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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Abstract

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

Introduction

In recent years more and more environmental pollutants have been proved neurotoxic1,2,3,4. However, the assessment of neurotoxicity in vivo after exposure to environmental pollutants is not as easy as that of endocrine disruption or developmental toxicity. In addition, early exposure to pollutants, especially at environmentally relevant doses, has attracted increasing attention in toxicity studies5,6,7,8.

Zebrafish is being established as an animal model fit for neurotoxicity studies during early development after exposure to environmental pollutants. Zebrafish are vertebrates that develop faster than other species after fertilization. The larvae do not need to be fed because the nutrients in the chorion are enough for sustain them for 7 days postfertilization (dpf)9. Larvae come out from the chorion at ~2 dpf and develop behaviors such as swimming and turning that can be observed, tracked, quantified, and analyzed automatically using behavior instruments10,11,12,13 starting at 3-4 dpf14,15,16,17,18. In addition, high-throughput tests can also be realized by behavior instruments. Thus, zebrafish larvae are an outstanding model for the neurobehavioral study of environmental pollutants19. Here, a protocol is offered using high-throughput monitoring to study the neurobehavioral toxicity of environmental pollutants on zebrafish larvae under light stimuli.

Our lab has studied the neurobehavioral toxicity of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47)20,21, 6'-Hydroxy/Methoxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH/MeO-BDE-47)22, deca-brominated diphenyl ether (BDE-209), lead, and commercial chlorinated paraffins23 using the presented protocol. Many labs also use the protocol to study the neurobehavioral effects of other pollutants on larvae or adult fish24,25,26,27. This neurobehavioral protocol was used to help provide mechanistic support showing that low-dose exposure to bisphenol A and replacement bisphenol S induced premature hypothalamic neurogenesis in embryonic zebrafish27. In addition, some researchers optimized the protocol to perform corresponding studies. A recent study eliminated the toxicity of amyloid beta (Aβ) in an easy, high-throughput zebrafish model using casein-coated gold nanoparticles (βCas AuNPs). It showed that βCas AuNPs in systemic circulation translocated across the blood-brain barrier of zebrafish larvae and sequestered intracerebral Aβ42, eliciting toxicity in a nonspecific, chaperone-like manner, which was supported by behavioral pathology28.

Locomotion, path angle, and social activity are three neurobehavioral indicators used to study the neurotoxicity effects of zebrafish larvae after exposure to pollutants in the presented protocol. Locomotion is measured by the swimming distance of larvae and can be damaged after exposure to pollutants. Path angle and social activity are more closely related with the function of the brain and the central nervous system29. The path angle refers to the angle of the path of animal motion relative to the swimming direction30. Eight angle classes from ~-180°-~+180° are set in the system. To simplify the comparison, six classes in the final outcome are defined as routine turns (-10° ~0°, 0° ~+10°), average turns (-10° ~-90°, +10° ~+90°), and responsive turns (-180° ~-90°, +90° ~+180°) according to our previous studies21,22. Two-fish social activity is fundamental of group shoaling behavior; here a distance of < 0.5 cm between two larvae valid is defined as social contact.

The protocol presented here demonstrates a clear process for studying neurobehavioral effects on zebrafish larvae and provides a way to reveal the neurotoxicity effects of various substances or pollutants. The protocol will benefit researchers interested in studying the neurotoxicity of environmental pollutants.

Protocol

The protocol is in accordance with guidelines approved by the Animal Ethics Committee of Tongji University.

1. Zebrafish embryo collection

  1. Put two pairs of healthy adult Tubingen zebrafish into the spawning box on the night before exposure, keeping the sex ratio at 1:1.
  2. Remove the adult fish back to the system 30-60 min after daylight the next morning.
  3. Remove the embryos out of the spawning box.
  4. Rinse the embryos with system water.
  5. Transfer the embryos into a glass Petri dish (9 cm diameter) with enough system water.
  6. Observe the embryos under the microscope and select healthy embryos for later exposure.
    NOTE: Healthy embryos are usually transparent with light golden color under the microscope. The unhealthy embryos are usually pale and clumped together as observed under the microscope.

