the behavioral arena or the zebrafish strain, AB wild type, AB Tübingen (AB/TU), or Tüpfel long-fin (TL), could also influence larval behavior. Following the single exposures, we co-exposed larvae to DMSO and either MB or two other compounds with known behavioral effects in larval zebrafish, flutamide and perfluorooctanesulfonic acid (PFOS). We found ≥0.55% DMSO and 0.0005% MB significantly affected larval behavior, but there was no effect of MeOH. Similarly, TL showed less movement compared to AB and AB/TU strains, whereas lower media volumes also significantly reduced larval movement. However, all strains responded similarly to DMSO and MB. In the co-exposure studies, we found either additive or interaction effects between DMSO and either MB, flutamide, or PFOS, depending on the behavioral endpoint measured. In addition, media volume had no effect on the DMSO concentration response curve, but again we observed additive effects on behavior. In conclusion, methodology can lead to alterations in baseline locomotor activity and compounds can have additive or interaction effects on behavioral endpoints. However, we found no evidence that strain effects should be a concern when deciding on solvents for a simple light/dark behavioral test in larval zebrafish.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S004896971934481X

Isoliquiritigenin triggers developmental toxicity and oxidative stressemediated apoptosis in zebrafish embryos/larvae via Nrf2-HO1/ JNK-ERK/mitochondrion pathway

Chemosphere

2019-12-24

Highlights

 

• ISL triggered developmental toxicity in zebrafish, caused death and malformations.
• ISL exposure instigated developmental toxicity in heart, liver, and nervous system.
• ISL triggered apoptosis in zebrafish via Nrf2-HO1/JNK-ERK/mitochondrion pathway.

 

Abstract

Isoliquiritigenin (ISL) is an emerging natural flavonoid found in the roots of licorice, exhibits antioxidant, anti-cancer, anti-inflammatory, anti-allergic, cardioprotective, hepatoprotective and neuroprotective properties. However, the effect of ISL in embryonic development is yet to be elucidated, and the mechanisms underlying its target-organ toxicity and harmful side effects are still unclear. In the present study, we employed zebrafish embryos to study the developmental toxicity effect of ISL and its underlying mechanisms. Zebrafish embryos upon treatment with either vehicle control (0.1% DMSO) or ISL solutions for 4–96 h post fertilization (hpf) showed that ISL exposure instigated severe developmental toxicity in heart, liver, and nervous system. Mortality and morphological abnormalities were also observed. High concentrations of ISL exposure resulted in abnormal phenotypes and embryonic malformations including pericardial edema, swim bladder defects, yolk retention, curved body shape and shortening of body length. Moreover, ISL exposure led to significant loss of dopaminergic neurons accompanied by reduced locomotor behaviour. Apoptotic cells were predominantly located in the heart area of 96 hpf embryo. Additionally, ISL significantly increased the levels of reactive oxygen species, lipid peroxidation content and decreased antioxidant enzyme activities. The expressions pattern of apoptosis-related genes BadCyto cCaspase-9Caspase-3 and Bax/Bcl-2 indicated that the oxidative stress–induced apoptosis triggered by ISL suggest involvement of Nrf2-HO1/JNK-ERK/mitochondrion pathways. In conclusion, here we provide first evidence that demonstrate ISL-induced dose-dependent developmental toxicity in zebrafish embryos. Furthermore, gene expression patterns in the embryos correlate the above and reveal potential genetic mechanisms of developmental toxicity.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653519329686

Phenotypic Characterization of Larval Zebrafish (Danio rerio) with Partial Knockdown of the cacna1a Gene

Molecular Neurobiology

2019-12-26

Abstract

The CACNA1A gene encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+ channels (Cav2.1). Mutations in this gene, among others, have been described in patients and rodents suffering from absence seizures and episodic ataxia type 2 with/without concomitant seizures. In this study, we aimed for the first time to assess phenotypic and behavioral alterations in larval zebrafish with partial cacna1aa knockdown, placing special emphasis on changes in epileptiform-like electrographic discharges in larval brains. Whole-mount in situ hybridization analysis revealed expression of cacna1aa in the optic tectum and medulla oblongata of larval zebrafish at 4 and 5 days post-fertilization. Next, microinjection of two antisense morpholino oligomers (individually or in combination) targeting all splice variants of cacna1aa into fertilized zebrafish eggs resulted in dose-dependent mortality and decreased or absent touch response. Over 90% knockdown of cacna1aa on protein level induced epileptiform-like discharges in the optic tectum of larval zebrafish brains. Incubation of morphants with antiseizure drugs (sodium valproate, ethosuximide, lamotrigine, topiramate) significantly decreased the number and, in some cases, cumulative duration of epileptiform-like discharges. In this context, sodium valproate seemed to be the least effective. Carbamazepine did not affect the number and duration of epileptiform-like discharges. Altogether, our data indicate that cacna1aa loss-of-function zebrafish may be considered a new model of absence epilepsy and may prove useful both for the investigation of Cacna1a-mediated epileptogenesis and for in vivo drug screening.

 

Link to the publication :

https://link.springer.com/article/10.1007/s12035-019-01860-x

Anti-Parkinson's disease activity of phenolic acids from Eucommia ulmoides Oliver leaf extracts and its autophagy activation mechanism

Food and Function

2019-12-30

Abstract

Although Parkinson’s disease (PD) is the second most common neurodegenerative disorder, the preventative or therapeutic agents for the treatment of PD are limited. Eucommia ulmoides Oliver (EuO) is widely used as traditional herb to treat various diseases. EuO bark extracts have been reported to possess anti-PD activity. Here, we investigated whether extracts of EuO leaves (EEuOL) also have therapeutic effect on PD since similar components and clinical application have been found between barks and leaves of this tree. We identified the chemical composition of EEuOL by HPLC-Q-TOF-MS and tested the anti-PD effect of EEuOL using zebrafish PD model. As a result, 28 compounds including 3 phenolic acids, 7 flavonoids, and 9 iridoids were identified. EEuOL significantly reversed the loss of dopaminergic neurons and neural vasculature as well as reduced the numbers of apoptotic cells in the zebrafish brain in a concentration-dependent manner. Moreover, EEuOL relieved locomotor impairments in MPTP-modeled PD zebrafish. We also investigated the underlying mechanism and found that EEuOL may activate autophagy, contributing to α-synuclein degradation, therefore alleviate PD-like symptoms. Molecular docking simulation implied the interaction between autophagy regulators (Pink1, Beclin1, Ulk2, and Atg5) and phenolic acids of EEuOL, affirming the involvement of autophagy in EEuOL-exerted anti-PD action. The overall results indicated the anti-PD effect of EEuOL, opening a possibility to use the extract in PD treatment.

 

Link to the publication :

https://pubs.rsc.org/en/content/articlelanding/2020/fo/c9fo02288k#!divAbstract

Toxicity of different zinc oxide nanomaterials and dose-dependent onset and development of Parkinson’s disease-like symptoms induced by zinc oxide nanorods

Environment International

2020-01-01

ABSTRACT

With the increasing applications in various fields, the release and accumulation of zinc oxide (ZnO) nano-materials ultimately lead to unexpected consequences to environment and human health. Therefore, toxicity comparison among ZnO nanomaterials with different shape/size and their adverse effects need better charac-terization. Here, we utilized zebrafish larvae and human neuroblastoma cells SH-SY5Y to compare the toxic effects of ZnO nanoparticles (ZnO NPs), short ZnO nanorods (s-ZnO NRs), and long ZnO NRs (l-ZnO NRs). We found their developmental- and neuro-toxicity levels were similar, where the smaller sizes showed slightly higher toxicity than the larger sizes. The developmental neurotoxicity of l-ZnO NRs (0.1, 1, 10, 50, and 100 μg/mL) was further investigated since they had the lowest toxicity. Our results indicated that l-ZnO NRs induced develop-mental neurotoxicity with hallmarks linked to Parkinsons disease (PD)-like symptoms at relatively high doses, including the disruption of locomotor activity as well as neurodevelopmental and PD responsive genes expres-sion, and the induction of dopaminergic neuronal loss and apoptosis in zebrafish brain. l-ZnO NRs activated reactive oxygen species production, whose excessive accumulation triggered mitochondrial damage and mito-chondrial apoptosis, eventually leading to PD-like symptoms. Collectively, the developmental- and neuro-toxicity of ZnO nanomaterials was identified, in which l-ZnO NRs harbors a remarkably potential risk for the onset and development of PD at relatively high doses, stressing the discretion of safe range in view of nano-ZnO exposure to ecosystem and human beings.

Link to the publication :

https://sci-hub.se/https://www.sciencedirect.com/science/article/pii/S0160412020321346

Ocean acidification does not impair the behaviour of coral reef fishes

Nature

2020-01-08

Abstract

The partial pressure of CO2 in the oceans has increased rapidly over the past century, driving ocean acidification and raising concern for the stability of marine ecosystems1,2,3. Coral reef fishes are predicted to be especially susceptible to end-of-century ocean acidification on the basis of several high-profile papers4,5 that have reported profound behavioural and sensory impairments—for example, complete attraction to the chemical cues of predators under conditions of ocean acidification. Here, we comprehensively and transparently show that—in contrast to previous studies—end-of-century ocean acidification levels have negligible effects on important behaviours of coral reef fishes, such as the avoidance of chemical cues from predators, fish activity levels and behavioural lateralization (left–right turning preference). Using data simulations, we additionally show that the large effect sizes and small within-group variances that have been reported in several previous studies are highly improbable. Together, our findings indicate that the reported effects of ocean acidification on the behaviour of coral reef fishes are not reproducible, suggesting that behavioural perturbations will not be a major consequence for coral reef fishes in high CO2 oceans.

