These studies were funded by grants received from the MEXT, JSPS KAKENHI Grant Numbers JP25290009, JP25650120, JP17K07494, and JP17H05984.

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope &#12288;
Note: If users plan on performing Ca imaging following ablation, use the UAShspzGCaMP6s line21. If users plan on performing behavioral recording following ablation, use the UAS:EGFP line, as the ablation of EGFP-positive cells is easier to perform than of GCaMP6s-expressing cells.
<ol>
	<li>Begin by setting up the mating of a Gal4 line that labels specific neurons to be studied and UAS:EGFP or UAShspzGCaMP6s. Be sure to use the nacre background for both parents so that nacre homozygotes can be obtained. 
	Note: Homozygotes of nacre contain no melanophores on the body surface (Figure 1B). In the present study, a Gal4 line gSAIzGFFM119B (which labels the pretectum) and another Gal4 line hspGFFDMC76A (which labels the ILH) are used21.</li>
	<li>On the following day of the mating setup, collect the zebrafish eggs, and raise them to the larval stage at which point the neurons of interest will express fluorescent reporters.</li>
	<li>Mount the zebrafish larva in 2% low melting point-agarose (Figure 1B).
	<ol>
		<li>Begin by preparing 2% agarose stock solution: dissolve 2 g of low melting-point agarose powder in 100 mL system water (i.e., the water used to maintain adult zebrafish in a circulating water system)22 or E3 water23, by bringing the water to the boiling point in a microwave, and mixing vigorously.</li>
		<li>Microwave the 2% low melting-point agarose stock until it boils. Pour 3.5-4.0 mL of the agarose onto the lid of a 6-cm Petri dish. Wait until the agarose cools so that it is warm to the touch before proceeding.</li>
		<li>Next, place a single larva (not anesthetized at this step) in the agarose. Keep adjusting the position and orientation of the larva so that it is upright, using a dissecting needle (Figure 1C), until the agarose solidifies to ensure the proper positioning of the larva. 
		Note: The larva will continue to swim around while the agarose solidifies.</li>
		<li>Once the larva is positioned, allow 5 min for the agarose to solidify completely.</li>
		<li>Pour 5 mL of tricaine solution (0.4% at 1x working concentration) over the agarose. 
		Note: To avoid movements by the larvae during laser ablation, the larvae must be kept anesthetized throughout the ablation procedure.</li>
	</ol>
	</li>
	<li>Ablate the larva using the bleaching function in a two-photon laser microscopy system.
	<ol>
		<li>Place the agarose-embedded larva under a two-photon laser scanning microscope and start the microscopy system.</li>
		<li>Start the image acquisition software and click the &#34;On&#34; button on the &#34;Laser&#34; tab to turn on the laser (Figure 1D). Wait for the &#34;Status&#34; of the laser to change from &#34;Busy&#34; to &#34;Mode-locked.&#34;</li>
		<li>On the &#34;Channels&#34; tab, set the laser&#39;s wavelength at 880 nm (maximum power: approximately 2,300 mW) for EGFP ablation, or at 800 nm (maximum power: approximately 2,600 mW) for GCaMP ablation.</li>
		<li>Select the 20X objective lens by manually moving the lens revolver. Click the &#34;Locate&#34; tab, click &#34;GFP&#34; to change the optical pathways, and directly view the fluorescence by eye.</li>
		<li>Locate the zebrafish larval brain at the center of view; then click the &#34;Acquisition&#34; tab to go back to the two-photon microscopy.</li>
		<li>As a record of the &#39;before ablation&#39; condition, take a z-stack image with the 20X objective lens at fast scanning-speed (to avoid heat-damage). Later compare the images taken before and after ablation experiments (Figure 2A). To obtain a z-stack, click &#34;Z-Stack&#34; to select the z-stack option and expand the tab. Set the lower limit (click the &#34;Set First&#34; button&#34;) after focusing on the ventral end of the larval brain, and set the upper limit (click the &#34;Set Last&#34; button) after focusing on the dorsal most-surface of the brain. Then, click the &#34;Start Experiment&#34; button to run the z-stack image acquisition.</li>
		<li>Select the 63X objective lens by manually moving the lens revolver.</li>
		<li>Click the &#34;Locate&#34; tab to switch to the epi-fluorescent microscopy, locate the cells to be ablated at the center of view by eye, then click the &#34;Acquisition&#34; tab to go back to laser scanning microscopy.</li>
	</ol>
	</li>
	<li>On the &#34;Acquisition Mode&#34; tab, set the &#34;Frame Size&#34; at 256 x 256. Click &#34;Live&#34; and observe the neurons of interest.</li>
	<li>Starting from the dorsal-most side, find a focal plane in which the cells to be ablated are in focus.</li>
	<li>Mark a small, circular area about one third the cell&#39;s size in diameter on each neuron as a region of interest (ROI) using the &#34;Regions&#34; function.</li>