2. Preparation before exposure

  1. Prepare the Hanks' solution according to the guidelines of the zebrafish book31.
    NOTE: The Hanks' solution includes 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4.2 mM NaHCO3.
  2. Dilute the Hanks' solution to 10% Hanks' solution using sterile water.
  3. Add 1 mL of DMSO into 999 mL of 10% Hanks' solution to make a control solution of 10% Hanks' solution including 0.1% DMSO.
    NOTE: The next steps use BDE-47 as an example of an exposure solution.
  4. Dissolve 5 mg of the neat BDE-47 in 1 mL of 100% DMSO to make a standard exposure solution of 5 mg/mL.
  5. Vortex the 5 mg/mL solution for 1 min to completely dissolve the BDE-47 in the DMSO.
  6. Transfer 10 µL of the 5 mg/mL solution to a 12 mL brown glass bottle.
  7. Add sterilized water to a final volume of 10 mL to make the concentration of BDE-47 exposure solution 5 mg/L and DMSO ratio at 0.1%, then vortex for 1 min.
  8. Transfer 10 µL and 100 µL of the 5 mg/L solution into two 100 mL volumetric flasks respectively.
  9. Add 10% Hanks' solution including 0.1% DMSO (prepared in step 2.3) to 100 mL to make the final concentrations of the BDE-47 exposure solutions 5 µg/L and 50 µg/L, respectively.
  10. Transfer the solutions into 100 mL brown glass bottles and store them at 4 °C.

3. Exposure of embryos

  1. Transfer ~50 embryos into each of the three glass Petri dishes (6 cm diameter) 3-5 hours post fertilization (hpf).
  2. Use a 1 mL pipette tip to blot the system water around the embryos.
  3. Use a pipette to transfer the control and two BDE-47 exposure solutions (control, 5 µg/L, 50 µg/L) into the three glass Petri dishes, respectively.
  4. Shake the glass Petri dishes gently one by one to make the embryos disperse in the bottom of the plate.
  5. Put the glass Petri dishes into the light incubator under 28.5 °C.
  6. Renew half of the exposure solutions every 24 h until 5 dpf.
  7. Check the dead embryos of every group on 1 dpf and 2 dpf and calculate the death rate.
  8. Check the incubated embryos of every group on 2 dpf and 3 dpf and calculate the hatchability.
  9. Check the deformity of the larvae every day after they come out from the chorion and calculate the deformity rate of every group.
    NOTE: The deformity indicators include pericardial cyst, spinal curvature, tail curvature, among others factors32.

4. Preparation for the behavior test

  1. Prepare a 48 well microplate for the locomotion and path angle test and three 6 well microplates for the social activity test on the morning of 5 dpf.
  2. Transfer 800 µL of exposure solution into every well of the 48 well microplate.
    NOTE: Use 16 wells for every group (i.e., the control solution, 5 µg/L, and 50 µg/L group).
  3. Use a 1 mL pipette tip to transfer 200 µL of exposure solution with one larva from the glass Petri dish into one well of the 48 well microplate.
  4. Transfer 4 mL of exposure solution into every well of the 6 well microplate.
    NOTE: Use one 6 well microplate for every group.
  5. Use a 1 mL pipette tip to transfer 200 µL of exposure solution with two larvae into each well of the 6 well microplate.
    NOTE: Every group has six repeating groups.
  6. Make sure the temperature of the test room is 28 °C 2 h before the test.
    NOTE: Behavioral tests are usually performed in the afternoon.

