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

Estos estudios fueron financiados por subvenciones recibidas del MEXT, JSP KAKENHI Grant números JP25290009, JP25650120, JP17K07494 y JP17H05984.

1. ablación de una subpoblación de neuronas utilizando un microscopio de dos fotones láser
Nota: Si los usuarios plan realizar ablación siguiente imagen Ca, utilice la línea de UAShspzGCaMP6s21. Si los usuarios plan realizar grabación comportamiento después de la ablación, use la línea de UAS:EGFP, como la ablación de las células positivas de EGFP es más fácil de realizar que la de las células GCaMP6s expresan.
<ol><li>Comenzar por estab...

<ol>
	<li>Feierstein, C. E., Portugues, R., Orger, M. 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. S., Fetcho, J. R., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+space+cadet+gene+controls+axonal+pathfinding+of+neurons+that+modulate+fast+turning+movements.">The zebrafish space cadet gene controls axonal pathfinding of neurons that modulate fast turning movements.</a> Development. 128 (11), 2131-2142 (2001).</li><li>Asakawa, K., 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+dissection+of+neural+circuits+by+Tol2+transposon-mediated+Gal4+gene+and+enhancer+trapping+in+zebrafish.">Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.</a> Proc Natl Acad Sci U S A. 105 (4), 1255-1260 (2008).</li><li>Sternberg, J. 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H., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+visually+guided+behavior+by+distinct+populations+of+spinal+projection+neurons.">Control of visually guided behavior by distinct populations of spinal projection neurons.</a> Nat Neurosci. 11 (3), 327-333 (2008).</li><li>Huang, K. H., Ahrens, M. B., Dunn, T. W., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+projection+neurons+control+turning+behaviors+in+zebrafish.">Spinal projection neurons control turning behaviors in zebrafish.</a> Curr Biol. 23 (16), 1566-1573 (2013).</li><li>Smear, M. C., 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=Vesicular+glutamate+transport+at+a+central+synapse+limits+the+acuity+of+visual+perception+in+zebrafish.">Vesicular glutamate transport at a central synapse limits the acuity of visual perception in zebrafish.</a> Neuron. 53 (1), 65-77 (2007).</li><li>Bianco, I. H., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+transformations+underlying+hunting+behavior+in+zebrafish.">Visuomotor transformations underlying hunting behavior in zebrafish.</a> Curr Biol. 25 (7), 831-846 (2015).</li><li>Trivedi, C. A., Bollmann, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually+driven+chaining+of+elementary+swim+patterns+into+a+goal-directed+motor+sequence:+a+virtual+reality+study+of+zebrafish+prey+capture.">Visually driven chaining of elementary swim patterns into a goal-directed motor sequence: a virtual reality study of zebrafish prey capture.</a> Front Neural Circuits. 7, 86 (2013).</li><li>Jouary, A., Haudrechy, M., Candelier, R., Sumbre, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+2D+virtual+reality+system+for+visual+goal-driven+navigation+in+zebrafish+larvae.">A 2D virtual reality system for visual goal-driven navigation in zebrafish larvae.</a> Sci Rep. 6, 34015 (2016).</li><li>Muto, A., Ohkura, M., Abe, G., Nakai, J., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+visualization+of+neuronal+activity+during+perception.">Real-time visualization of neuronal activity during perception.</a> Curr Biol. 23 (4), 307-311 (2013).</li><li>Del Bene, F., 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=Filtering+of+visual+information+in+the+tectum+by+an+identified+neural+circuit.">Filtering of visual information in the tectum by an identified neural circuit.</a> Science. 330 (6004), 669-673 (2010).</li><li>Semmelhack, J. L., 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=A+dedicated+visual+pathway+for+prey+detection+in+larval+zebrafish.">A dedicated visual pathway for prey detection in larval zebrafish.</a> Elife. 3, 04878 (2014).</li><li>Preuss, S. J., Trivedi, C. A., vom Berg-Maurer, C. M., Ryu, S., Bollmann, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+object+size+in+retinotectal+microcircuits.">Classification of object size in retinotectal microcircuits.</a> Curr Biol. 24 (20), 2376-2385 (2014).</li><li>Romano, S. 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=Spontaneous+Neuronal+Network+Dynamics+Reveal+Circuit's+Functional+Adaptations+for+Behavior.">Spontaneous Neuronal Network Dynamics Reveal Circuit's Functional Adaptations for Behavior.</a> Neuron. 85 (5), 1070-1085 (2015).</li><li>Barker, A. J., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+decision+making+in+the+zebrafish+tectum.">Sensorimotor decision making in the zebrafish tectum.</a> Curr Biol. 25 (21), 2804-2814 (2015).</li><li>Ewert, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroethology: an Introduction to the Neurophysiological Fundamentals of Behavior. , (1980).</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=Activation+of+the+hypothalamic+feeding+centre+upon+visual+prey+detection.">Activation of the hypothalamic feeding centre upon visual prey detection.</a> Nat Commun. 8, 15029 (2017).</li><li>Muto, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Imaging+of+Neuronal+Activity+in+Free-Swimming+Larval+Zebrafish.">Calcium Imaging of Neuronal Activity in Free-Swimming Larval Zebrafish.</a> Methods Mol Biol. 1451, 333-341 (2016).</li><li>Westerfield, 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> THE ZEBRAFISH BOOK, 5th Edition. , (2007).</li><li>. 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Aunque el régimen de dos fotones láser tiene una excelente resolución espacial a ablar específicamente las neuronas individuales, gran debe tener precaución para evitar daños no deseados en el tejido cerebral debido a la calor. El paso más importante en el experimento de ablación es determinar la cantidad óptima de la irradiación del laser. Insuficiente irradiación no mata a las neuronas. Demasiada irradiación será daño por calor el tejido circundante, que darán lugar a efectos no deseados. El rango óptim...