 

Link to the publication : 

https://www.nature.com/articles/s41586-019-1903-y

Acute and chronic effects of environmental realistic concentrations of clofibric acid in Danio rerio: Behaviour, oxidative stress, biotransformation and lipid peroxidation endpoints

Environmental Toxicology and Pharmacology

2020-05-15

Highlights

 

• Clofibric acid exposure in zebrafish affected behaviour through hypoactivity.
• Clofibric acid properties caused an increase in all biochemical biomarkers.
• The metabolism of this antihyperlipidemic seems to have modulated behavioral and biochemical results.
• No evidences were found in terms of histology endpoints being altered by clofibric acid.

Abstract

Due to their widespread use, pharmaceuticals can be metabolized, excreted and ultimately discarded in the environment, thereby affecting aquatic organisms. Lipid-regulating drugs are among the most prescribed medications around the world, controlling human cholesterol levels, in more than 20 million patients. Despite this growing use of lipid-regulating drugs, particularly those whose active metabolite is clofibric acid, the potential toxicological effects of these pharmaceuticals in the environment is not fully characterized. This work intended to characterize the toxicity of an acute (120 hours post-fertilization) and chronic (60 days post-fertilization) exposures to clofibric acid in concentrations of 10.35, 20.7, 41.4, 82.8, and 165.6 μg L-1 in zebrafish (Danio rerio). The concentrations which were implemented in both exposures were based on predicted environmental concentrations for Portuguese surface waters. The acute effects were analysed focusing on behavioural endpoints (small and large distance travelled, swimming time and total distance travelled), biomarkers of oxidative stress (activity of the enzymes superoxide dismutase, Cu/Zn- and Mn SOD; catalase, CAT; glutathione peroxidase, Se- and total GPx), biotransformation (activity of glutathione S-transferases, GSTs) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Chronically exposed individuals were also histologically analysed for sex determination and gonadal developmental stages. In terms of acute exposure, significant alterations were reported, in terms of behavioural alterations (hypoactivity), followed by an overall increase in all tested biomarkers. Chronically exposed organisms did not show alterations in terms of sex ratio and maturation stages, suggesting that clofibric acid did not act as an endocrine disruptor. Moreover, the metabolism of clofibric acid resulted in increased levels of both forms of SOD activity, especially for animals exposed to higher levels of this drug. An increase of CAT activity was observed in fish exposed to low levels, and a decrease in those exposed to higher amounts of clofibric acid. Both GPx forms had their activities increased. The enzyme of biotransformation GSTs were increased at low levels of clofibric acid but inhibited at higher amounts of this substance. Lipid peroxidation levels were also changed, with an induction of this parameter with increasing amounts of clofibric acid. Changes also occurred in behavioural endpoints and patterns for control organisms and for those exposed to clofibric acid were significantly distinct, for all types (light and darkness) of exposure, and for the two analysed endpoints (small and large distance). Results from this assay allow inferring that clofibric acid can have an ecologically relevant impact in living organisms exposed to this substance, with putative effects on the metabolism of individuals, affecting their behaviour and ultimately their survival.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S1382668920301447

Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

2020-06-12

Abstract

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefits from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafish panx1a gene using TALEN technology. Here, RNA-seq analysis of 6 days post fertilization larvae were confirmed by Real-Time PCR and paired with testing visual-motor behavior and in vivo electrophysiology. Results demonstrated that loss of panx1a specifically affected the expression of gene classes representing the development of the visual system and visual processing. Abnormal swimming behavior in the dark and the expression regulation of pre-and postsynaptic biomarkers suggested changes in dopaminergic signaling. Indeed, altered visuomotor behavior in the absence of functional Panx1a was evoked through D1/D2-like receptor agonist treatment and rescued with the D2-like receptor antagonist Haloperidol. Local field potentials recorded from superficial areas of the optic tectum receiving input from the retina confirmed abnormal responses to visual stimuli, which resembled treatments with a dopamine receptor agonist or pharmacological blocking of Panx1a. We conclude that Panx1a functions are relevant at a time point when neuronal networks supporting visual-motor functions undergo modifications preparing for complex behaviors of freely swimming fish.

Introduction

Pannexin 1 (Panx1) is an integral membrane glycoprotein forming ATP release channels in different tissues and cell types1,2,3,4,5, including neurons6,7,8. In the CNS, evidence for physiological functions of Panx1 points at roles in the processing of sensory signals and learning and memory9,10,11. For example, in Panx1 knockout mice, altered retinal contrast sensitivity12 and hearing loss have been found13,14. Also, performance in spatial learning and memory abilities such as object recognition and fear conditioning tasks are decreased9,15,16. Intellectual disabilities, severe hearing loss, primary ovarian failure, kyphoscoliosis, and difficulties navigating in darkness were found in the first human patient identified with a homozygous Panx1 mutation17.

To form a better view of Panx1 functions in the processing of sensory information, we used the zebrafish as model organisms. Two Panx1 genes, panx1a and panx1b, originated from partial genome duplications during early teleost evolution18,19. Although the two genes have been separated for more than 200 million years, principal channel functions are comparable to rodent or human Panx120. In the retina, the panx1a protein is expressed in horizontal cells19,20 and plays essential roles in feedback from horizontal cells to cones in adult zebrafish21,22,23. Here the panx1a gene was edited using transcription activator-like effector nucleases (TALEN). A loss of function mutation allowed to investigate Panx1a in 6 dpf old zebrafish larvae at a developmental stage when neuronal networks for visually guided locomotor behaviors were functional. Transcriptome analysis detected noteworthy expression differences of genes associated with eye development and vision-related processes. When followed up, altered dopaminergic signaling affecting both pre- and postsynaptic proteins were found. The molecular evidence was supported by measuring abnormal responsiveness of panx1a−/− larvae to darkness, or after the abrupt loss of illumination. Pharmacological activation of D1/D2-like dopamine receptors simulated this phenotype. Blocking D2-like receptors with Haloperidol rescued the phenotype. In vivo electrophysiological recording of local field potentials (LOF) from the larval optic tectum in a region receiving input from the retina demonstrated that the dynamic transition from low to higher-frequency brain waves in light and darkness was compromised in panx1a−/− larvae. This phenotype was reproduced by pharmacological blocking of Panx1a, or by treatment with the D1/D2 receptor agonist apomorphine. This research delivers a novel association between Panx1a in the integration of sensory-motor behavior through modulation of dopaminergic signaling.

 

Link to the publication :

https://www.nature.com/articles/s41598-020-66378-y

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ‐aminobutyric acidergic anticonvulsant

Epilepsia

2020-06-14

Abstract

Objective

Current medicines are ineffective in approximately one‐third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high‐throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms.

Methods

We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action.

Results

Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC‐37, a worm γ‐aminobutyric acid type A (GABAA) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6‐Hz 44‐mA model of pharmacoresistant seizures.

Significance

These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

 

Key Points

 

  • Compound library screening in zebrafish identifies chlorothymol as an anticonvulsant
  • Genetic screening in nematodes reveals that chlorothymol acts on GABAA receptors
  • Electrophysiological recording from mouse and human neurons confirms chlorothymol's GABAergic mechanism of action
  • Chlorothymol is protective in several mouse seizure assays, including the 6‐Hz 44‐mA model of pharmacoresistant seizures

Link to the publication :

https://onlinelibrary.wiley.com/doi/full/10.1111/epi.16644

Role of GH/IGF axis in arsenite-induced developmental toxicity in zebrafish embryos

Ecotoxicology and Environmental Safety

2020-06-15

Highlights

 

• Arsenite induced abnormal development of zebrafish.
• Arsenite increased the level of GH in zebrafish.
• Arsenite down-regulated the expression of GH/IGF axis related genes.
• Arsenite caused locomotor behavior disorder by affecting abnormal neurodevelopment.