	<li>Set the scan speed to 13.93 s/256 x 256 pixels (134.42 &#181;m x 134.42 &#181;m), which corresponds to a laser dwell time of approximately 200 &#181;s/pixel, or 200 &#181;s/0.5 &#181;m. Also, set 4 repetitions (&#39;Iteration cycle: 4&#39;). Alternatively, optimize the number of iterations and scanning speed so that sufficient ablation is achieved.</li>
	<li>Perform the &#39;Bleaching&#39; function for ablation, in combination with the &#39;Time series (2 cycles)&#39; to image the sample (before and after ablation) and &#39;Regions&#39; (to set the ROIs) or equivalent functions in the software used (Figure 1D).</li>
	<li>By comparing the first image (before ablation) and the second image (after ablation) in the Time series cycle, ensure that after bleaching, the fluorescence in the targeted cells decreases to that observed at the background level (Figure 2B).</li>
	<li>If fluorescence is still present after ablation, increase the number of iterations.</li>
	<li>Next, move the focal plane slightly deeper (i.e., towards the ventral side), and choose the next focal plane where un-ablated cells appear.</li>
	<li>Perform the bleaching function as described above (step 1.9). Repeat these steps until all the cells in the neural structure of interest have been ablated.</li>
	<li>After going through all the focal planes covering the neural structures to be ablated, check the cells for abolished fluorescence. If some cells are found to still be fluorescent due to insufficient ablation, laser-irradiate them again as described above.</li>
	<li>If the larva needs to be recovered from agarose after laser irradiation, do not try to force it out of the agarose because this might damage the larva. Instead, carefully make tiny cuts in the agarose around the larva perpendicularly to the surface of its body. Then, wait for the larva to swim out of agarose on its own when the anesthetic wears off.</li>
	<li>Proceed to the next larva for ablation or perform control experiments using another population of neurons that are uninvolved in the behavior under investigation. 
	Note: Here, as a control, olfactory bulb neurons in UAS:EGFP; gSAIzGFFM119B larvae were laser-ablated using the same protocol described above (Figure 2A, right).</li>
	<li>Allow several hours or 1 day for recovery before proceeding to Ca imaging (Section 2) or behavioral recording (Section 3). During this recovery time, check the larval health (i.e., normal spontaneous swimming, no apparent heat-damage around the ablated area, etc.) and remove larvae showing any signs of poor health.</li>
</ol>
2. Calcium Imaging to Record Prey-evoked Neuronal Activity in the Pretectum-ablated Zebrafish Larvae
<ol>
	<li>To prepare a recording chamber, put a secure-seal hybridization chamber gasket on a glass slide, with the adhesive side facing on the glass, and remove the top seal. 
	Note: This creates a small recording chamber that is 9 mm in diameter and 0.8 mm in depth (Figure 3A).</li>
	<li>Next, place a nylon mesh (size of opening: 32 &#181;m) on a 50-mL tube that has the bottom cut open. Use the cap to attach the mesh on the tube (Figure 3B).</li>
	<li>Prepare the paramecia (which is the prey to be used as the visual stimulus for larva). 
	Note: Prepare the Paramecium culture (steps 2.3.1 and 2.3.2) beforehand.
	<ol>
		<li>Obtain paramecia seed culture from commercial sources or academic bio-resource centers24 in advance.</li>
		<li>Culture the paramecium for several days in water containing autoclaved rice straw and a pellet of dry beer-yeast22 (Figure 3C).</li>
		<li>Pour the paramecium culture through 2 sieves successively (one with a 150-&#181;m aperture, followed by another with a 75-&#181;m aperture) to remove debris from the culture.</li>
		<li>Collect paramecia in the flow-through from the previous step on a nylon mesh (13-&#181;m aperture).</li>
		<li>Re-suspend the paramecia on the nylon mesh into a glass beaker using a squeeze bottle of system water (Figure 3D).</li>
	</ol>
	</li>
	<li>Rinse the mesh (Figure 3B) in system water 2x (Figure 3E). 
	Note: The purpose of this step is to remove any possible odorants and tastants contained in the paramecium culture medium, as the goal of the present study is to observe visually driven neuronal activity.</li>
	<li>Place a zebrafish larva in the tricaine solution to anesthetize.</li>
	<li>Mount a single larva in 2% low melting-point agarose.
	<ol>
		<li>First, microwave 2% low melting-point agarose and add 1/10 volume of 10x concentrated tricaine to the agarose.</li>
		<li>Place one drop of the tricaine-containing agarose onto the recording chamber (Figure 3A).</li>
		<li>After confirming the agarose is not too hot, place the anesthetized larva (from step 2.5) into the agarose, immediately removing any excess agarose so that the surface of the agarose looks slightly convex.</li>
		<li>Quickly orient the larva into an upright position with a dissecting needle (Figure 1C). 
		Note: These procedures must be completed within several seconds before the agarose solidifies.</li>
