1. Assessment of Behavioral Consequences Following Laser Ablation
<ol>
	<li>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).</li>
	<li>Optimize the lighting condition so that the&#160;paramecium&#160;can be detected by image processing. For example, use a ring white LED light with a spacer (Figure 1B). 
	Note:&#160;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).</li>
	<li>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).</li>
	<li>Collect 50&#160;paramecia&#160;suspended in water from the beaker using a micropipette and place them in the recording chamber.</li>
	<li>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:&#160;In this step, some water with&#160;paramecia&#160;will overflow from the recording chamber. The remaining number of&#160;paramecia&#160;in the recording chamber will be approximately 30-40.</li>
	<li>Place the recording chamber (containing a larva and&#160;paramecia) in the lighting system (Figure 1D).</li>
	<li>Start the time-lapse recording at 10 fps for 11 min (6,600 frames).</li>
	<li>(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:&#160;Even after irradiation, some fluorescent cells can still be observed owing to later onset of Gal4 gene expression in cells that differentiated after ablation.</li>
	<li>After recording the larva&#39;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 &#39;Process&#39;, &#39;Image Calculator&#39;, &#39;Operation: Divide&#39;, &#39;32-bit(float)result&#39;, and &#39;OK&#39;. To make an average image, click &#39;Image&#39;, &#39;Stacks&#39;, &#39;Z-Project&#39;, &#39;Average intensity&#39;, and &#39;OK&#39; (Figure 1E, F).</li>
	<li>Count the number of&#160;paramecia&#160;left in the chamber by clicking &#39;Analyze&#39;, &#39;Analyze Particles&#39;, setting an area range that covers all the&#160;paramecia, checking the &#39;Show:Masks&#39; checkbox, and clicking &#39;OK&#39;. The &#39;Show:Masks&#39; option will provide an extracted&#160;paramecia&#160;image (Figure 1G), with a list of the number of particles (paramecia) in each frame.</li>
	<li>Plot the number of&#160;paramecia&#160;left in the recording chamber over time. Because the estimates of the number of&#160;paramecia&#160;are noisy, we recommend performing a moving average on each point in a range of 600 frames (1 min) on the spreadsheet.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Imageing Chambers</td>
			<td>Grace Bio-Labs</td>
			<td>CoverWell Imaging Chambers PCIA-2.5</td>
			<td>Used as a behavioral recording chamber</td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>Model:C11440-22CU&#160;</td>
			<td>A scientific CMOS camera</td>
		</tr>
		<tr>
			<td>SZX7</td>
			<td>Olympus</td>
			<td></td>
			<td>Stereoscope</td>
		</tr>
		<tr>
			<td>A ring LED light</td>
			<td>CCS&#160;&#160;</td>
			<td>Model: LDR2-100SW2-LA</td>
			<td>White LED</td>
		</tr>
		<tr>
			<td>Secure-Seal Hybridization Chamber Gasket, 8 chambers, 9 mm diameter x 0.8 mm depth</td>
			<td>Molecular Probes</td>
			<td>Catalogue # S-24732</td>
			<td>Used as a recording chamber in Ca imaging</td>
		</tr>
	</tbody>
</table>

prey capture assay: a method to study the prey capture behavior of zebrafish larva

Source: Muto, A., et al. <a href="https://www.jove.com/v/57485/" target="_blank">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae</a>. &#160;J. Vis. Exp.&#160;(2018)
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.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20172/20172fig1.jpg" /> 
Figure 1. Prey capture assay and representative data in laser-ablated zebrafish larvae.&#160;(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...

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

Source: Muto, A., et al. Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in ...

- To begin, place a zebra fish larva in a recording chamber, such as a glass-bottomed well, in which a larva can swim. Collect paramecia, the prey, with the help of a micropipette, and place them in the recording chamber. Next, put a small drop of water at the center of the cover slip and place it over the chamber to avoid an air bubble.
Now place the chamber on the lighting system equipped with a white LED ring light to eliminate shadows and make the organisms clearly visible. Observe the prey capture behavior using a behavioral recording system. As soon as the prey comes in sight, the larva converges its eyes and takes a J turn to orient its body towards the prey and captures it. We can quantify the prey capture behavior by recording the number of paramecia remaining in the chamber over a period of time. In the following protocol, we will assess the prey capture behavior of the zebra fish larva after the laser ablation of specific neurons.
- After setting up the behavioral recording system and placing a larva into a recording chamber according to the text protocol, use a micropipette to collect 50 paramecia and place them in the recording chamber. To place a cover slip on top of the recording chamber, put a small drop of water on the cover slip before placing it on the water surface of the recording chamber to avoid introducing air in the chamber.
Place the recording chamber in the lighting system. Then start the time lapse recording at 10 frames per second for 11 minutes. Finally, carry out analysis according to the text protocol.

prey-capture-assay-method-to-study-prey-capture-behavior-zebrafish

Research

JoVE Encyclopedia of Experiments

Biology

Danio rerio (zebrafish)

Larva

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.

Overview

Protocol

<img title="figure-results-25" alt="figure-results-25" class="xfigimg" src="/files/ftp_upload/20172/20172fig1.jpg" /> 
Figure 1. Prey capture assay and representative data in laser-ablated zebrafish larvae.&#160;(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...

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

In This Article

Overview

Protocol

Results

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