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

Abstract

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 Zebrafish Larvae. J. Vis. Exp. (2018)

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.

Protocol

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope

1. 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 (Lobe of the hypothalamus) are used.

2. 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.

3. Mount the zebrafish larva in 2% low melting point-agarose (Figure 1B).

1. 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.

   1. 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.

   2. 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.

   3. Once the larva is positioned, allow 5 min for the agarose to solidify completely.

   4. 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.

4. Ablate the larva using the bleaching function in a two-photon laser microscopy system.

1. Place the agarose-embedded larva under a two-photon laser scanning microscope and start the microscopy system.

2. 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."

3. 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.

4. 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.

5. Locate the zebrafish larval brain at the center of view; then click the "Acquisition" tab to go back to the two-photon microscopy.

6. 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.

7. Select the 63X objective lens by manually moving the lens revolver.

8. 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.

5. On the "Acquisition Mode" tab, set the "Frame Size" at 256 x 256. Click "Live" and observe the neurons of interest.

6. Starting from the dorsal-most side, find a focal plane in which the cells to be ablated are in focus.

7. 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.

8. Set the scan speed to 13.93 s/256 x 256 pixels (134.42 µm x 134.42 µm), which corresponds to a laser dwell time of approximately 200 µs/pixel, or 200 µs/0.5 µm. Also, set 4 repetitions ('Iteration cycle: 4'). Alternatively, optimize the number of iterations and scanning speed so that sufficient ablation is achieved.

9. 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) .

10. 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).

11. If fluorescence is still present after ablation, increase the number of iterations.

12. Next, move the focal plane slightly deeper (i.e., towards the ventral side), and choose the next focal plane where un-ablated cells appear.

13. Perform the bleaching function. Repeat these steps until all the cells in the neural structure of interest have been ablated.

14. 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.

15. 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.

16. 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).

17. 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.

Results

Materials

List of materials used in this article

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