Abstract

Growth hormone (GH)/insulin-like growth factor (IGF) axis plays a critical role in fetal development. However, the effect of arsenite exposure on the GH/IGF axis and its toxic mechanism are still unclear. Zebrafish embryos were exposed to a range of NaAsO2 concentrations (0.0–10.0 mM) between 4 and 120 h post-fertilization (hpf). Development indexes of survival, malformation, hatching rate, heart rate, body length and locomotor behavior were measured. Hormone levels, GH/IGF axis-related genes, and nerve-related genes were also tested. The results showed that survival rate, hatching rate, heart rate, body length and locomotor behavior all decreased, while deformity increased. At 120 hpf, the survival rate of zebrafish in 1.5 mM NaAsO2 group was about 70%, the deformity rate exceeded 20%, and the body length shortened to 3.35 mm, the movement distance of zebrafish decreased approximately 63.6% under light condition and about 52.4% under dark condition. The level of GH increased and those of IGF did not change significantly, while the expression of GH/IGF axis related genes (ghra, ghrb, igf2r, igfbp3, igfbp2a, igfbp5b) and nerve related genes (dlx2, shha, ngn1, elavl3, gfap) decreased. In 1.5 mM NaAsO2 group, the decrease of igfbp3 and igfbp5b was almost obvious, about 78.2% and 72.2%. The expression of nerve genes in 1.5 mM NaAsO2 group all have declined by more than 50%. These findings suggested that arsenite exerted disruptive effects on the endocrine system by interfering with the GH/IGF axis, leading to zebrafish embryonic developmental toxicity.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S014765132030659X

Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

Molecular Ecology

2020-06-15

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefts from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafsh panx1a gene using TALEN technology. Here, RNA-seq analysis of 6 days post fertilization larvae were confrmed by Real-Time PCR and paired with testing visual-motor behavior and in vivo electrophysiology. Results demonstrated that loss of panx1a specifcally afected the expression of gene classes representing the development of the visual system and visual processing. Abnormal swimming behavior in the dark and the expression regulation of pre-and postsynaptic biomarkers suggested changes in dopaminergic signaling. Indeed, altered visuomotor behavior in the absence of functional Panx1a was evoked through D1/D2-like receptor agonist treatment and rescued with the D2-like receptor antagonist Haloperidol. Local feld potentials recorded from superfcial areas of the optic tectum receiving input from the retina confrmed abnormal responses to visual stimuli, which resembled treatments with a dopamine receptor agonist or pharmacological blocking of Panx1a. We conclude that Panx1a functions are relevant at a time point when neuronal networks supporting visualmotor functions undergo modifcations preparing for complex behaviors of freely swimming fsh.

 

Link to the publication :

https://search.proquest.com/openview/f0d3636e8d437810609cff88aa0b955a/1?pq-origsite=gscholar&cbl=2041939

The multi-dimensional embryonic zebrafish platform predicts flame retardant bioactivity

Reproductive Toxicology

2020-06-15

Highlights

 

• Identified the bioactivity of 61 flame retardant chemicals (FRCs).
• The zebrafish assay detects bioactivity of 12 of the 10 known developmental neurotoxic FRCs.
• Classification model using physicochemical properties and 3 zebrafish assays yields a balanced accuracy of 91.7%.

 

Abstract

Flame retardant chemicals (FRCs) commonly added to many consumer products present a human exposure burden associated with adverse health effects. Under pressure from consumers, FRC manufacturers have adopted some purportedly safer replacements for first-generation brominated diphenyl ethers (BDEs). In contrast, second and third-generation organophosphates and other alternative chemistries have limited bioactivity data available to estimate their hazard potential. In order to evaluate the toxicity of existing and potential replacement FRCs, we need efficient screening methods. We built a 61-FRC library in which we systemically assessed developmental toxicity and potential neurotoxicity effects in the embryonic zebrafish model. Data were compared to publicly available data generated in a battery of cell-based in vitro assays from ToxCast, Tox21, and other alternative models. Of the 61 FRCs, 19 of 45 that were tested in the ToxCast assays were bioactive in our zebrafish model. The zebrafish assays detected bioactivity for 10 of the 12 previously classified developmental neurotoxic FRCs. Developmental zebrafish were sufficiently sensitive at detecting FRC structure-bioactivity impacts that we were able to build a classification model using 13 physicochemical properties and 3 embryonic zebrafish assays that achieved a balanced accuracy of 91.7%. This work illustrates the power of a multi-dimensional in vivo platform to expand our ability to predict the hazard potential of new compounds based on structural relatedness, ultimately leading to reliable toxicity predictions based on chemical structure.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0890623820301969

Environmental levels of venlafaxine impact larval behavioural performance in fathead minnows

Chemosphere

2020-06-18

Highlights

 

• Environmental levels of venlafaxine reduce larval growth and activity.
• Larval minnows exhibit altered behavioural responses in light and darkness.
• Thigmotaxis is a reliable measure of larval anxiogenic/anxiolytic behaviour in fathead minnows.
• Venlafaxine did not affect thigmotaxis in larval fathead minnows.

 

Abstract

Venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is one of the most abundant antidepressants in municipal wastewater effluents (MWWE). The early life stages are particularly sensitive to contaminant exposure, but few studies have examined whether persistent exposure to venlafaxine impart adverse developmental outcomes. The fathead minnow (Pimephales promelas) is a widely used model for ecotoxicological studies, and this fish is native to Alberta, Canada. We tested the hypothesis that environmental levels of venlafaxine compromises early developmental behavioural performances in fathead minnows. Embryos were exposed to waterborne venlafaxine at either 0, 0.06, 0.33, 0.66, 1.37 or 3 μg L−1 concentration for 7 days. Environmental levels of venlafaxine did not impact the survival, hatch rate or heart rate of fathead minnow embryos and larvae but reduced the growth of larvae even at concentrations as low as 0.06 μg L−1. We validated thigmotaxis as a screen for anxiolytic and anxiogenic behaviour in fathead minnow larvae by exposing them to concentrations of ethanol and caffeine, respectively. Behavioural analyses revealed that early developmental exposure to venlafaxine does not alter thigmotaxis but reduced the activity of fathead minnows. The larval behavioural assays reported here for fathead minnow have the potential to be used as screening tools for the risk assessment of neurotoxic contaminants in MWWE. Overall, we demonstrate for the first time that exposure to environmental levels of venlafaxine during the critical early developmental window does not elicit an anxiogenic response but may adversely affect the larval growth performance of fathead minnows.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0045653520316313

Impacts of bisphenol A analogues on zebrafish post‐embryonic brain

Journal of Neuroendocrinology

2020-06-21

Abstract

Bisphenol A (BPA) is a widely studied and well‐recognised endocrine‐disrupting chemical, and one of the current issues is its safe replacement by various analogues. Using larva zebrafish as a model, the present study reveals that moderate and chronic exposure to BPA analogues such as bisphenol S, bisphenol F and bisphenol AF may also affect vertebrate neurodevelopment and locomotor activity. Several parameters of embryo‐larval development were investigated, such as mortality, hatching, number of mitotically active cell, as defined by 5‐bromo‐2'‐deoxyuridine incorporation and proliferative cell nuclear antigen labelling, aromatase B protein expression in radial glial cell and locomotor activity. Our results show that exposure to several bisphenol analogues induced an acceleration of embryo hatching rate. At the level of the developing brain, a strong up‐regulation of the oestrogen‐sensitive Aromatase B was also detected in the hypothalamic region. This up‐regulation was not associated with effects on the numbers of mitotically active progenitors nor differentiated neurones in the preoptic area and in the nuclear recessus posterior of the hypothalamus zebrafish larvae. Furthermore, using a high‐throughput video tracking system to monitor locomotor activity in zebrafish larvae, we show that some bisphenol analogues, such as bisphenol AF, significantly reduced locomotor activity following 6 days of exposure. Taken together, our study provides evidence that BPA analogues can also affect the neurobehavioural development of zebrafish.

 

Link to the publication : 

https://onlinelibrary.wiley.com/doi/abs/10.1111/jne.12879

Inhibitory effects of polystyrene microplastics on caudal fin regeneration in zebrafish larvae

Environmental Pollution

2020-06-21

Highlights

 

• Microplastic exposure inhibited the caudal fin regeneration in zebrafish larvae.
• Regeneration-related signalings were altered after microplastic exposure.
• Changes of ROS signaling and immune response contributed to the inhibitory effects.
• Transcriptomic analyses suggested the metabolic pathways were also involved.
• Microplastics might impair the capacity for repair and regeneration in injured fish.

 

Abstract

Microplastic pollution is pervasive in aquatic environments, but the potential effects of microplastics on aquatic organisms are still under debate. Given that tissue damage is unavoidable in fish and the available data mostly concentrate on healthy fish, there is a large chance that the ecotoxicological risk of microplastic pollution is underrated. Therefore, in this study, the effects of microplastics on the regenerative capacity of injured fish were investigated using a zebrafish caudal fin regeneration model. After fin amputation at 72 h post fertilization, the larvae were exposed to polystyrene microplastics (0.1–10 mg/L) with diameters of 50 or 500 nm. Microplastic exposure significantly inhibited fin regeneration, both morphologically and functionally. Furthermore, the signaling networks that regulate fin regeneration, as well as reactive oxygen species signaling and the immune response, both of which are essential for tissue repair and regeneration, were altered. Transcriptomic analyses of the regenerating fin confirmed that genes related to fin regeneration were transcriptionally modulated in response to microplastic exposure and that metabolic pathways were also extensively involved. In conclusion, this study demonstrated for the first time that microplastic exposure could disrupt the regenerative capacity of fish and might eventually impair their fitness in the wild.

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0269749120301524

Freezing activity brief data from a new FUS mutant zebrafish line

Data in Brief

2020-06-24

Abstract

The data presented in this paper are related to the research article “Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS”. In this model the lack of fus causes reduced lifespan as well as impaired motor abilities associated with a decrease of motor neurons axons lenght and an increase of neuromuscular junctions fragmentation.

