1. Touch-evoked Response Assay
<ol>
	<li>Preparation of 2 dpf Embryos for Touch-evoked Response Assay
	<ol>
		<li>Ensure that the time of day at which the test is conducted is consistent between experiments because activity can vary dramatically throughout the day. 
		&#8203;NOTE: The experiment should be performed blinded and the order of testing randomized to minimize experimental artifacts.</li>
		<li>Assign the fish strains a number, which is unknown to the individual carrying out the experiment. Following this, using freely available online tools generate a random list that dictates the order of testing.</li>
		<li>At least one hour prior to testing, dechorionate embryos by ripping a hole in the chorion and gently pulling the chorion apart using a pair of fine tweezers. Remove any debris from the petri dish before returning to the 28 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Performing Touch-evoked Response Assay
	<ol>
		<li>Heat stage to 28 &#176;C at least 15 min prior to starting the testing. 
		&#8203;NOTE: This stage is temperature controlled and will remain at 28&#160;&#176;C for the duration of the testing. Temperature will affect activity and it is therefore important to maintain a constant temperature. If a heated stage is not available then the temperature of the water should be monitored and all experiments should be conducted at the same temperature.</li>
		<li>Place a petri dish filled with embryo medium (5 mM NaCl, 0.17mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4&#160;in water) onto an illuminated stage and mount the high-speed camera over the petri dish.</li>
		<li>Launch the video camera recording software (such as Stream Pix 5, described here) and under the &#34;workspace&#34; tab select 1,000 frame per sec (1,000 fps) as the capture speed to ensure that fast swimming action of the fish is recorded.</li>
		<li>Working with one embryo at a time, place the embryo in the middle of the petri dish with the zebrafish clearly visible in the field of view. 
		&#8203;NOTE: If the embryo swims away prior to commencement of the experiment replace it with another, as recapturing and positioning of the embryo may result in it becoming desensitized to the stimulus and repeated burst responses may promote muscle weakness in some disease models.</li>
		<li>Begin recording by clicking on the &#34;record&#34; button and, deliver the mechanosensory stimulus to the embryo by touching it gently with a blunt needle on the top of the head.</li>
		<li>Stop the recording after the embryo has swum out of the field of view or returned to rest. 
		&#8203;NOTE: The acceleration peaks within the first 0.2 sec of the burst escape response following the mechanical stimulus. Therefore, ensure that at least during the first 0.2 sec of the escape response recording the fish is in the field of view. Using the software Stream Pix 5, the data will automatically be saved as a&#160;.avi&#160;video file. Alternative video capture software such as Free Video Capture or Softonic, both of which are freely available for download, could also be used.</li>
		<li>Return the embryo to a new petri dish and select another embryo for testing. Perform testing on a minimum of 15 fish.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>90 mm Petri Dishes</td><td>&amp;nbsp;Pacific Laboratory Products</td><td>PT S90001</td><td></td></tr><tr><td>Tweezers, style 8</td><td>&amp;nbsp;ProSciTech</td><td>T04-821</td><td></td></tr><tr><td>Incubator</td><td>&amp;nbsp;Thermoline Scientific&amp;nbsp;</td><td>TEI-43L</td><td></td></tr><tr><td>High Speed Camera</td><td>&amp;nbsp;Baumer</td><td>HXC20</td><td></td></tr><tr><td>StreamPix5&amp;nbsp;</td><td>NorPix</td><td></td><td>Step 1.2.3</td></tr><tr><td>Temperature Control Unit</td><td>&amp;nbsp;Viewpoint</td><td></td><td></td></tr><tr><td>http://www.randomization.com</td><td>&amp;nbsp;N/A</td><td></td><td>Steps 1.1.2</td></tr></tbody></table>

escape response assay: a method to study the response of zebrafish larva to touch stimuli

Source: Sztal, T. E., et al. <a href="https://www.jove.com/v/54431/" target="_blank">Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish</a>.&#160;J. Vis. Exp.&#160;(2016)
This video describes the touch-evoke response assay, which measures the response of the zebrafish larva to touch stimuli.

Escape Response Assay: A Method to Study the Response of Zebrafish Larva to Touch Stimuli

Source: Sztal, T. E., et al. Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish.&#160;J. Vis. ...

- To begin, gently transfer a few embryos into a Petri dish containing embryo medium. Now hold the chorion with forceps and make a tear in it with another forceps.
Next, gently push the embryo through the tear. Removing the chorion from the embryo accelerates its growth and movement. To get rid of the debris, transfer the embryos to another Petri dish containing embryo medium using a Pasteur pipette.
Place the Petri dish in an incubator, and let the embryos grow at 28 degrees Celsius. Set an illuminated water filled microscope stage to 28 degrees Celsius prior to the experiment to avoid any temperature related deviations in larval behavior.
Place the Petri dish on the stage and mount in the camera above the dish. Next, center the embryo in the camera&#39;s field of view. Now gently touch the embryo with a blunt needle, and record the larval response on the video camera recording software.
Upon stimulus, the embryo exhibits a powerful escape response-- a burst swim along with sharp turns. Measure the time between the touch and the larvae&#39;s peak acceleration during its escape.
In the example protocol, we will observe the response of zebrafish larvae to external stimuli.
- To perform the assay, place a Petri dish filled with approximately 25 milliliters of embryo medium onto an illuminated temperature controlled stage set to approximately 28 degrees Celsius. Mount the high speed camera above the dish. Launch the video recording software, and set the capture speed to 1,000 frames per second to ensure fast swimming speeds can be captured.
Position the embryo in the center of the Petri dish, clearly visible in the field of view. Press record and then deliver a mechanosensory stimulus by gently touching the embryo with a blunt needle on top of the head.
Stop the recording once the embryo has swum out of the field of view or returned to rest.
- Repeated testing of the same larvae may lead to habituation or promote muscle weakness in some disease models, resulting in reduced response to the tactile stimulus. Therefore, each embryo should only be tested once.

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JoVE Encyclopedia of Experiments

Biology

Danio rerio (zebrafish)

Larva

2.5K Views. - To begin, gently transfer a few embryos into a Petri dish containing embryo medium. Now hold the

Abstract

Protocol

<table><tbody><tr><td>90 mm Petri Dishes</td><td>&nbsp;Pacific Laboratory Products</td><td>PT S90001</td><td></td></tr><tr><td>Tweezers, style 8</td><td>&nbsp;ProSciTech</td><td>T04-821</td><td></td></tr><tr><td>Incubator</td><td>&nbsp;Thermoline Scientific&nbsp;</td><td>TEI-43L</td><td></td></tr><tr><td>High Speed Camera</td><td>&nbsp;Baumer</td><td>HXC20</td><td></td></tr><tr><td>StreamPix5&nbsp;</td><td>NorPix</td><td></td><td>Step 1.2.3</td></tr><tr><td>Temperature Control Unit</td><td>&nbsp;Viewpoint</td><td></td><td></td></tr><tr><td>http://www.randomization.com</td><td>&nbsp;N/A</td><td></td><td>Steps 1.1.2</td></tr></tbody></table>

Escape Response Assay: A Method to Study the Response of Zebrafish Larva to Touch Stimuli

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Name	Company	Catalog Number	Comments
90 mm Petri Dishes	Pacific Laboratory Products	PT S90001
Tweezers, style 8	ProSciTech	T04-821
Incubator	Thermoline Scientific	TEI-43L
High Speed Camera	Baumer	HXC20
StreamPix5	NorPix		Step 1.2.3
Temperature Control Unit	Viewpoint
http://www.randomization.com	N/A		Steps 1.1.2