		<li>Once the larva is properly positioned, allow 5 min for the agarose to solidify completely. Then, pour a drop of 1x tricaine solution on the agarose.</li>
	</ol>
	</li>
	<li>Remove the agarose around the head of the zebrafish larva and make space for the paramecium to swim using a surgical knife. To do this, first, make a few small cuts in the agarose (Figure 3F). Then, carefully remove the excess agarose by gently pouring system water on the chamber. 
	Note: At this step, the tricaine will be washed out.</li>
	<li>Next, put one paramecium in the recording chamber to serve as a visual stimulus. 
	Note: When the paramecium swims near the head of the zebrafish larva, it will evoke a neuronal response (Figure 3G).</li>
	<li>Place the recording chamber under an epi-fluorescence microscope equipped with a scientific CMOS camera (Figure 3H) and select a 2.5X objective lens (Figure 3I).</li>
	<li>Collect time-lapse recordings of the GCaMP fluorescence signals.
	<ol>
		<li>Start the image acquisition application for the equipped camera.</li>
		<li>Click &#34;Sequence Pane&#34; and select &#34;Hard Disk Record&#34; on the pane. In the Scan Settings section, set the &#34;Frame Count&#34; to 900 or any other total frame number desired. 
		Note: If available, use time-lapse recording software with a module (here, &#34;Hard Disk Record&#34;) that enables efficient saving of the data onto a hard drive.</li>
		<li>In the &#34;Capture&#34; pane, set the exposure time at 30-ms (i.e., 33.3 fps), and Binning 2 x 2.</li>
		<li>On the microscope controlling touch panel, choose a filter set for GFP, as the GCaMP has similar excitation/emission spectra to GFP.</li>
		<li>Click &#34;Live&#34; on the Capture pane to locate the larva in the camera view and focus (Figure 4A), and then click &#34;Stop Live&#34; and click the &#34;Start&#34; button. Save the movie in .cxd or any other format that holds information about the acquisition condition.</li>
	</ol>
	</li>
	<li>After the recordings, analyze the GCaMP6s fluorescent signals (Ca signals) by obtaining mean pixel values for the ROI set on a brain structure in the time-lapse movie using the free software Fiji25. 
	Note: Fiji is a version of ImageJ with many useful plug-in&#39;s preinstalled and can read .cxd format image files with the Bio-Formats plug-in.</li>
	<li>If the larva move slightly during the recording, perform image registration by running the TurboReg plug-in26 in Fiji. From the menu, select &#39;Plugins&#39;, &#39;Registration&#39;, and &#39;TurboReg&#39;.</li>
	<li>Consider how to analyze the data according to the objective of the study. The following is an example.
	<ol>
		<li>Calculate the F/F0 (fluorescence intensity at each time, divided by the fluorescence intensity at rest or before any Ca transient has occurred) as follows:</li>
		<li>Set ROIs on the pretectum or the ILH using the ROI Manager: in the menu, select &#34;Analyze,&#34; &#34;Tools,&#34; &#34;ROI Manager&#34;, and &#34;Add&#34; to the list of the ROIs (Figure 4A).</li>
		<li>Calculate the mean pixel values for the ROIs (i.e., fluorescence intensities of GCaMP6s) by selecting on the ROI Manager panel: &#34;More,&#34; &#34;Multi-Measure&#34;, and &#34;OK.&#34; Save the Results as a spreadsheet file.</li>
		<li>As the signals are noisy, average the signals for 11 time-points (moving average) using an application that can handle spreadsheet data.</li>
		<li>Divide F (fluorescence intensities over time) by F0 (fluorescence intensity at the basal level) to calculate the normalized fluorescence intensity. As an example of data visualization, changes in normalized GCaMP6s fluorescence intensity in the pretectum and the ILH are color-coded and mapped on the paramecium trajectories (Figure 4B - D).</li>
	</ol>
	</li>
</ol>
3. 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 5A).</li>
	<li>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 5B). 
	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 5C).</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 5D).</li>
	<li>Collect 50 paramecia from the culture (step 2.3.5) 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: 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.</li>
	<li>Place the recording chamber (containing a larva and paramecia) in the lighting system (Figure 5D).</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: Even after irradiation, some fluorescent cells can still be observed owing to later onset of Gal4 gene expression in cells that differentiated after ablation27.</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 5E, F).</li>
	<li>Count the number of paramecia left in the chamber by clicking &#39;Analyze&#39;, &#39;Analyze Particles&#39;, setting an area range that covers all the paramecia, checking the &#39;Show:Masks&#39; checkbox, and clicking &#39;OK&#39;. The &#39;Show:Masks&#39; option will provide an extracted paramecia image (Figure 5G), with a list of the number of particles (paramecia) in each frame.</li>
	<li>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.</li>
</ol>