5. Behavioral test

  1. Locomotion and path angle test
    1. Click the launcher icon on the computer desk to open the software (see Table of Materials) that controls the high-throughput monitoring enclosure to start the program.
    2. Choose the "Tracking, Rotations, Path Angles" module to enter the operating interface.
    3. Transfer the 48 well microplate prepared in step 4.3 to the recording platform and pull down the cover.
    4. Click the "File" "Generate Protocol" button in turn in the software to begin generating a new protocol.
    5. Input "48" in the "Location count" position and click the "OK" button.
    6. Click the "Parameters" "Protocol Parameters" "Time" button in turn in the software. Set the experiment duration for 1 h and 10 min and set the integration period to 60 s33.
    7. Draw the detected areas.
      1. Select the elliptical shape and draw the first circle around the first top left well.
      2. Select the circle, click the "Copy" "Top-Right Mark" "Paste" "Select" button in turn, and use the mouse to drag the copied circle to the top right well.
      3. Select the circle, click the "Copy" "Bottom Mark" "Paste" "Select" button in turn, and use the mouse to drag the copied circle to the bottom right well.
      4. Click the "Build" "Clear marks" button in turn.
        NOTE: The system will automatically draw every other well of the plate. The newly created areas should perfectly fit each well between the actual fish and its reflection on the side of the well.
    8. Click the "Draw Scale" button, draw a calibration line on the screen (a diagonal path or parallel to the side of the microplate), enter its length and set the "Unit". Then click the "Apply to group" button.
    9. Set the animal color at "Black" in the software.
    10. Set the detection threshold at 16-18 to allow the detection of the animals.
    11. Input inactive/small and small/large speed at 0.5 cm/s and 2.5 cm/s respectively.
    12. Set the path angle classes. Input "-90, -30, -10, 0, 10, 30, and 90" to make path angle classes from -180°-+180°.
    13. Set the light conditions
      1. Click the "Parameters" "Light driving" "Uses one of the 3 triggering methods below" "Enhanced stimuli" button in turn to set the light conditions.
      2. Choose the "Edge" button, then set a dark period of 10 min, followed by three cycles of alternating 10 min light and dark periods.
    14. Save the protocol and turn down the light of the test room.
    15. Acclimate the larvae in the system for 10 min and click the "Experiment" "Execute" button in turn, then choose the folder where the experiment files are saved and enter the result name.
    16. Click the "Background" "Start" button in turn to start the test.
    17. Click the "Experiment" "Stop" button in turn to stop the experiment when the test ends.
      NOTE: The system shows the data tested when the system stops. The data include the tracked distance at three speed classes and path angle numbers at eight angle classes of every minute. For the locomotion test in the presented example, the total distance in every light period (10 min) is calculated and the difference between the control group and the treatment groups compared.
    18. Transfer the 48 well microplate back to the light incubator for other experiments.
  2. Social activity test
    1. Click on the launcher icon on the computer desk to open the software that controls the high-throughput monitoring enclosure to start the program.
    2. Choose the "Social Interactions" module to enter the operating interface.
    3. Transfer the prepared 6 well microplate (control group) in step 4.4 to the recording platform and pull down the cover.
    4. Click the "File" "Generate Protocol" button in turn in the software to begin generating a new protocol.
    5. Input "6" in the "Location count" position and click the "OK" button.
    6. Click the "Parameters" "Protocol Parameters" "Time" button in turn in the software. Set the experiment duration for 1 h and 10 min and set the integration period to 60 s.
    7. Draw the detected areas.
      1. Select the elliptical shape and draw the first circle around the first top left well.
      2. Select the circle, click the "Copy" "Top-Right Mark" "Paste" "Select" button in turn, and use the mouse to drag the copied circle to the top-right well.
      3. Select the circle, click the "Copy" "Bottom Mark" "Paste" "Select" button in turn, and use the mouse to drag the copied circle to the bottom right well.
      4. Click the "Build" "Clear marks" button in turn.
        NOTE: The newly created areas should fit each well perfectly and between the actual larvae and its reflection on the side of the well.
    8. Click the "Draw Scale" button, draw a calibration line in the screen (a diagonal path or parallel to the side of the microplate), enter its length and set the "Unit". Then click the "Apply to group" button.
    9. Set the detection threshold at 16-18 to allow the detection of the animals.
    10. Click the "Black animal" button in the software.
    11. Choose the "Distance Threshold" button and input "5" in the software.
    12. Set the light conditions.
      1. Click the "Parameters" "Light driving" "Uses one of the 3 triggering methods below" "Enhanced stimuli" button in turn to set the light conditions.
      2. Choose the "Edge" button, then set a dark period of 10 min, followed by three cycles of alternating 10 min light and dark periods.
    13. Save the protocol and turn down the light of the room.
    14. Acclimate the larvae in the system for 10 min and click the "Experiment" "Execute" button in turn, then choose the folder where the experiment files are saved and enter the result name.
    15. Click the "Background" "Start" button in turn to start the test.
    16. Click the "Experiment" "Stop" button in turn to stop the experiment in the software when the test ends.
      NOTE: The system shows the data tested when the system stops.
    17. Transfer the 6 well microplate (control group) back to the light incubator for other experiments.
    18. Transfer the 6 well microplates (5 µg/L and 50 µg/L groups) in turn to the recording platform and repeat the steps from 5.2.4 to 5.2.17 by ordinal.

