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Environment

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Published: February 5, 2020 doi: 10.3791/60818
Automatic Translation
1,2, 2, 3, 2,4, 2,4
1State Key Laboratory of Marine Geology, Tongji University, 2Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, 3Shanghai Collaborative Innovation Centre for WEEE Recycling, WEEE Research Center of 10 Shanghai Polytechnic University, 4Shanghai Institute of Pollution Control and Ecological Security

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.

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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.

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Representative 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 and 6-OH/MeO-BDE-47. The third study presents the effects of four commercial chlorinated paraffins on social behavior.

The setup of the 48 well microplate and the movement locus of the larvae in the locomotion and path angle test.
Three groups, including one control group and two treatment groups, were used in the protocol. Because every group can have 16 animals, the system can be used to perform high-throughput tests of locomotion and path angle in one microplate. Figure 1 shows one larva treated with the control solution, 5 µg/L solution, and 50 µg/L solution in each well of the first, middle, and last two rows, respectively.

Figure 1 also shows all movement loci of the larvae in the locomotion and path angle tests. The system tracked the locomotion of the larvae and calculated the swimming distance at different speed classes. The system calculated the path angle numbers of larvae at different path angle classes. Researchers can analyze the data recorded by the system in their own ways.

Figure 1
Figure 1: The setup of the 48 well microplate and the movement loci of the larvae in the locomotion and path angle test. A1-A8, B1-B8 = the control group; C1-C8, D1-D8 = the 5 µg/L group; E1-E8, F1-F8 = the 50 µg/L group. The black color tracking line means inactivity or small movements; the green color tracking line means normal movements; and the red color tracking line means large movements. Please click here to view a larger version of this figure.

The 6 well microplate in the social activity test.
Figure 2 shows a 6 well microplate in the social activity testing process. Every well had two larvae, and the system recorded the distance between the two larvae during the whole testing process. The system recorded the social activity numbers and duration in the set testing time (1 min in this protocol).

Figure 2
Figure 2: The 6 well microplate in the social activity test. Every well had two larvae. The yellow line means the distance between two animals is < 0.5 cm; the red line means the distance between two animals is > 0.5 cm. Please click here to view a larger version of this figure.

BDE-47 exposure affected locomotion in zebrafish larvae at 5 dpf.
As shown in Figure 3, the highest concentration group of BDE-47 produced pronounced hypoactivity during the dark period. However, there were no observed changes due to BDE-47 exposure during the light periods.

Figure 3
Figure 3: Effects of BDE-47 exposure on locomotion of larval zebrafish at 5 dpf. Locomotion (distance moved measured in cm) was recorded in alternating periods of darkness and light for a total duration of 70 min. Solid and open bars at the bottom indicate dark and light periods, respectively. Data are presented as mean ± SEM (*p < 0.05 compared with the control group). This figure has been modified from Zhao et al.17 with permission. Please click here to view a larger version of this figure.

6-OH/MeO-BDE-47 exposure affected the path angles of zebrafish larvae at 5 dpf.
As shown in Figure 4, the high concentration group of 6-OH-BDE-47 performed fewer routine turns and average turns at 5 dpf. However, more responsive turns were induced by 6-MeO-BDE-47 exposure groups.

Figure 4
Figure 4: Effects of 6-OH/MeO-BDE-47 on the path angle of larval zebrafish during the dark period. Data are presented as the mean ± SEM (*p < 0.05 compared with control). This figure has been modified from Zhang et al.18 with permission. Please click here to view a larger version of this figure.

CPs exposure affected social activity of zebrafish larvae at 5 dpf.
As shown in Figure 5, the social behaviors of zebrafish larvae were influenced by three CP products. The social activity was stimulated by CP-70 and the short-chain CP-52b. The long chain CP-52a shortened the duration per contact of the larvae.

Figure 5
Figure 5: Effects of CPs on the average social duration per contact in different light/dark periods. (A) CP-42, (B) CP-52a, (C) CP-52b, (D) CP-70. The data are presented as the mean ± SEM (*p < 0.05 compared with the control). This figure has been modified from Yang et al.19 with permission. Please click here to view a larger version of this figure.

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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.

For the culture environment, zebrafish embryos and larvae live under a stable temperature of ~28 °C. In this work, a light incubator that can set the light conditions automatically and keep the temperature stable is used to house the embryos and larvae. The embryos do not come out from the chorion at 1 dpf and 2 dpf, so care should be taken to avoid damaging the unhatched embryos when renewing the exposure solution. Also, the ratio of DMSO in the solution should be under 0.1%34,35, and the fresh exposure solution should be at 28 °C before it is used for renewal.

The process of selecting embryos before exposure is also a key factor for the success of the experiment. Choosing healthy embryos developing concurrently for every group guarantees the accuracy of toxicity assessment. Zebrafish can live without food during the first 7 days after fertilization, so it is best to not feed the embryos or larvae during the whole exposure period because food could influence the final result. Also, it is best to prepare the exposure solution fresh when needed.

During the behavior test, it is essential to offer the larvae enough time to adapt to the environment of the high-throughput monitoring enclosure. Before the test, every step of the tested protocol should be checked carefully, including the light condition, testing time, etc. The testing room should be kept completely quiet and dark in order to not disturb the animals.

The protocol presented offers a fundamental frame to study the neurobehavioral toxicity of environmental pollutants. There are also other types of behaviors used when studying neurobehavioral effects, such as color-preference tests36, bottom dwelling tests37, light/dark preference tests38,39, etc. However, these tests mainly use adult zebrafish, which are not fit for high-throughput tests. In addition, Weichert et al. videotaped to the behavior of spontaneous tail movements which could be quantified just after 24 h exposure40. The evaluation of neurobehavioral toxicity also includes mechanism studies on the function of the brain and the central nervous system. The fundamental neurobehavioral indicators are introduced here and can form the basis for more complex indicators using other behavior instruments. Ultimately, the development of new neurobehavioral indicators accompanied with this study mechanism can be used in future studies.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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

Name Company Catalog Number Comments
48-well-microplate Corning 3548 Embyros housing
6-well-microplate Corning 3471 Embyros housing
BDE-47 AccuStandard 5436-43-1 Pollutant
DMSO Sigma 67-68-5 Cosolvent
Microscope Olympus SZX 16 Observation instrument
Pipette Eppendorf 3120000267 Transfer solution
Zebrabox Viewpoint ZebraBox Behavior instrument
Zebrafish Shanghai FishBio Co., Ltd. Tubingen Zebrafish supplier
ZebraLab Viewpoint ZebraLab Behavior software

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Neurobehavioral Effects Environmental Pollutants Zebrafish Larvae Protocol Substances Pollutants Neurotoxicity High Throughput Testing Allele-neurobehavioral Effects Chemical Management Public Health Problems Psychotropic Drugs Environmental Toxicology Research Experiment Room Temperature Exposure Solution Microplate Locomotion Experiment Path Angle Experiment Social Behavior Experiments
Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae
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Zhang, B., Yang, X., Zhao, J., Xu,… More

Zhang, B., Yang, X., Zhao, J., Xu, T., Yin, D. Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae. J. Vis. Exp. (156), e60818, doi:10.3791/60818 (2020).

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