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Fiji Available from: <a target="_blank" href="https://fiji.sc">https://fiji.sc</a> (2017)</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7 (1), 27-41 (1998).</li><li>Mueller, T., Wullimann, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU-,+neuroD+(nrd)-+and+Hu-studies+reveal+unusual+non-ventricular+neurogenesis+in+the+postembryonic+zebrafish+forebrain.">BrdU-, neuroD (nrd)- and Hu-studies reveal unusual non-ventricular neurogenesis in the postembryonic zebrafish forebrain.</a> Mech Dev. 117 (1-2), 123-135 (2002).</li><li>Muto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+genetic+analysis+of+visual+behavior+in+zebrafish.">Forward genetic analysis of visual behavior in zebrafish.</a> PLoS Genet. 1 (5), 66 (2005).</li><li>Chen, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity.">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li></ol>

The authors have no conflicts of interest to report.

Although the two-photon laser has an excellent spatial resolution to specifically ablate individual neurons, great caution should be taken to avoid any undesired damage on the brain tissue owing to heat. The most important step in the ablation experiment is to determine the optimal amount of laser irradiation. Insufficient irradiation fails to kill the neurons. Too much irradiation will heat-damage the surrounding tissue, which will result in undesired effects. The optimal range of laser irradiation (areas of the ROIs, n...