Data in this article describes the global locomotor activity data at 3, 4 and 5 days post fertilization in WT, fus heterozygous (fus+/−) and fus homozygous (fus−/−) zebrafish embryos as a response to visual light stimulation, with particular attention on the freezing respose.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S2352340920308155

Toxicity of different polycyclic aromatic hydrocarbons (PAHs) to the freshwater planarian Girardia tigrina

Environmental Pollution

2020-07-03

Highlights

 

• Several endpoints can be evaluated in planarians in response to chemical stress.
• Phenanthrene, but not pyrene or B[a]P, caused mortality in planarians.
• Planarian sub-lethal endpoints were more sensitive to pyrene and B[a]P exposures.
• Behavioral endpoints showed the greatest sensitivity for all tested PAHs.
• Planarians can be useful experimental animals for ecotoxicological studies.

 

Abstract

Freshwater planarians have been gaining relevance as experimental animals for numerous research areas given their interesting features, such as high regeneration potential, shared features with the vertebrates’ nervous system or the range of endpoints that can be easily evaluated in response to contaminants. Ecotoxicological research using these animals has been steadily increasing in the past decades, as planarians’ potentialities for this research area are being recognized. In this work, we used polycyclic aromatic hydrocarbons (PAHs) as model contaminants and evaluated effects of exposure to phenanthrene, pyrene and benzo[a]pyrene (B[a]P) in planarians. The freshwater planarian Girardia tigrina was chosen and mortality, cephalic regeneration (during and post-exposure), behavioral endpoints and presence of PAHs in tissues, were evaluated. Mortality was only observed in planarians exposed to phenanthrene, with an estimated LC50 of 830 μg L−1. Results indicate that planarian behavioral endpoints were very sensitive in response to sub-lethal concentrations of PAHs, showing a greater sensitivity towards B[a]P and pyrene. Briefly, post-exposure locomotion and post-exposure feeding were significantly impaired by sub-lethal concentrations of all compounds, whereas regeneration of photoreceptors was only significantly delayed in planarians exposed to pyrene. Moreover, levels of PAH-type compounds in planarian tissues followed a concentration-dependent increase, showing uptake of compounds from experimental solutions. The present results highlight the importance of studying alternative and complementary endpoints, such as behavior, not only because these may be able to detect effects at lower levels of contamination, but also due to their ecological relevance. The simplicity of evaluating a wide range of responses to contaminants further demonstrates the utility of freshwater planarians for ecotoxicological research.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0269749120302244

Lrrk2 modulation of Wnt signaling during zebrafish development

Journal of Agricultural and food chemistry

2020-07-05

Abstract

Mutations in leucine‐rich repeat kinase 2 (lrrk2) are the most common genetic cause of Parkinson's disease. Difficulty in elucidating the pathogenic mechanisms resulting from disease‐associated Lrrk2 variants stems from the complexity of Lrrk2 function and activities. Lrrk2 contains multiple protein–protein interacting domains, a GTPase domain, and a kinase domain. Lrrk2 is implicated in many cellular processes including vesicular trafficking, autophagy, cytoskeleton dynamics, and Wnt signaling. Here, we generated a zebrafish lrrk2 allelic series to study the requirements for Lrrk2 during development and to dissect the importance of its various domains. The alleles are predicted to encode proteins that either lack all functional domains (lrrk2sbu304), the GTPase, and kinase domains (lrrk2sbu71) or the kinase domain (lrrk2sbu96). All three lrrk2 mutants are viable, morphologically normal, and display wild‐type‐like locomotion. Because Lrrk2 modulates Wnt signaling in some contexts, we assessed Wnt signaling in all three mutant lines. Analysis of Wnt signaling by studying the expression of target genes using whole mount RNA in situ hybridization and a transgenic Wnt reporter revealed wild‐type domains of Wnt activity in each of the mutants. However, we found that Wnt pathway activation is attenuated in lrrk2sbu304/sbu304, which lacks both scaffolding and catalytic domains, but not in the other alleles during late embryogenesis. This supports a model in which Lrrk2 scaffolding functions are key to a context‐dependent role in promoting canonical Wnt signaling.

 

Link to the publication : 

https://onlinelibrary.wiley.com/doi/abs/10.1002/jnr.24687

Impacts of high dose 3.5 GHz cellphone radiofrequency on zebrafish embryonic development

Plos One

2020-07-09

Abstract

The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. In this study, we used the embryonic zebrafish model to assess the impacts of a 3.5 GHz RFR on biology- a frequency typically used by 5G-enabled cell phones and lies within the 4G and 5G bandwidth. We established a plate-based exposure setup for RFRs, exposed developing zebrafish to 3.5 GHz RFR, specific absorption rate (SAR) ≈ 8.27 W/Kg from 6 h post fertilization (hpf) to 48 hpf, and measured a battery of morphological and behavioral endpoints at 120 hpf. Our results revealed no significant impacts on mortality, morphology or photomotor response and a modest inhibition of startle response suggesting some levels of sensorimotor disruptions. This suggests that the cell phone radiations at low GHz-level frequencies are likely benign, with subtle sensorimotor effects. Through this assessment, we have established a robust setup for zebrafish RFR exposures readily amenable to testing various powers and frequencies. Future developmental exposure studies in zebrafish will evaluate a wider portion of the radio frequency spectrum to discover the bioactive regions, the potential molecular targets of RFR and the potential long-term effects on adult behavior.

 

Link to the publication : 

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235869

Ponatinib-induced ischemic stroke in larval zebrafish for drug screening

European Journal of Pharmacology

2020-07-12

Highlights

 

• We developed and characterized a larval zebrafish ischemic stroke model using Ponatinib as an inducer under an optimized exposure concentration and treatment period. The larval zebrafish ischemic stroke was further validated with 6 known human ischemic stroke therapeutics. The pathophysiology of this zebrafish ischemic stroke is closely similar to that of human ischemic stroke and could be used for rapidly identifying the preventive and therapeutic agents.

 

Absract

Conventional mammalian ischemic stroke models for drug screening are technically challenging, laborious and time-consuming. In this study, using Ponatinib as an inducer, we developed and characterized a zebrafish ischemic stroke model. This zebrafish ischemic stroke had the cerebral vascular endothelial injury, thrombosis, reduced blood flow, inflammation and apoptosis as well as the reduced motility. The zebrafish ischemic stroke model was validated with 6 known human therapeutic drugs of ischemic stroke (Aspirin, Clopidogrel, Naoxintong capsules, Edaravone, Xingnaojing injection, Shuxuening injection). The mRNA levels of the neovascularization-related gene (vegfaa) and vascular endothelial growth factor receptor gene (VEGFR), neurodevelopment related genes (mbp and α1-tubulin), brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) were significantly downregulated; whereas apoptosis-related genes (caspase-3caspase-7caspase-9 and bax/bcl-2), and inflammatory factor genes (IL-1βIL-6IL-10TNF-α and NF-κB) were remarkably upregulated in the model. These results suggest that the pathophysiology of Ponatinib-induced zebrafish ischemic stroke is similar to that of human ischemic stroke patients and this whole animal model could be used to study the complex cellular and molecular pathogenesis of ischemic stroke and to rapidly identify therapeutic agents.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0014299920303848

Xanthotoxin reverses Parkinson’s disease-like symptoms in zebrafish larvae and mice models: a comparative study

Pharmacological Reports

2020-07-22

Abstract

Background

The aim of this study is to preliminary evaluate the antiparkinsonian activity of furanocoumarin—xanthotoxin, in two behavioral animal models, zebrafish larvae treated with 6-hydroxydopamine and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in order to compare both models.

Methods

Xanthotoxin was isolated from Pastinaca sativa L. (Apiaceae) fruits. Then, the compound was administered by immersion to zebrafish 5 days after fertilization (dpf) larvae or intraperitoneally to male Swiss mice, as a potential therapeutic agent against locomotor impairments.

Results

Acute xanthotoxin administration at the concentration of 7.5 µM reversed locomotor activity impairments in 5-dpf zebrafish larvae. In mice model, acute xanthotoxin administration alleviated movement impairments at the concentration of 25 mg/kg.

Conclusions

The similar activity of the same substance in two different animal models indicates their compatibility and proves the potential of in vivo bioassays based on zebrafish models. Results of our study indicate that xanthotoxin may be considered as a potential lead compound in the discovery of antiparkinsonian drugs.

 

Link to the publication : 

https://link.springer.com/article/10.1007/s43440-020-00136-9

Dopaminergic neurons regenerate following chemogenetic ablation in the olfactory bulb of adult Zebrafish (Danio rerio)

Nature

2020-07-30

Abstract

Adult zebrafish have the ability to regenerate cells of the central nervous system. However, few neuronal regeneration studies in adult zebrafish addressed their ability to regenerate specific types of neurons following cell specific ablation. We show here that treatment of transgenic Tg(dat:CFP-NTR) adult zebrafish with the prodrug metronidazole (Mtz) according to our administration regimen predominantly ablates dopamine (DA) neurons within the olfactory bulb (OB) of adult fish. Loss of DA neurons was accompanied by an impaired olfaction phenotype, as early as 1-week post-treatment, in which fish were unable to sense the presence of the repulsive stimulus cadaverine. The olfactory impairment was reversed within 45 days and coincided with the recovery of DA neuron counts in the OB. A multi-label pulse-chase analysis with BrdU and EdU over the first seventeen days-post Mtz exposure showed that newly formed DA neurons were recruited within the first nine days following exposure and led to functional and morphological recovery of the OB.