Para entender cómo el comportamiento surge de la actividad neuronal en el cerebro, es necesario identificar los circuitos neuronales que intervienen en la generación de ese comportamiento. En la etapa larval, el pez cebra proporciona un modelo animal ideal para estudiar la función cerebral asociado con el comportamiento porque sus cerebros pequeños, transparentes permiten investigar la actividad neuronal en una resolución de celular en una zona amplia de la cerebro mientras observa el comportamiento1. Proyección de imagen de la actividad neuronal en las neuronas específicas ha sido posible a través de la invención de indicadores genético codificados calcio (Ca) (GECIs) como GCaMP2. Pez cebra transgénico GCaMP han demostrado para ser útiles para asociar el circuito neuronal funcional con un comportamiento mediante la realización de proyección de imagen de Ca en comportamiento de animales3.
Mientras que la proyección de imagen de Ca puede demostrar las correlaciones entre la actividad neuronal y el comportamiento, para demostrar causalidad, supresión de la actividad neuronal y su consequence(s) sobre el comportamiento de la prueba son pasos importantes. Hay varias maneras de lograrlo: uso de la mutación genética que altera circuitos neurales específicos4, expresión de neurotoxinas en neuronas específicas5,6, uso de herramientas optogenetic como halorodopsina7, y laser de ablación de las neuronas específicas8,9. Ablación láser es especialmente adecuada para la eliminación de la actividad en un número relativamente pequeño de neuronas específicas. Eliminación irreversible de la actividad neuronal matando neuronas facilita la evaluación de consecuencias conductuales.
Un comportamiento interesante que se puede observar en la etapa larval en el pez cebra es la captura de presas (figura 1A). Este comportamiento guiado visualmente, intencionadas ofrece un favorable sistema experimental para el estudio de la agudeza visual10, transformación visuomotor11,12,13, percepción visual y reconocimiento de objetos14,15,16,17,18y19de la toma de decisiones. Cómo presa es reconocida por depredadores y cómo detección de presa lleva a la presa catching comportamiento ha sido una cuestión central en Neuroetología20. En este artículo, nos centramos en el papel del circuito hipotalámico pretecto formado por proyecciones de un núcleo en el pretectum (núcleo pretectalis superficialis equipara magnocellularis, en adelante, simplemente observado como el pretectum) al ILH. Ablación del laser del pretectum fue demostrada para reducir la actividad de captura de presas y suprimen la actividad neuronal en el ILH asociado a la percepción visual de la presa del21. Aquí, los protocolos para la realización de láser ablación y probar su efecto en Ca2 + la proyección de imagen y grabación de comportamiento describen a las larvas de pez cebra.