To understand how behavior arises from neuronal activity in the brain, it is necessary to identify the neural circuits that are involved in the generation of that behavior. At the larval stage, the zebrafish provides an ideal animal model for studying the brain function associated with the behavior because their small, transparent brains make it possible to investigate neuronal activity at a cellular resolution in a broad area of the brain while observing the behavior1. Imaging of neuronal activity in specific neurons has become possible through the invention of genetically encoded calcium (Ca) indicators (GECIs) such as GCaMP2. GCaMP transgenic zebrafish have proven to be useful for associating the functional neural circuit with behavior by conducting Ca imaging in behaving animals3.
While Ca imaging can demonstrate correlations between neuronal activity and behavior, to show causality, suppression of neuronal activity and testing its consequence(s) on behavior are important steps. There are various ways to achieve this: use of genetic mutation that alters specific neural circuits4, expression of neurotoxins in specific neurons5,6, use of optogenetic tools such as halorhodopsin7, and laser ablation of targeted neurons8,9. Laser ablation is particularly suited for eliminating activity in a relatively small number of specific neurons. Irreversible elimination of neuronal activity by killing neurons facilitates assessing behavioral consequences.
One interesting behavior that can be observed at the larval stage in zebrafish is prey capture&#160;(Figure 1A). This visually-guided, goal-directed behavior provides a favorable experimental system for the study of visual acuity10, visuomotor transformation11,12,13, visual perception and recognition of objects14,15,16,17,18, and decision making19. How prey is recognized by predators and how prey detection leads to prey catching behavior has been a central question in neuroethology20. In this paper, we focus on the role of the pretecto-hypothalamic circuit formed by projections of a nucleus in the pretectum (nucleus pretectalis superficialis pars magnocellularis, hereafter, simply noted as the pretectum) to the ILH. Laser-ablation of the pretectum was shown to reduce prey capture activity and abolish neuronal activity in the ILH that is associated with the visual prey perception21. Here, protocols for performing laser ablation and testing its effect using Ca2+ imaging and behavioral recording in zebrafish larvae are described.

<table><tbody><tr><td>NuSieve GTG Agarose</td><td>Lonza</td><td>Cat.#50080</td><td>low-melting temperature agarose</td></tr><tr><td>6 cm petri dish</td><td>FALCON</td><td>Product#:351007</td><td></td></tr><tr><td>dissecting needle</td><td>AS ONE Corporation</td><td>Cat. No. 2-013-01</td><td>https://keystone-lab.com/en/item/detail/404142</td></tr><tr><td>LSM7MP</td><td>Carl Zeiss</td><td></td><td>two-photon laser scanning microscope</td></tr><tr><td>W Plan-Apochromat 63x/1.0</td><td>Carl Zeiss</td><td></td><td>63X objective lens</td></tr><tr><td>Imager.Z1</td><td>Carl Zeiss</td><td></td><td>an epi-fluorescence microscope</td></tr><tr><td>ZEN</td><td>Carl Zeiss</td><td></td><td>Image acquisition software for confocal microscopes</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><tr><td>Imageing Chambers</td><td>Grace Bio-Labs</td><td>CoverWell Imaging Chambers PCI-A-2.5</td><td>Used as a behavioral recording chamber</td></tr><tr><td>surgical knife</td><td>MANI</td><td>Ophthalmic knife MST15</td><td></td></tr><tr><td>ORCA-Flash4.0</td><td>Hamamatsu Photonics</td><td>model:C11440-22CU</td><td>a scientific CMOS camera</td></tr><tr><td>HCImage</td><td>Hamamatsu Photonics</td><td></td><td>image acuisition software</td></tr><tr><td>Hard Disk Recording module</td><td>Hamamatsu Photonics</td><td></td><td>An software module that enables saving the movie files onto a hard disc drive in a short time</td></tr><tr><td>SZX7</td><td>Olympus</td><td></td><td>stereoscope</td></tr><tr><td>DF PL 0.5X</td><td>Olympus</td><td></td><td>objective lens for SZX7</td></tr><tr><td>Point Grey Grasshopper3 4.1 MP Mono USB3 Visio</td><td>FLIR Systems, Inc.</td><td>Product No. GS3-U3-41C6NIR-C</td><td>CMOS camera</td></tr><tr><td>XIMEA xiQ camera</td><td>XIMEA</td><td>Product No. MQ042RG-CM</td><td>CMOS camera</td></tr><tr><td>a ring LED light</td><td>CCS</td><td>Model: LDR2-100SW2-LA</td><td>White LED</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Nylon mesh 32&amp;micro;m</td><td>Tokyo Screen</td><td>N-No.380T</td><td>http://www.tokyo-screen.com/cms/sta20347/</td></tr><tr><td>Nylon mesh 13&amp;micro;m</td><td>Tokyo Screen</td><td>N-No. 508T-K</td><td>http://www.tokyo-screen.com/cms/sta20347/</td></tr><tr><td>Metal seive 150 micron aperture</td><td>Tokyo Screen</td><td></td><td>http://www.tokyo-screen.com/cms/sta20341/#ami</td></tr><tr><td>Metal seive 75 micron aperture</td><td>Tokyo Screen</td><td></td><td>http://www.tokyo-screen.com/cms/sta20341/#ami</td></tr><tr><td>EBIOS</td><td>Asahi Food &amp;amp; Healthcare, Co. Ltd.</td><td></td><td>dry beer yeast</td></tr><tr><td>LabVIEW</td><td>National Instruments</td><td></td><td>an integrated development environment for programming</td></tr><tr><td>Mai-Tai HP</td><td>Spectra Physics&amp;nbsp;</td><td></td><td>two-photon laser&amp;nbsp;</td></tr></tbody></table>