Introduction

Over the last decade, the zebrafish has emerged as a strong model for the study of neurogenesis and brain regeneration as previously reviewed1,2,3. Unlike mammals, which have two main proliferation zones—the subgranular and subventricular zones4,5, the zebrafish holds 16 different zones of proliferation in the brain6,7. This ability to hold numerous cells in a proliferating stage allows for the recruitment of stem-progenitor cells to multiply and migrate following damage to brain tissue in order to allow cells to then remodel the brain environment in a regenerative fashion8,9. While much of our knowledge about the regenerative capacity of the adult zebrafish brain have come from invasive stab lesion studies8,9,10,11, few studies have investigated the zebrafish ability to regenerate a specific neuronal subtype through a conditional and specific ablation approach in the adult brain.

Neurotoxins such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and 6-OHDA (oxidopamine) have been classically used for the ablation of DA neurons in animal models of Parkinson’s disease (PD)12,13,14,15. Some of these drug treatments failed to produce changes in TH-positive neuronal population arrangement16,17. Others showed variable ability of DA neurons to regenerate15.

Dopaminergic neurons have been described in the olfactory bulb, pretectum, telencephalon, preoptic area and diencephalon of the adult zebrafish brain. Whereas some clusters of dopaminergic neurons of the teleost diencephalon are thought to regulate movement similarly to the mammalian striatum; DA neurons of the OB carry sensory response to odor. Overall, brain DA is involved in the regulation of movement, emotion, reward, memory, attention, motivation and hormonal secretion.

Chemogenetic ablation has emerged as an alternative to the ablation of DA neurons with neurotoxins, which are toxic to the animals and provided variable results in zebrafish. The chemogenetic ablation method based on expression of nitroreductase (NTR) allows for the conditional and specific induction of cell death mediated via the caspase-activated cascade18,19. The technique relies on the enzyme NTR, expressed under the control of regulatory elements from genes expressed in a tissue-specific manner. We and others have used NTR to successfully ablate DA neurons in juvenile zebrafish20,21,22,23,24. There has been evidence for the recovery of lost cells in juvenile zebrafish following chemogenetic ablation but so far, the ability of adult zebrafish to regenerate DA neurons had not been demonstrated. Here, we use the Tg(dat:CFP-NTR) transgenic line that we previously used to ablate DA neurons of juveniles22 and show that administration of the NTR substrate metronidazole (Mtz) to adult zebrafish leads to a large depletion of DA neurons in the olfactory bulb. This neuronal loss is accompanied by decreases in dopamine neurotransmitter content and impaired olfactory behavior. Both cell counts and phenotype are recovered after removal of Mtz, indicative of regeneration of DA neurons.

Pulse chase lineage tracing experiments showed that recruitment of progenitor cells occurs within the first nine days post exposure, showing that upon injury there is an immediate recruitment of cells with the ability to give rise to functional DA neurons in the adult zebrafish OB.

 

Link to the publication : 

https://www.nature.com/articles/s41598-020-69734-0

Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD)

Reproductive Toxicology

2020-08-04

Highlights

 

• Embryonic EtOH exposure induced morphological defects in the zebrafish FASD model.
• FA prevented defects on morphology, swimming, thigmotaxis, and optomotor response in larvae.
• EtOH exposure affected the sleep pattern, inducing several arousal periods in zebrafish.
• EtOH produced developmental defects, and FA reduced partially these defects.

 Abstract

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6–7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0890623820301878

Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD)

Reproductive Toxicology

2020-08-04

Highlights

 

• Embryonic EtOH exposure induced morphological defects in the zebrafish FASD model.
• FA prevented defects on morphology, swimming, thigmotaxis, and optomotor response in larvae.
• EtOH exposure affected the sleep pattern, inducing several arousal periods in zebrafish.
• EtOH produced developmental defects, and FA reduced partially these defects.

 

Abstract

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6–7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0890623820301878

Parental exposure to environmental concentrations of tris(1,3-dichloro-2- propyl)phosphate induces abnormal DNA methylation and behavioral changes in F1 zebrafish larvae

Environmental Pollution

2020-08-11

Highlights

 

Residues of TDCIPP were detected in F1 eggs after exposure of parents.

Parental exposure had effect on motor behaviors of F1 individuals.

Parental exposure induced DNA methylation alterations in F1 larvae.

Parental exposure downregulated the expression of genes related to neuronal cell body.

Downregulation of slc1a2b was positively correlated with reduced swimming speed of F1 larvae.

 

Abstract

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been demonstrated to be transferred from parental animals to their offspring. However, whether parental exposure to environmental concentrations of TDCIPP show neurodevelopmental toxicity in the F1 generation and the possible underlying mechanism remain unclear. Therefore, in this study, zebrafish embryos were exposed to environmental concentrations of TDCIPP (3, 30 and 300 ng L−1) for 120 days. The effects of exposure on motor behaviors, neurotransmitter levels, DNA methylation, and gene expression of F1 larvae were investigated. Parental exposure left TDCIPP residues in F1 eggs as well as reduced body length of F1 larvae. Moreover, parental exposure significantly reduced swimming activity in F1 5 dpf larvae, although it did not significantly alter serotonin, dopamine, 3,4-dihydroxyphenylacetic acid, γ-aminobutyrate, and acetylcholine levels. Genes encoding DNA methylation transferases (dnmt3aa and dnmt1) were downregulated in F1 larvae. Reduced representation bisulfite sequencing analysis revealed 446 differentially methylated regions and enriched neuronal cell body Gene Ontology term in F1 generation. Correlation analysis between the expression of genes related to neural cell body and swimming speed indicated that solute carrier family 1 member 2b (slc1a2b) downregulation might be responsible for the inhibition of motor behaviors. Furthermore, bisulfite amplicon sequencing analysis confirmed hypermethylation of the promoter region of slc1a2b in F1 larvae following parental exposure to 300 ng L−1 TDCIPP, which might have led to significant downregulation of gene expression and, in turn, influenced the motor behaviors. These results indicate that parental exposure to environmental concentrations of TDCIPP alters DNA methylation, downregulates gene expressions and, thus inducing developmental neurotoxicity, in F1 larvae.

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0269749120359935

Parental exposure to environmental concentrations of tris(1,3-dichloro-2-propyl)phosphate induces abnormal DNA methylation and behavioral changes in F1 zebrafish larvaeParental exposure to environmental concentrations of tris(1,3-dichloro-2-propyl)phosphate induces abnormal DNA methylation and behavioral changes in F1 zebrafish larvae

Environmental Pollution

2020-08-11

Highlights

 

• Residues of TDCIPP were detected in F1 eggs after exposure of parents.
• Parental exposure had effect on motor behaviors of F1 individuals.
• Parental exposure induced DNA methylation alterations in F1 larvae.
• Parental exposure downregulated the expression of genes related to neuronal cell body.
• Downregulation of slc1a2b was positively correlated with reduced swimming speed of F1 larvae.

 

Abstract

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been demonstrated to be transferred from parental animals to their offspring. However, whether parental exposure to environmental concentrations of TDCIPP show neurodevelopmental toxicity in the F1 generation and the possible underlying mechanism remain unclear. Therefore, in this study, zebrafish embryos were exposed to environmental concentrations of TDCIPP (3, 30 and 300 ng·L-1) for 120 days. The effects of exposure on motor behaviors, neurotransmitter levels, DNA methylation, and gene expression of F1 larvae were investigated. Parental exposure left TDCIPP residues in F1 eggs as well as reduced body length of F1 larvae. Moreover, parental exposure significantly reduced swimming activity in F1 5 dpf larvae, although it did not significantly alter serotonin, dopamine, 3,4-dihydroxyphenylacetic acid, γ-aminobutyrate, and acetylcholine levels. Genes encoding DNA methylation transferases (dnmt3aa and dnmt1) were downregulated in F1 larvae. Reduced representation bisulfite sequencing analysis revealed 446 differentially methylated regions and enriched neuronal cell body Gene Ontology term in F1 generation. Correlation analysis between the expression of genes related to neural cell body and swimming speed indicated that solute carrier family 1 member 2b (slc1a2b) downregulation might be responsible for the inhibition of motor behaviors. Furthermore, bisulfite amplicon sequencing analysis confirmed hypermethylation of the promoter region of slc1a2b in F1 larvae following parental exposure to 300 ng·L-1 TDCIPP, which might have led to significant downregulation of gene expression and, in turn, influenced the motor behaviors. These results indicate that parental exposure to environmental concentrations of TDCIPP alters DNA methylation, downregulates gene expressions and, thus inducing developmental neurotoxicity, in F1 larvae.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0269749120359935

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ‐aminobutyric acidergic anticonvulsant

Epilepsia

2020-08-14

Abstract

Objective:

Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms.

Methods:

We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action.

Results:

Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures.

 

Link to the publication : 

https://onlinelibrary.wiley.com/doi/full/10.1111/epi.16644

Acute and chronic effects of environmental realistic concentrations of clofibric acid in Danio rerio: Behaviour, oxidative stress, biotransformation and lipid peroxidation endpoints

Environmental Toxicology and Pharmacology

2020-08-14

Highlights

• Clofibric acid exposure in zebrafish affected behaviour through hypoactivity.
• Clofibric acid properties caused an increase in all biochemical biomarkers.
• The metabolism of this antihyperlipidemic seems to have modulated behavioral and biochemical results.
• No evidences were found in terms of histology endpoints being altered by clofibric acid.