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	<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>
		</tr>
		<tr height="40">
			<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>
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			<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>
		</tr>
		<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>
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		<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>
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		<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

Para identificar el papel de una subpoblación de neuronas en el comportamiento, es esencial para poner a prueba las consecuencias de bloquear su actividad en animales vivos. Ablación láser de las neuronas es un método eficaz para este propósito cuando las neuronas son selectivamente marcadas con sondas fluorescentes. En el presente estudio, se describen los protocolos para láser ablación una subpoblación de neuronas utilizando un microscopio de dos fotones y la prueba de sus consecuencias funcionales y conductuales. En este estudio, el comportamiento de captura de presas en las larvas de pez cebra se utiliza como un modelo de estudio. El circuito hipotalámico pretecto se conoce para ser la base de esta presa visualmente impulsado por captura de comportamiento. Pez cebra pretectum fue quitado por ablación láser, y la actividad neuronal en el lóbulo inferior del hipotálamo (ILH; el objetivo de la proyección pretectal) fue examinada. Comportamiento de captura de presas después de la ablación pretectal también fue probado.

Neuronas específicas fueron etiquetadas genéticamente con EGFP o GCaMP6s, cuya expresión fueron conducidos en líneas de Gal4. Un gSAIzGFFM119B de la línea de Gal4 fue utilizada para etiquetar un núcleo en el área pretectal (núcleo pretectal superficial magnocelulares) y una subpoblación de neuronas del bulbo olfatorio. Otra línea de Gal4, hspGFFDMC76A, fue utilizado para etiquetar el ILH. Laser-ablación las neuronas pretectal bilateral (panel de la izquierda<strong class="xfig"...

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

Aquí, presentamos un protocolo para ablación de una subpoblación genéticamente marcada de neuronas por un láser de dos fotones de larvas de pez cebra.

Ablación de una población Neuronal utilizando un láser de dos fotones y su evaluación mediante grabación comportamiento y proyección de imagen de calcio en las larvas de pez cebra

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

Automatizados análisis de comportamiento de alto rendimiento en larvas de pez cebra

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Investigación

Neurociencias

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De dos fotones<em&gt; In vivo</em&gt; Imágenes de las espinas dendríticas en la corteza del ratón Usando un Diluido-cráneo Preparación

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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

La incubación prolongado de aguda neuronal de tejidos para la Sección de Electrofisiología y calcio de imágenes

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Proyección de imagen de calcio de dos fotones en las dendritas neuronales en rebanadas de cerebro

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

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Cuantificación De La Ingesta De Macronutrientes En Una Pantalla Neuronal Termogenética Usando Larvas De Drosophila

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 Imágenes de calcio en la aceituna inferior de ratón

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

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

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In vivo Imágenes de calcio de campo amplio y dos fotones de un ratón usando una ventana craneal grande

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 ...

Seguimiento del comportamiento y patrón de actividad neuronal en el prosencéfalo del pez cebra

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

Autor destacado: Avances en la investigación del cerebro del pez cebra adulto 

Imágenes de calcio de dos fotones de la actividad del prosencéfalo en peces cebra adultos que se comportan

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