ablation of a neuronal population using a two-photon laser and its assessment using calcium imaging and behavioral recording in zebrafish larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Specific neurons were genetically labeled with either EGFP or GCaMP6s, whose expression were driven in Gal4 lines. A Gal4 line gSAIzGFFM119B was used to label a nucleus in the pretectal area (magnocellular superficial pretectal nucleus), and a subpopulation of olfactory bulb neurons. Another Gal4 line, hspGFFDMC76A, was used to label the ILH. We laser-ablated the pretectal neurons bilaterally (Figure 2A left panel) and also ablated neurons in...

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Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

ablation-neuronal-population-using-two-photon-laser-its-assessment

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.7K Views.  SOKENDAI (The Graduate University for Advanced Studies). Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

Video: Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Biology

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Developmental Biology

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns.

Embryonic development requires large-scale coordination of cell motion. Two-photon excitation mediated laser ablation allows the spatially controlled 3-dimensional ablation of large groups of deep cells. In addition, this technique can probe the reaction of collectively migrating cells in vivo to perturbations in their mechanical environment.

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Laser Ablation Method to Study Bone Regeneration in Zebrafish Larvae

Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using different methods such as fin amputation, scale plucking, skull trepanation, and microscopic approaches. Using a confocal laser scanning setup equipped with a two-photon laser, a laser ablation method was developed as a lesion paradigm to ablate bone-forming cells (osteoblasts) in the developing opercle of zebrafish larvae. The method described here allows the ablation of cells in a precise manner, as the area, shape, and depth can be finely adjusted. In addition, this method allows imaging of the area before and just after the ablation, so that short-term effects of the injury can be analyzed. In this experimental setup, the immune response after ablation of osteoblasts in the injured area was studied. An increase in the recruitment of macrophages was observed after ablation, indicating the relevance of their presence during bone regeneration.

Author Spotlight: Immune-Mediated Bone Regeneration &#8212; The Critical Role of Macrophages and Controlled Immunomodulation in Restorative Processes

This protocol describes a two-photon laser ablation approach carried out in zebrafish larvae, which serves as a model to study bone regeneration and the effects of the immune response to ablation.

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using ...

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

Author Spotlight: Advancements in Adult Zebrafish Brain Research 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

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

Source: Muto, A., et al. <a href="https://jove.unicartagenaproxy.elogim.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.

Test chwytania zdobyczy: metoda badania zachowania larw danio pręgowanego podczas chwytania zdobyczy

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 ...

JoVE Encyclopedia of Experiments

Danio rerio (zebrafish)

Larva

Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Source: Muto, A. et al.,&#160; <a href="https://appjove.unicartagenaproxy.elogim.com/v/57485">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.</a> J. Vis. Exp. (2018)
This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

Analiza aktywności neuronalnej wywołanej przez zdobycz w transgenicznej larwie danio pręgowanego

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope
NOTE: If users plan on performing Ca imaging following ablation, use the ...