Abstract

Due to their widespread use, pharmaceuticals can be metabolized, excreted and ultimately discarded in the environment, thereby affecting aquatic organisms. Lipid-regulating drugs are one of the most prescribed medications around the world, controlling human cholesterol levels, in more than 20 million patients. Despite this growing use of lipid-regulating drugs, particularly those whose active metabolite is clofibric acid, the potential toxicological effects of these pharmaceuticals in the environment is not fully characterized. This work intended to characterize the toxicity of an acute (120 hours post-fertilization) and chronic (60 days post-fertilization) exposures to clofibric acid in concentrations of 10.35, 20.7, 41.4, 82.8 and 165.6 µg L-1 in zebrafish (Danio rerio). The concentrations which were implemented in both exposures were based on predicted environmental concentrations for Portuguese surface waters. The acute effects were analysed focusing on behavioural endpoints (small and large distance travelled, swimming time and total distance travelled), biomarkers of oxidative stress (activity of the enzymes superoxide dismutase, Cu/Zn- and Mn SOD; catalase, CAT; glutathione peroxidase, Se- and total GPx), biotransformation (activity of glutathione S-transferases, GSTs) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Chronically exposed individuals were also histologically analysed for sex determination and gonadal developmental stages. In terms of acute exposure, significant alterations were reported, in terms of behavioural alterations (hypoactivity), followed by an overall increase in all tested biomarkers. Chronically exposed organisms did not show alterations in terms of sex ratio and maturation stages, suggesting that clofibric acid did not act as an endocrine disruptor. Moreover, the metabolism of clofibric acid resulted in increased levels of both forms of SOD activity, especially for animals exposed to higher levels of this drug. An increase of CAT activity was observed in fish exposed to low levels, and a decrease in those exposed to higher amounts of clofibric acid. Both GPx forms had their activities increased. The enzyme of biotransformation GSTs were increased at low levels of clofibric acid but inhibited at higher amounts of this substance. Lipid peroxidation levels were also changed, with an induction of this parameter with increasing amounts of clofibric acid. Changes also occurred in behavioural endpoints and patterns for control organisms and for those exposed to clofibric acid were significantly distinct, for all types (light and darkness) of exposure, and for the two analysed endpoints (small and large distance). Results from this assay allow inferring that clofibric acid can have an ecologically relevant impact in living organisms exposed to this substance, with putative impact on the metabolism of individuals, affecting their behaviour and ultimately their survival.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S1382668920301447

Developmental neurotoxicity fingerprint of silica nanoparticles at environmentally relevant level on larval zebrafish using a neurobehavioral-phenomics-based biological warning method

Science of the total environment

2020-08-21

Highlights

 

• Silica NPs at environmental level induced the negligible embryonic teratogenicity.
• Silica NPs at environmental level altered behavioral profiles determined by NBP.
• NBP are more sensitive and effective than FET to determine sub-teratogenic toxicity.

Abstract

Background

Larval zebrafish (Danio rerio) is not only an ideal vertebrate applied in Fish Embryos Toxicity (FET) test but also a well-accepted model in behavioral neurotoxicity research. By applying the commercial standard behavioral tracking system (Zebrabox), the locomotion profiles (neurobehavioral-phenomics) of larval zebrafish can be comprehensively monitored and systematically analyzed to probe ecotoxicological neurotoxicity of nano-pollutants at environmental relevant concentration level.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0048969720354073

Multiple Screening of Pesticides Toxicity in Zebrafish and Daphnia Based on Locomotor Activity Alterations

Biomolecules

2020-08-23

Abstract:

Pesticides are widely used to eradicate insects, weed species, and fungi in agriculture. The half-lives of some pesticides are relatively long and may have the dire potential to induce adverse effects when released into the soil, terrestrial and aquatic systems. To assess the potential adverse effects of pesticide pollution in the aquatic environment, zebrafish (Danio rerio) and Daphnia magna are two excellent animal models because of their transparent bodies, relatively short development processes, and well-established genetic information. Moreover, they are also suitable for performing high-throughput toxicity assays. In this study, we used both zebrafish larvae and water flea daphnia neonates as a model system to explore and compare the potential toxicity by monitoring locomotor activity. Tested animals were exposed to 12 various types of pesticides (three fungicides and 9 insecticides) for 24 h and their corresponding locomotor activities, in terms of distance traveled, burst movement, and rotation were quantified. By adapting principal component analysis (PCA) and hierarchical clustering analysis, we were able to minimize data complexity and compare pesticide toxicity based on locomotor activity for zebrafish and daphnia. Results showed distinct locomotor activity alteration patterns between zebrafish and daphnia towards pesticide exposure. The majority of pesticides tested in this study induced locomotor hypo-activity in daphnia neonates but triggered locomotor hyper-activity in zebrafish larvae. According to our PCA and clustering results, the toxicity for 12 pesticides was grouped into two major groups based on all locomotor activity endpoints collected from both zebrafish and daphnia. In conclusion, all pesticides resulted in swimming alterations in both animal models by either producing hypo-activity, hyperactivity, or other changes in swimming patterns. In addition, zebrafish and daphnia displayed distinct sensitivity and response against different pesticides, and the combinational analysis approach by using a phenomic approach to combine data collected from zebrafish and daphnia provided better resolution for toxicological assessment.

Vape flavourants dull sensory perception and cause hyperactivity in developing zebrafish embryos

Biology Letters

2020-08-24

E-cigarette use (vaping) during pregnancy has been increasing, and the potential exists for the developing brain in utero to be exposed to chemical constituents in the vape. Vapes come in over 7000 unique flavours with and without nicotine, and while nicotine is a known neurotoxicant, the effects of vape flavouring alone, in the absence of nicotine, on brain function are not well understood. Here, we performed a screen of vape aerosol extracts (VAEs) to determine the potential for prenatal neurotoxicity using the zebrafish embryo photomotor response (PMR)—a translational biosensor of neurobehavioural effects. We screened three commonly used aerosolized vape liquids (flavoured and flavourless) either with or without nicotine. No neurobehavioural effects were detected in flavourless, nicotine-free VAEs, while the addition of nicotine to this VAE dulled sensory perception. Flavoured nicotine-free VAEs also dulled sensory perception and caused hyperactivity in zebrafish embryos. The combination of flavour and nicotine produced largely additive effects. Flavoured VAEs without nicotine had similar neuroactive potency to nicotine. Together, using zebrafish PMR as a high throughput translational behavioural model for prenatal exposure, our results demonstrate that e-cigarette flavourants that we screened elicit neurobehavioural effects worthy of further investigation for long-term neurotoxic potential and also have the potential to modulate nicotine impact on the developing brain.

 

Link to the publication : 

https://royalsocietypublishing.org/doi/abs/10.1098/rsbl.2020.0361

Lobetyolin Efficiently Promotes Angiogenesis and Neuronal Development in Transgenic Zebrafish

Natural Product Communications

2020-08-31

Studies have shown that lobetyolin (LBT), a component of traditional Chinese herbal medicine, has many very good biological activities and functions. However, its side effects and toxicities have not been evaluated adequately. In this work, we investigated the effects of LBT in transgenic zebrafish. LBT treatments promoted angiogenesis and led to vascular morphological malformation in zebrafish embryos, although they were normal in appearance. Interestingly, our results indicated that LBT has a function of promoting nerve growth in the embryonic stage of zebrafish. We also treated the zebrafish with combretastatin A-4 (which resulted in neuronal apoptosis) and LBT simultaneously and found that LBT promoted nerve growth at higher concentrations. Taken together, our findings clearly display that LBT efficiently promotes angiogenesis, leading to vascular morphological malformation, has low toxicity, and notably promotes neuronal development in zebrafish.

 

Link to the publication : 

https://journals.sagepub.com/doi/full/10.1177/1934578X20937174

Genetic Risk Factors in prediction and treatment of Chronic Post-Surgical Pain

Universiteit Maastricht

2020-09-01

Concluding remarks :

This dissertation aimed to study the role of genetics in prediction and treatment of chronic postsurgical pain. In view of this, we started genetic screening and provided a genome-wide insight into the genetics of CPSP. This initial step identified the first potential risk loci and candidates for follow up studies. Furthermore, we have shown the important role of genetic variations related to dopaminergic neurotransmission in CPSP and preformed first steps in genetic and pharmacological screening in a zebrafish nociception model. With this dissertation a framework is provided for future studies and a combined and focussed clinical /pre-clinical approach aimed to develop personalized medicine in treatment of CPSP.

 

Link to the publication : 

https://www.researchgate.net/profile/Roel_Van_Reij/publication/343850803_Genetic_Risk_Factors_in_prediction_and_treatment_of_Chronic_Post-Surgical_Pain/links/5f44bf9992851cd302283738/Genetic-Risk-Factors-in-prediction-and-treatment-of-Chronic-Post-Surgical-Pain.pdf#page=137

Loss-of-function of the NOMO1 gene causes neuropsychiatric disorder-related phenotypes

Research Square

2020-09-01

Abstract Background:

Clinical genome-wide analysis identified NOMO1 in human chromosome 16p13.11 as a candidate gene associated with neuropsychiatric disorders such as autism, schizophrenia and epilepsy. However, the important contributions underlying NOMO1 deficiency resulting in neuropsychiatric disorders is not understand, and the molecular and pathogenesis mechanisms of nomo1 gene are unclear. Therefore, it is necessary to construct animal models to systematically study the effects of nomo1 deficiency on neuropsychiatric system and explore pathogenic molecular mechanism of diseases.

