Name: ablation of a neuronal population using a two-photon laser and its assessment using calcium imaging and behavioral recording in zebrafish larvae
Uploaded: 2018-06-02T13:00:00
Description: Watch this Scientific Journal Video about Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae at JoVE.com

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 t...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+whole+picture:+A+comprehensive+imaging+approach+to+functional+mapping+of+circuits+in+behaving+zebrafish.">Seeing the whole picture: A comprehensive imaging approach to functional mapping of circuits in behaving zebrafish.</a> Neuroscience. 296, 26-38 (2015).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19 (2), 137-141 (2001).</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=Genetic+visualization+with+an+improved+GCaMP+calcium+indicator+reveals+spatiotemporal+activation+of+the+spinal+motor+neurons+in+zebrafish.">Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish.</a> Proc Natl Acad Sci U S A. 108 (13), 5425-5430 (2011).</li><li>Lorent, K., Liu, K. 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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.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">NuSieve GTG Agarose</td>
			<td style="width:223px;">Lonza</td>
			<td style="width:361px;">Cat.#50080</td>
			<td style="width:383px;">low-melting temperature agarose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6 cm petri dish</td>
			<td>FALCON</td>
			<td>Product#:351007</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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>
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			<td height="40" style="height:40px;">LSM7MP</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>two-photon laser scanning microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">W Plan-Apochromat 63x/1.0</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>63X objective lens</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Imager.Z1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>an epi-fluorescence microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ZEN</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Image acquisition software for confocal microscopes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">surgical knife</td>
			<td>MANI</td>
			<td>Ophthalmic knife MST15</td>
			<td></td>
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			<td height="40" style="height:40px;">ORCA-Flash4.0</td>
			<td>Hamamatsu Photonics</td>
			<td>model:C11440-22CU</td>
			<td>a scientific CMOS camera</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:316px;">HCImage</td>
			<td>Hamamatsu Photonics</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">image acuisition software</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Hard Disk Recording module</td>
			<td>Hamamatsu Photonics</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">An software module that enables saving the movie files onto a hard disc drive in a short time</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SZX7</td>
			<td>Olympus</td>
			<td></td>
			<td>stereoscope</td>
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		<tr height="40">
			<td height="40" style="height:40px;">DF PL 0.5X</td>
			<td>Olympus</td>
			<td></td>
			<td>objective lens for SZX7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">XIMEA xiQ camera</td>
			<td>XIMEA</td>
			<td>Product No. MQ042RG-CM</td>
			<td>CMOS camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">a ring LED light</td>
			<td>CCS</td>
			<td>Model: LDR2-100SW2-LA</td>
			<td>White LED</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;"></td>
			<td style="width:223px;"></td>
			<td style="width:361px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nylon mesh 32&#181;m</td>
			<td>Tokyo Screen</td>
			<td>N-No.380T</td>
			<td>http://www.tokyo-screen.com/cms/sta20347/</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Nylon mesh 13&#181;m</td>
			<td>Tokyo Screen</td>
			<td>N-No. 508T-K</td>
			<td style="width:383px;">http://www.tokyo-screen.com/cms/sta20347/</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:316px;">Metal seive 150 micron aperture</td>
			<td>Tokyo Screen</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">http://www.tokyo-screen.com/cms/sta20341/#ami</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">Metal seive 75 micron aperture</td>
			<td>Tokyo Screen</td>
			<td></td>
			<td>http://www.tokyo-screen.com/cms/sta20341/#ami</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">EBIOS</td>
			<td>Asahi Food &#38; Healthcare, Co. Ltd.</td>
			<td></td>
			<td>dry beer yeast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">LabVIEW</td>
			<td>National Instruments</td>
			<td></td>
			<td>an integrated development environment for programming</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;">Mai-Tai HP</td>
			<td>Spectra Physics&#160;</td>
			<td></td>
			<td>two-photon laser&#160;</td>
		</tr>
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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

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...

Watch this Scientific Journal Video about Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae at JoVE.com

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 ...

