targeted neuronal ablation using two-photon microscopy in a zebrafish larava

Targeted Neuronal Ablation Using Two-Photon Microscopy in a Zebrafish Larava

Place the agarose-embedded larva under a 2-photon laser-scanning microscope and start the microscopy system. Open the image acquisition software, and under the Laser tab, click the on button to turn on the laser. Wait for the status of the laser to change from Busy to Mode Locked.On the Channels tab, set the laser's wavelength to 880 nanometers for EGFP ablation, or 800 nanometers for GcAMP ablation. Select the 20x objective lens by manually moving the lens revolver. Then, click the Locate tab. Click GFP to change the optical pathways, and directly view the fluorescence by eye. Center the zebrafish larval brain in the field of view. Then, click the Acquisition tab to go back to two-photon microscopy and set the laser power and gain.As a record of the before ablation condition, click Z-Stack to select the Z-Stack option. Then, after focusing on the ventral end of the larval brain, click the Set First button to set the lower limit. And after focusing on the dorsal most surface of the brain, click the Set Last button to set the upper limit. Finally, click the Start experiment button to run the Z-Stack image acquisition.Next, select the 63x objective lens by manually moving the lens revolver. Then, click the Locate tab to switch to epifluorescence microscopy. Center the cells to be ablated by eye. Then, click the Acquisition tab to go back to laser scanning microscopy.In the Acquisition Mode tab, set the frame size to 256 by 256. Click Live and observe the neurons of interest. Then, starting from the dorsal-most side, find a focal plane in which the cells to be ablated are in focus. Using the Regions function, mark a small circular area about one-third the cell size in diameter on each neuron as an ROI.Set the scan speed to 13.93 seconds per 256 by 256 pixels, which corresponds to a laser dwell time of approximately 200 microseconds per pixel, or 200 microseconds per half micron. Also, set the iteration cycle to 4 repetitions. Click Start Experiment to execute the time series and bleaching functions. Compare the image before ablation to the image after ablation in the time series cycle, to ensure that the fluorescence in the targeted cells is gone.Next, move the focal plane slightly deeper, and choose the next focal plane where unablated cells appear. Carry out the bleaching function as just demonstrated, and repeat the process until all the cells in the neural structure of interest have been ablated. Following ablation, check the cells for abolished fluorescence, and repeat the laser irradiation if necessary.

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<div style='background-color:white;border:1.0pt none #4B4B4B;margin-left:18.0pt;margin-right:0cm;padding:0cm;'><ol><li>&#160;Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope&#160;</li></ol></div><div style='background-color:white;border:1.0pt none #1A202C;margin-left:92.0pt;margin-right:0cm;padding:0cm;'><ol style='padding-left:24px;'><li>Begin by setting up the mating of a Gal4 line that labels specific neurons to be studied and UAS:EGFP or UAShspzGCaMP6s. Be&#160;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 (Lobe of the hypothalamus) are used.</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).</li></ol></div><div style='border:1.0pt none #1A202C;margin-left:166.0pt;margin-right:0cm;padding:0cm;'><ol style='padding-left:24px;'><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) or E3 water, by bringing the water to the boiling point in a microwave, and mixing vigorously.<ol style='list-style-type:decimal;padding-left:0px;'><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></ol></div><div style='border:1.0pt none #1A202C;margin-left:54.0pt;margin-right:0cm;padding:0cm;'><ol style='padding-left:24px;' start='4'><li>Ablate the larva using the bleaching function in a two-photon laser microscopy system.</li></ol></div><div style='border:1.0pt none #1A202C;margin-left:130.0pt;margin-right:0cm;padding:0cm;'><ol style='list-style-type:decimal;padding-left:24px;'><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 "On" button on the "Laser" tab to turn on the laser (Figure 1D). Wait for the "Status" of the laser to change from "Busy" to "Mode-locked."</li><li>On the "Channels" tab, set the laser's wavelength at 880 nm (maximum power: approximately 2,300 mW) for EGFP (Enhanced Green Fluorescent Protein) 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 "Locate" tab, click "GFP" (Green Fluorescent Protein) 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 "Acquisition" tab to go back to the two-photon microscopy.</li><li>As a record of the 'before ablation' condition, take a z-stack image with the 20X objective lens at a 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 "Z-Stack" to select the z-stack option and expand the tab. Set the lower limit (click the "Set First" button") after focusing on the ventral end of the larval brain, and set the upper limit (click the "Set Last" button) after focusing on the dorsal most-surface of the brain. Then, click the "Start Experiment" 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 "Locate" tab to switch to epi-fluorescent microscopy, locate the cells to be ablated at the center of view by the eye, then click the "Acquisition" tab to go back to laser scanning microscopy.</li></ol></div><div style='background-color:white;border:1.0pt none #1A202C;margin-left:54.0pt;margin-right:0cm;padding:0cm;'><ol style='padding-left:24px;' start='5'><li>On the "Acquisition Mode" tab, set the "Frame Size" at 256 x 256. Click "Live" 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's size in diameter on each neuron as a region of interest (ROI) using the "Regions" 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 ('Iteration cycle: 4'). Alternatively, optimize the number of iterations and scanning speed so that sufficient ablation is achieved.</li><li>Perform the 'Bleaching' function for ablation, in combination with the 'Time series (2 cycles)' to image the sample (before and after ablation) and 'Regions' (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. 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 or behavioral recording. 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></div>

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/57485/ablation-neuronal-population-using-two-photon-laser-its-assessment'><span style='-webkit-text-decoration-skip:none;font-family:Roboto,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Muto, A.,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Roboto,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Roboto,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2018)</a> &#160;In this video, a two-photon laser ablation procedure is demonstrated, enabling the selective destruction of fluorescent neurons in agarose-immobilized zebrafish larvae. High-resolution imaging before and after the procedure confirms efficient neuronal damage, as observed by the absence of fluorescence.

Source:&#160;Muto, A.,&#160;et al. Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral ...

Begin with an anesthetized transgenic zebrafish larva immobilized in agarose and placed in a Petri dish containing an anesthetic solution.Position it under a two-photon laser microscope, which uses two photons for detailed imaging.Focus on the top and bottom layers of the brain to set imaging boundaries.Capture images at various depths to observe neurons before laser treatment.Adjust the focus on the region of interest and adjust the frame size to refine the focus on neurons.Select and irradiate the neurons using the two-photon laser.The Laser induces localized heat, resulting in neuronal death with minimal damage to surrounding tissue. Capture the image.Compare pre and post-treatment images to assess fluorescence changes.Refocus neurons in deeper planes and repeat the irradiation to treat all neurons in the region of interest..Recapture the image and compare it with the pre-treatment image. The loss of fluorescence after laser treatment confirms successful neuronal ablation.

9 ビュー. Targeted Neuronal Ablation Using Two-Photon Microscopy in a Zebrafish Larava

ビデオ: Targeted Neuronal Ablation Using Two-Photon Microscopy in a Zebrafish Larava - 実験

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.

ゼブラフィッシュインターニューロマスト細胞における共焦点顕微鏡ベースレーザーアブレーションと再生アッセイ

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

Targeted Neuronal Ablation Using Two-Photon Microscopy in a Zebrafish Larava

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Targeted Neuronal Ablation Using Two-Photon Microscopy in a Zebrafish Larava