Methods:

We developed a viable vertebrate model of loss-of-function of nomo1 using CRISPR/Cas9 and studied the characterization of nomo1 mutant zebrafish. Phenotypic research was performed in developing nomo1 mutant zebrafish, including morphological measurements, behavioral tests, and functional mechanism analyses.

 

Link to the publication : 

https://assets.researchsquare.com/files/rs-54088/v1_stamped.pdf

Neurobehavioral effects of cyanobacterial biomass field extracts on zebrafish embryos and potential role of retinoids

Aquatic Toxicology

2020-09-06

Highlights

 

• Field cyanobacterial biomass extracts cause behavioural changes in zebrafish.
• The effects were localized in fractions with retinoid compounds content.
• The behavioural effect increased after combination of retinoids containing fractions.
• The effects on behaviour did not occur after exposure to the ATRA alone.
• Several genes related to the retinoid pathway were affected by the biomass extracts.

 

Abstract

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L−1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.

 

Link to the publication : https://www.sciencedirect.com/science/article/abs/pii/S0166445X20303635

Biomass-derivedcellulosenanoparticlesdisplayconsiderableneurotoxicity in zebrafish

International Journal of Biological MacroMolecules

2020-10-05

Abstract

The widespread use of nanomaterials poses a great threat to human living environments. Among them, biomass-derived cellulose nanoparticle (CN) is one of the widely used nanomaterials. To date, the toxicity of CNs during embryonic development remains undetermined. In this study, we exposed zebrafish embryos to cellulose nanofibrils (CNFs) and cellulose nanocrystals (CNCs) to evaluate the toxicity of these CNs. Exposure to CNFs or CNCs below 30 mg/ml exhibited no dose-dependent increases in malformation and mortality in zebrafish embryos. Then we demonstrated that CNs were highly enriched in zebrafish embryo via imaging analyses of embryos treated with FITC-coupled CNCs. In addition, we found that CNF or CNC exposure resulted in compromised motor ability of zebrafish larva. Furthermore, it was revealed that the differentiation and the morphogenesis of motor neurons were significantly interrupted. While, blood vessels were normally patterned, suggesting the specific neurotoxicity of these nanomaterials. Transcriptome sequencing assay showed that the neurotoxicity of CNs in the motor neurons might be attributed to the expression alteration of neural genes. In summary, we discovered the neurotoxicity of CNs for the first time.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0141813020346559

Zebrafish and mouse models for anxiety evaluation – A comparative study with xanthotoxin as a model compound

Brain Research Bulleting

2020-10-10

Highlights

• Xanthotoxin modifies anxiety-related behavior in two animal models: zebrafish and mice.
• Xanthotoxin exerted reversed U-shape effect on anxiety behaviors in both models.
• The predictive power of zebrafish for behavioral research of natural compounds was proven.

Abstract

The ever-present trend for introducing new drugs of natural origin with anxiolytic properties meets healthcare needs of the population, whose almost 34 % struggles with anxiety-related disorders. At the same time, animal assays that could serve as fast and reliable models of anxiety-like behaviors are of great interest to scientists. Thus, the aim of the present study was to evaluate the utility of the zebrafish model for assessing the influence of natural compounds on anxiety in comparison with the well-known mouse model. Secondly, this study is also the first attempt to investigate the influence of a naturally occurring metabolite, i.e. xanthotoxin, on anxiety-related behaviors. The anxiety level in zebrafish was assessed by measuring thigmotaxis, a specific animal behavior to move closer to the boundaries of an open area and to avoid its center. In mice, the elevated plus maze test was chosen to study anxiety-related behaviors. Our results show that xanthotoxin exerted reversed U-shape effect on anxiety behaviors in both models. The similar pattern of xanthotoxin-induced anxiety-related behaviors in both animal models not only confirms the pharmacological properties of xanthotoxin but also proves the predictive power of the zebrafish model for behavioral research of natural compounds.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0361923020306596

Rods Contribute to Visual Behavior in Larval Zebrafish

Investigative Ophthalmology & Visual Science - Retina

2020-10-13

Abstract

Purpose: Although zebrafish rods begin to develop as early as 2 days postfertilization (dpf), they are not deemed anatomically mature and functional until 15 to 21 dpf. A recent study detected a small electroretinogram (ERG) from rods in a cone mutant called no optokinetic response f (nof) at 5 dpf, suggesting that young rods are functional. Whether they can mediate behavioral responses in larvae is unknown.

Methods: We first confirmed rod function by measuring nof ERGs under photopic and scotopic illumination at 6 dpf. We evaluated the role of rods in visual behaviors using two different assays: the visual-motor response (VMR) and optokinetic response (OKR). We measured responses from wild-type (WT) larvae and nof mutants under photopic and scotopic illuminations at 6 dpf.

Results: Nof mutants lacked a photopic ERG. However, after prolonged dark adaptation, they displayed scotopic ERGs. Compared with WT larvae, the nof mutants displayed reduced VMRs. The VMR difference during light onset gradually diminished with decreased illumination and became nearly identical at lower light intensities. Additionally, light-adapted nof mutants did not display an OKR, whereas dark-adapted nof mutants displayed scotopic OKRs.

Conclusions: Because the nof mutants lacked a photopic ERG but displayed scotopic ERGs after dark adaptation, the mutants clearly had functional rods. WT larvae and the nof mutants displayed comparable scotopic light-On VMRs and scotopic OKRs after dark adaptation, suggesting that these responses were driven primarily by rods. Together, these observations indicate that rods contribute to zebrafish visual behaviors as early as 6 dpf.

 

Link to the publication :

https://iovs.arvojournals.org/article.aspx?articleid=2770905

Systematic Assessment of Exposure Variations onObserved Bioactivity in Zebrafish Chemical Screening

Toxics

2020-10-14

Abstract:

The embryonic zebrafish is a powerful tool for high-throughput screening of chemicals.While this model has significant potential for use in safety assessments and chemical prioritization,a lack of exposure protocol harmonized across laboratories has limited full model adoption. To assessthe potential that exposure protocols alter chemical bioactivity, we screened a set of eight chemicalsand one 2D nanomaterial across four different regimens: (1) the current Tanguay laboratory’sstandard protocol of dechorionated embryos and static exposure in darkness; (2) exposure withchorion intact; (3) exposure under a 14 h light: 10 h dark cycle; and (4) exposure with daily chemicalrenewal. The latter three regimens altered the concentrations, resulting in bioactivity of the testagents compared to that observed with the Tanguay laboratory’s standard regimen, though notdirectionally the same for each chemical. The results of this study indicate that with the exceptionfor the 2D nanomaterial, the screening design did not change the conclusion regarding chemicalbioactivity, just the nominal concentrations producing the observed activity. Since the goal of tierone chemical screening often is to differentiate active from non-active chemicals, researchers couldconsider the trade-offs regarding cost, labor, and sensitivity in their study design without alteringhit rates. Taken further, these results suggest that it is reasonably feasible to reach agreement on astandardized exposure regiment, which will promote data sharing without sacrificing data content

 

See Publication : https://www.mdpi.com/2305-6304/8/4/87/htm

Backbone Cyclization Turns a Venom Peptide into a Stable andEquipotent Ligand at Both Muscle and Neuronal Nicotinic Receptors

Journal of Medicinal Chemistry

2020-10-16

Abstract

Abstract Image

Venom peptides are promising drug leads, but their therapeutic use is often limited by stability and bioavailability issues. In this study, we designed cyclic analogues of α-conotoxin CIA, a potent muscle nicotinic acetylcholine receptor (nAChR) blocker with a significantly lower affinity at the neuronal α3β2 subtype. Remarkably, all analogues retained the low nanomolar activity of native CIA toward muscle-type nAChRs but showed greatly improved resistance to degradation in human serum and, surprisingly, displayed up to 52-fold higher potency for the α3β2 neuronal nAChR subtype (IC50 1.3 nM). Comparison of nuclear magnetic resonance-derived structures revealed some differences that might explain the gain of potency at α3β2 nAChRs. All peptides were highly paralytic when injected into adult zebrafish and bath-applied to zebrafish larvae, suggesting barrier-crossing capabilities and efficient uptake. Finally, these cyclic CIA analogues were shown to be unique pharmacological tools to investigate the contribution of the presynaptic α3β2 nAChR subtype to the train-of-four fade.