6. Data analysis

  1. Open the spreadsheet file in the locomotion and path angle results.
  2. Select the three distance columns (inadist, smldist, lardist) and add them up.
    NOTE: The data of inadist, smldist, and lardist mean different distances recorded by the system in different speed classes (inactive/small/large), respectively.
  3. For every 10 minute light period sum up the distance of every well, calculate the average distance of 16 wells, and compare the data of the three groups under light stimuli.
  4. For every 10 minute light period sum up the angle number of every well in every light duration from cl01 to cl08 in turn, and compare the data of the three groups under light stimuli.
    NOTE: The data of columns from cl01 to cl08 mean different path angle numbers recorded by the system in different path angles, respectively.
  5. Open the spreadsheet file in the social activity results.
  6. Select the contct and contdur columns, and for every 10 min light period sum up the social times and their duration for every well.
  7. Calculate the average social times and duration of one group in every light duration and compare the data of the three groups under light stimuli.

Results

Here, we describe a protocol for studying the neurobehavioral effects of environmental pollutants using zebrafish larvae under light stimuli. The locomotion, path angle, and social activity tests are defined in the introduction. The setup of the microplates in the locomotion and path angle tests and the images of the software are shown below. In addition, our own research results are presented as examples. Two studies present the locomotion and path angle effects after exposure to BDE-47 ...

Discussion

This work provides a detailed experimental protocol to evaluate the neurotoxicity of environmental pollutants using zebrafish larvae. Zebrafish go through the process from embryos to larvae during the exposure period, which means that good care of the embryos and larvae is essential. Anything that affects the development of the embryos and larvae can influence the final result. Here the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the whole assay.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors are grateful for the financial support by the National Natural Science Foundation of China (21876135 and 21876136), the National Major Science and Technology Project of China (2017ZX07502003-03, 2018ZX07701001-22), the Foundation of MOE-Shanghai Key Laboratory of Children's Environmental Health (CEH201807-5), and Swedish Research Council (No. 639-2013-6913).

Materials

NameCompanyCatalog NumberComments
48-well-microplateCorning3548Embyros housing
6-well-microplateCorning3471Embyros housing
BDE-47AccuStandard5436-43-1Pollutant
DMSOSigma67-68-5Cosolvent
MicroscopeOlympusSZX 16Observation instrument
PipetteEppendorf3120000267Transfer solution
ZebraboxViewpointZebraBoxBehavior instrument
ZebrafishShanghai FishBio Co., Ltd.TubingenZebrafish supplier
ZebraLabViewpointZebraLabBehavior software

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