The overall goal of this experiment is to test the effect of laser ablation of certain neurons on circuit activity and behavior. With this experimental technique we can examine the necessity of neurons in a circuit underlying behavior. This method can help answer our key questions in the behavior and neuroscience field such as how prey is perceived and how prey captured behavior is generated by neural activity.The main advantage of this technique is that neurons can be specifically ablated because of the genetic labeling of these neurons and also high spatial resolution of the laser. After raising larvae and mounting them in 2%low melting agarose according to the text protocol place the agarose embedded larvae under a two 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 subjective 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 zebra fish 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 subjective 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 1/3 the cell's size in diameter on each neuron as an ROI.Set the scan speed to 13.9 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 four 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. Carryout 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. Following irradiation the larvae needs to be recovered. Carefully make tiny perpendicular cuts in the agarose around the larvae.Then, wait for the fish to swim out of the agarose on its own when the anesthetic wears off. After culturing paramecia and anesthetizing a zebra fish larva according to the text protocol place the larvae into 2%low melting point agarose and immediately remove any excess agarose so that the surface of the agarose looks slightly convex. Using a dissecting needle quickly orient the larvae in an upright position.Then, using a surgical knife make a few small cuts in the agarose to remove the agarose around the zebra fish head which makes space for the paramecium to swim. Carefully remove the residual agarose by gently pouring system water on the chamber. Next, put one paramecium in the recording chamber to serve as a visual stimulus.Then place the recording chamber under an epifluorescence microscope equipped with a scientific CMOS camera and select a 2.5X objective lens. Start the image acquisition application for the equipped camera. Click sequence pane and select hard disk record on the pane.In the scan setting section set the frame count to 900 or any other total frame number desired. In the capture pane set the exposure time at 30 milliseconds and binning to two by two. Click live on the capture pane to locate the larvae in the camera view and focus and then click stop live and click the start button.Save the movie in cxd format and analyze the data according to the text protocol. After setting up the behavioral recording system and placing a larva into a recording chamber according to the text protocol use a micro pipette 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, carryout analysis according to the text protocol.In this experiment with the Gal4 line that labels nuclei in the pretectal area and a subpopulation of olfactory bulb neurons. The pretectal neurons were laser ablated bilaterally. Olfactory neurons were ablated as a control.The results show that the two photon laser can ablate targeted cells while leaving adjacent neurites unaffected. In this experiment the functional connectivity of pretectal neurons that project their axons toward the ILH ipsilaterally were investigated. Both the pretectum and the ILH showed neuronal activity in the proximal presence of prey suggesting that both neuronal activities are visually driven.Calcium imaging was also used to observe neuronal activity in the pretectum and the ILH in pretectal neuron ablated larvae. The left pretectum that was laser ablated showed residual to no neuronal activity. In addition, the ipsilateral ILH displayed dramatically displaced neuronal activity suggesting that the major input to the ILH comes from the ipsilateral pretectum.In contrast the ILH on the other side of the laser ablated pretectum showed neuronal activity comparable to the neuronal activity in the ipsilateral pretectum which suggests that the right ILH is receiving inputs from the right pretectum. Once mastered laser ablation can be done in about one hour for each zebra fish larva. While attempting this procedure it's important to remember to ablate only the targeted neurons with proper laser irradiation avoiding heat damage by the laser.Following this procedure ablation of neurons in other brain areas like forebrain or hindbrain can be performed in order to answer additions questions like how these neurons are involved in cognitive functions or motor functions. After its development this technique paved the way for researchers in the field of behavioral neuroscience to explore functional neural circuits in zebra fish models. After watching this video you should have a good understanding of how to specifically laser ablate neurons in the zebra fish larvae brain and assess the functional and behavioral consequences.Don't forget that working with a laser can be extremely hazardous and always comply with the manufacturer's instructions while preforming this procedure.

Research

JoVE Journal

Neuroscience

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

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Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

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Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

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Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

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Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

In vivo Calcium Imaging in Mouse Inferior Olive

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In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

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

Author Spotlight: Advancements in Adult Zebrafish Brain Research 

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