Prey Capture Assay: A Method to Study the Prey Capture Behavior of Zebrafish Larva

Overview

This video describes the prey capture assay. The video discusses the method to study prey capture behavior of zebrafish larvae after the laser ablation of specific neurons.

Protocol

1. Assessment of Behavioral Consequences Following Laser Ablation

1. Set up a behavioral recording system that consists of a stereoscope equipped with a video camera. Use a camera with an appropriate image acquisition software, commercial or custom-made (Figure 1A).
2. Optimize the lighting condition so that the paramecium can be detected by image processing. For example, use a ring white LED light with a spacer (Figure 1B).
   Note: The distance from, and the angle of, the LED affects the appearance of the sample (i.e., bright in dark background or dark in bright background) and are, therefore, critical for successful image processing. Determine the best height of the spacer (Figure 1C).
3. Place a zebrafish larva (recovered from the laser-ablation experiment described in Section 1) in a behavioral recording chamber (which was attached to a glass slide) with the original top seal removed (Figure 1D).
4. Collect 50 paramecia suspended in water from the beaker using a micropipette and place them in the recording chamber.
5. Place a cover slip on top of the recording chamber. Put a small drop of water on the cover slip before placing on the water surface of the recording chamber to avoid introducing air in the chamber.
   Note: In this step, some water with paramecia will overflow from the recording chamber. The remaining number of paramecia in the recording chamber will be approximately 30-40.
6. Place the recording chamber (containing a larva and paramecia) in the lighting system (Figure 1D).
7. Start the time-lapse recording at 10 fps for 11 min (6,600 frames).
8. (Optional) For each larva that was tested for behavior, observe the fluorescence of the laser-ablated areas by fluorescent microscopy to confirm the effectiveness of the laser-ablation (i.e., absence, or significantly reduced number of, fluorescent cells in the laser-irradiated area).
   Note: Even after irradiation, some fluorescent cells can still be observed owing to later onset of Gal4 gene expression in cells that differentiated after ablation.
9. After recording the larva's behavior, to compensate for the lack of uniformity in the background, perform a pixel-by-pixel division of each frame by an average image in Fiji: click 'Process', 'Image Calculator', 'Operation: Divide', '32-bit(float)result', and 'OK'. To make an average image, click 'Image', 'Stacks', 'Z-Project', 'Average intensity', and 'OK' (Figure 1E, F).
10. Count the number of paramecia left in the chamber by clicking 'Analyze', 'Analyze Particles', setting an area range that covers all the paramecia, checking the 'Show:Masks' checkbox, and clicking 'OK'. The 'Show:Masks' option will provide an extracted paramecia image (Figure 1G), with a list of the number of particles (paramecia) in each frame.
11. Plot the number of paramecia left in the recording chamber over time. Because the estimates of the number of paramecia are noisy, we recommend performing a moving average on each point in a range of 600 frames (1 min) on the spreadsheet.

Representative Results

Figure 1. Prey capture assay and representative data in laser-ablated zebrafish larvae. (A) Behavioral recording system. The stereomicroscope was equipped with a CMOS camera. Images were acquired using a custom-made script. (B) Components of the lighting system. A grey-colored paper, a glass diffuser, white LED ring light, diffuser, a custom-made spacer (cardboard), and diffuser with a hole at the center. (C) The lighting system assembled from the parts shown in B. (D) Recording chamber for the prey capture assay. A chamber (diameter: 20 mm; depth: 2.5 mm) was placed on a glass slide. The original top seal of the chamber was peeled off. A glass-cover was put on the recording chamber. (E) Raw image from a frame of the recorded movie. (F) A frame divided (pixel-by-pixel) by an average image in a movie. Note that the non-uniform background in lighting can be compensated by this image processing. (G) Paramecia extracted from a frame using the "Particle Analysis" function in Fiji. (H) Example of image in Fiji with an applied threshold. Paramecia appear bright, whereas the eyes of the zebrafish larva appear dark, and the background is grey. Changes in eye positions (i.e., angles with respect to the body axis) can be calculated, if necessary. (I) Representative experimental data of paramecium consumption in an olfactory-bulb-ablated control larva (OB) and a pretectum-ablated larva (PT). Pretectum ablation significantly reduced prey hunting ability while olfactory bulb ablation did not affect it. 

Materials

Name	Company	Catalog Number	Comments
Imageing Chambers	Grace Bio-Labs	CoverWell Imaging Chambers PCIA-2.5	Used as a behavioral recording chamber
ORCA-Flash4.0	Hamamatsu Photonics	Model:C11440-22CU	A scientific CMOS camera
SZX7	Olympus		Stereoscope
A ring LED light	CCS	Model: LDR2-100SW2-LA	White LED
Secure-Seal Hybridization Chamber Gasket, 8 chambers, 9 mm diameter x 0.8 mm depth	Molecular Probes	Catalogue # S-24732	Used as a recording chamber in Ca imaging