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

这些研究由下个、jsp KAKENHI 赠款编号 JP25290009、JP25650120、JP17K07494 和 JP17H05984 提供的赠款资助。

1. 用双光子激光显微镜消融神经元亚群
注意:如果用户计划在消融后执行 Ca 映像, 请使用 UAShspzGCaMP6s 行21。如果用户计划在消融后执行行为记录, 使用无人参与: EGFP 线, 因为 EGFP 阳性细胞的消融比 GCaMP6s-expressing 细胞更容易执行。
<ol><li>首先, 建立一个 Gal4 线的交配, 标签特定的神经元要研究和无人操作: EGFP 或 UAShspzGCaMP6s。确保对两个父级都使用...

<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. 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. R., 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=Optimization+of+a+Neurotoxin+to+Investigate+the+Contribution+of+Excitatory+Interneurons+to+Speed+Modulation+In+Vivo.">Optimization of a Neurotoxin to Investigate the Contribution of Excitatory Interneurons to Speed Modulation In Vivo.</a> Curr Biol. , (2016).</li><li>Arrenberg, A. B., Del Bene, F., 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=Optical+control+of+zebrafish+behavior+with+halorhodopsin.">Optical control of zebrafish behavior with halorhodopsin.</a> Proc Natl Acad Sci U S A. 106 (42), 17968-17973 (2009).</li><li>Orger, M. B., Kampff, A. R., Severi, K. E., Bollmann, J. 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. 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虽然双光子激光器有一个很好的空间分辨率来专门消融单个神经元, 但应谨慎地避免因热而对脑组织造成任何不应有的损伤。在烧蚀实验中, 最重要的一步是确定激光辐照的最佳量。辐射不足无法杀死神经元。太多的辐照会对周围的组织造成热损伤, 这将导致不想要的效果。激光辐照的最佳范围 (ROIs 区域、迭代次数和扫描速度) 似乎很窄。影响消融效率的因素有几个。
首先, 使...

为了理解大脑中神经元活动的行为是如何产生的, 有必要识别出这种行为产生的神经回路。在幼虫阶段, 斑马鱼提供了一个理想的动物模型来研究与行为相关的大脑功能, 因为它们的小而透明的大脑使人们有可能在一个广泛的区域内研究细胞分辨率的神经元活动。大脑, 同时观察行为1。通过基因编码钙 (Ca) 指标 (GECIs) (如 GCaMP2) 的发明, 可以使特定神经元的神经元活动成像成为可能。GCaMP 转基因斑马鱼已证明是有用的关联功能性神经回路与行为通过进行 Ca 成像在行为动物3。
虽然 Ca 成像可以证明神经元活动和行为之间的相关性, 显示因果关系, 抑制神经元活动和测试其后果的行为是重要的步骤。有多种方法可以实现这一点: 利用基因突变改变特定的神经回路4, 在特定神经元5,6中表达毒素, 使用 optogenetic 工具 (如 halorhodopsin7) 和靶向神经元的激光消融8,9。激光消融特别适合于在相对少量的特定神经元中消除活性。通过杀死神经元不可逆转地消除神经元活动有助于评估行为后果。
在斑马鱼幼虫阶段可以观察到的一个有趣的行为是猎物捕获 (图 1A)。这种视觉引导, 目标导向的行为提供了一个良好的实验系统的视觉敏锐度的研究10, visuomotor 转换11,12,13, 视觉感知和识别对象14,15,16,17,18, 决策19。在神经行为学20 中, 捕食者如何识别猎物以及猎物检测如何导致猎物捕获行为一直是一个中心问题。在本文中, 我们关注的作用, 形成的 pretecto-下丘脑的 pretectum (核 pretectalis superficialis, 此后, 简单地指出 magnocellularis) 的核投射到 pretectum。激光消融的 pretectum 被证明可以减少猎物捕获活动, 并废除与视觉猎物知觉21相关的 ILH 中的神经元活动。这里介绍了使用 Ca2 +成像和行为记录在斑马鱼幼虫中进行激光消融和测试效果的协议。

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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>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">surgical knife</td>
			<td>MANI</td>
			<td>Ophthalmic knife MST15</td>
			<td></td>
		</tr>
		<tr height="40">
			<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>
		</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>
	</tbody>
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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

为了确定神经元的亚群在行为中的作用, 必须测试阻断其在活动物中活动的后果。当神经元选择性地用荧光探针标记时, 激光消融神经元是一种有效的方法。在本研究中, 介绍了用双光子显微镜对神经元进行亚群的激光烧蚀及其功能和行为后果的测试。本研究采用斑马鱼幼虫捕食行为作为研究对象。pretecto-下丘脑电路是已知的基础上这种视觉驱动的猎物捕捉行为。斑马鱼 pretectum 激光消融, 下丘脑下叶 (ILH; pretectal 投射的目标) 进行了神经活性检查。pretectal 消融后的猎物捕获行为也进行了测试。

特定神经元的基因标记为 EGFP 或 GCaMP6s, 其表达在 Gal4 线驱动。Gal4 线 gSAIzGFFM119B 用于标记 pretectal 区 (巨浅 pretectal 核) 的细胞核, 以及嗅球神经元的亚群。另一个 Gal4 线, hspGFFDMC76A, 被用来标记 ILH。我们对 pretectal 神经元进行了双侧激光消融 (图 2A左面板), 并在 Gal4 线的斑马鱼幼虫中以双边方式将嗅球中的神经元消融 (图 2</s...

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

在这里, 我们提出一个协议, 以消融基因标记亚群的神经元由双光子激光从斑马鱼幼虫。

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

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 ...

Research

JoVE Journal

Neuroscience

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

在斑马鱼幼虫的高通量自动化的行为分析

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 ...

科研

神经科学

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

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

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

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

对于电生理和钙成像急性神经组织的长时间培养

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

脑切片神经元树突的双光子钙成像

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

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

在体内 小鼠劣质橄榄中的钙成像

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 ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

使用双光子全息显微镜评估和操纵神经活动

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

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

体内 使用大颅窗的小鼠宽场和双光子钙成像

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 ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

使用双光子显微镜对闭合颅骨创伤性脑损伤和颅窗小鼠荧光蛋白表达进行活体成像

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

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

Advancements in Adult Zebrafish Brain Research (Video) | JoVE

成年斑马鱼前脑活动的双光子钙成像

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