 

Link to the publication :

https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.0c00957#

Bioconcentration and developmental neurotoxicity of novelbrominatedflame retardants, hexabromobenzene andpentabromobenzene in zebrafish

Environmental Pollution

2020-10-21

abstract

Theflame retardants hexabromobenzene (HBB) and pentabromobenzene (PBB) have been extensivelyused and become ubiquitous pollutants in the aquatic environment and biota, but their potential toxiceffects on wildlife remained unknown. In this study, by using zebrafish (Danio rerio) as a model, thebioconcentration and developmental neurotoxicity were investigated. Zebrafish embryos were exposedto HBB and PBB (0, 30, 100 and 300mg/L) from 2 until 144 h post-fertilization (hpf). Chemical analysisshowed bioconcentrations of both chemicals, while HBB is readily metabolized to PBB in zebrafish larvae.Embryonic exposure to both chemicals did not cause developmental toxicity, but induced locomotorbehavioral anomalies in larvae. Molecular docking results indicated that both chemicals could bind tozebrafish acetylcholinesterase (AChE). Furthermore, HBB and PBB significantly inhibited AChE activities,accompanied by increased contents of acetylcholine and decreased choline in larvae. Downregulation ofthe genes associated with central nervous system (CNS) development (e.g.,mbp,a1-tubulin,gfap,shha)aswell as the corresponding proteins (e.g., Mbp,a1-Tubulin) was observed, but gap-43 was upregulated atboth gene and protein levels. Together, our results indicate that both HBB and PBB exhibit developmentalneurotoxicity by affecting various parameters related to CNS development and indications for futuretoxicological research and risk assessment of the novel brominatedflame retardants.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0269749120365842

The oxidative stress responses caused by phthalate acid esters increases mRNA abundance of base excision repair (BER) genes in vivo and in vitro

Ecotoxicology and Environmental Safety

2020-10-22

Highlights

• PAEs effects the development of zebrafish embryos.
• PAEs decreases cell viability in HEK293T cells.
• PAEs increases the production of ROS and decreases SOD activity in vivo and in vitro.
• PAEs increases pro-apoptotic gene expression and decreases anti-apoptotic gene expression.
• BER pathway were activated in response to PAEs exposure (with the exceptions of larvae at 50 μM MEHP) in vivo and vitro.

Abstract

The base excision repair (BER) pathway is an important defense response to oxidative DNA damage. It is known that exposures to phthalate esters (PAEs), including Dibutyl phthalate (DBP), Mono-(2-ethylhexyl) phthalate (MEHP), and Di-(2-ethylhexyl) phthalate (DEHP), cause reactive oxygen species-induced DNA damage and oxidative stress. Here, we determined the mRNA levels of BER pathway-related genes (ogg1, nthl1, apex1, parp1, xrcc1, lig3, ung, pcna, polb, pold, fen1, and lig1), pro-apoptotic gene (bax), and apoptotic suppressor gene (bcl2) in different PAEs-exposed zebrafish larvae and HEK293T cells. Further investigations were performed to examine reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD) activity, developmental toxicity, and cell viability after PAEs exposure in vivo and in vitro. The results showed that PAEs exposure can induce developmental abnormalities in zebrafish larvae, and inhibit cell viability in HEK293T cells. Additionally, we found that PAEs exposure results in the accumulation of ROS and the inhibition of SOD activation in vivo and in vitro. Notably, the mRNA levels of BER pathway-related genes (OGG1, NTHL1, APEX1, XRCC1, UNG, POLB, POLD, FEN1) were significantly upregulated after DBP or MEHP exposure, whereas the mRNA levels of NTHL1, UNG, POLB, POLD, and FEN1 were significantly altered in DEHP-treated HEK293T cells. In zebrafish, the mRNA levels of ogg1, pcna, fen1 and lig1 genes were increased after DBP or DEHP exposure, whereas the mRNA levels of nthl1, apex1, parp1, lig3, pcna and polb were decreased after MEHP exposure, respectively. Thus, our findings indicated that PAEs exposure can induce developmental toxicity, cytotoxicity, and oxidative stress, as well as activate BER pathway in vivo and in vitro, suggesting that BER pathway might play critical roles in PAEs-induced oxidative stress through repairing oxidative DNA damage.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651320313622

Assessing the effects of textile leachates in fish using multiple testing methods: From gene expression to behavior

Ecotoxicology and Environmental Safety

2020-10-24

ABSTRACT

The textile industry, while of major importance in the world economy, is a toxic industry utilizing and emitting thousands of chemical substances into the aquatic environment. The aim of this project was to study the potentially harmful effects associated with the leaching of chemical residues from three different types of textiles: sportswear, childrens bath towels, and denim using different fish models (cell lines, fish larvae and juvenile fish). A combination of in vitro and in vivo test systems was used. Numerous biomarkers, ranging from gene expression, cytotoxicity and biochemical analysis to behavior, were measured to detect effects of leached chemicals. Principle findings indicate that leachates from all three types of textiles induced cytotoxicity on fish cell lines (RTgill-W1). Leachates from sportswear and towels induced mortality in zebrafish embryos, and chemical residues from sportswear reduced locomotion responses in developing larval fish. Sportswear leachate increased Cyp1a mRNA expression and EROD activity in liver of exposed brown trout. Leachates from towels induced EROD activity and VTG in rainbow trout, and these effects were mitigated by the temperature of the extraction process. All indicators of toxicity tested showed that exposure to textile leachate can cause adverse reactions in fish. These findings suggested that chemical leaching from textiles from domestic households could pose an ecotoxicological threat to the health of the aquatic environment.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651320313609

Increased anxiety was found in serpini1 knockout zebrafish larval

Biochemical and Biophysical Research Communications

2020-11-06

Highlights

• Neuroserpin deficient zebrafish showed increased anxiety.
• Neuroserpin deficient zebrafish displayed extension of motoneurons axon defect at 24hpf.
Serpini1 knockout zebrafish resulted in affected expression of neurodegeneration-related genes.

Abstract

Serpini1, which encodes neuroserpin, has been implicated in the development and normal function of the nervous system. Mutations in serpini1 cause familial encephalopathy, a rare neurodegenerative disorder characterized with neuroserpin inclusion bodies. However, function of neuroserpin in the nervous system is not fully understood. In this study, we generated a novel serpini1 mutant zebrafish model to investigate the loss of function of neuroserpin. Serpini1- deficient mutation was created with the CRISPR/Cas9 technique. No severe morphological characteristics were found in serpini1- deficient zebrafish. Serpini1−/− zebrafish larvae did not cause locomotor defects but displayed anxiety-like behavior. Extension of motoneurons axon defect was observed in serpini1−/− zebrafish. Furthermore, RNA-sequencing analysis revealed that loss of serpini1 resulted in affected expression of neurodegeneration-related genes.

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0006291X20319653

Waterborne uranium causes toxic effect and thyroid disruption in zebrafish larvae

Ecotoxicology and Environmental Safety

2020-11-09

Highlights

• The effects of uranium on development and thyroid system of zebrafish were assessed.
• Uranium could bioaccumulate in zebrafish bodies.
• Uranium affected the development and locomotor behavior of zebrafish.
• The levels of thyroid hormones in zebrafish were changed after uranium exposure.
• Negative feedback loops through HPT axis-related genes regulated thyroid hormones.

Abstract

Uranium is a radioactive element that is widely present in aquatic environment. However, limited knowledge is available about the effect of uranium on thyroid system, which plays a key role in the development of animals. In this study, zebrafish embryos were exposed to different environmentally relevant concentrations of uranium (2, 20 and 100 μg/L) for 120 h. The bioaccumulation, developmental toxicities, changes of thyroid hormones (THs) and key genes related to the hypothalamic–pituitary–thyroid (HPT) axis in larvae were analyzed after exposure. Results showed that uranium could bioaccumulate in zebrafish larvae, with the bioconcentration factors ranging from 49.6 to 523. Consequently, significant developmental toxicities and changes in locomotor activities were observed with a concentration-dependent manner. The levels of triiodothyronine (T3) levels in larvae were substantially decreased, whereas those of thyroxine (T4) were increased in fish bodies. The levels of THs were regulated by the negative feedback loops through HPT axis related genes, most of which (NIS, Deio1, Deio2, TRα, TSHβ and UGT1ab) were significantly depressed after exposure to uranium. Our results suggest the potential toxicities and thyroid disruption of uranium on zebrafish, which would provide baseline data set for better understanding the impact of waterborne uranium on aquatic organisms and the associated mechanisms. This study also highlights the key role of thyroid disruption in the ecological risk assessment of uranium pollution.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651320314226

Zygotic Venlafaxine Exposure Impacts Behavioral Programming by Disrupting Brain Serotonin in Zebrafish

Environmental Science and Technology

2020-11-17

Abstract

Abstract Image

The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is present in surface waters downstream of wastewater treatment plants. We previously showed that zygotic venlafaxine deposition alters larval behavior in zebrafish (Danio rerio), but the mechanisms were unknown. Here we tested the hypothesis that venlafaxine disrupts central serotonergic development, leading to impaired behavioral responses in zebrafish larvae. This was tested by microinjecting embryos with venlafaxine immediately after fertilization and performing spatial distribution of serotonin immunoreactivity, as well as characterizing target genes involved in serotonin turnover in the zebrafish brain. We provide evidence that venlafaxine exposure reduces serotonin immunoreactivity and tyrosine hydroxylase-positive cell populations in specific larval brain regions, and this corresponded with reduced larval activity observed in the drug-exposed group. Lowered serotonin was not due to either reduced synthesis or increased breakdown capacity. However, co-injection of serotonin alongside venlafaxine in embryos recovered brain serotonin immunoreactivity, tyrosine hydroxylase-positive cell populations, and rescued venlafaxine-mediated behavioral changes. Overall, our results demonstrate for the first time that early life exposure to venlafaxine perturbs brain development, which may be due to reduced serotonin, leading to altered larval behavior in zebrafish.

Link to the publication :

https://pubs.acs.org/doi/abs/10.1021/acs.est.0c06032