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

これらの研究は、文部科学省、日本学術振興会科研費助成番号 JP25290009、JP25650120、JP17K07494、および JP17H05984 から受け取った補助金によって賄われました。

1。 2 光子レーザー顕微鏡を用いたニューロンの亜種のアブレーション
注:Ca イメージング次アブレーションを実行する場合は、UAShspzGCaMP6s ライン21を使用します。アブレーションに続く行動の記録を実行する場合は、EGFP 陽性細胞の切除は GCaMP6s 発現細胞のよりも簡単に UAS:EGFP 線を使用します。
<ol><li>調査される特定のニューロン、UAS:EGFP ...

<ol>
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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. 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2 光子レーザーには、具体的には個々 のニューロンをアブレーションする優れた空間分解能がありますが、偉大な用心は、暑さで脳組織上の任意の不要な損傷を避けるために取られるべき。アブレーションの実験で最も重要なステップは、レーザー照射の最適な量を決定することです。不十分な照射は、ニューロンを殺すために失敗します。あまりにも多くの照射、熱損傷周囲の組織は、望?...

動作が脳の神経細胞の活動から発生する方法を理解するには、行動の生成に関与している神経回路を識別するために必要です。幼虫の段階で、ゼブラフィッシュは、理想的な動物モデル小さく、透明な脳の広範な領域での携帯電話の解像度で神経細胞の活動を調査することが可能になるため、動作に関連付けられている脳機能を勉強して、1の動作を観察しながら脳。特定のニューロンの神経活動のイメージングは、GCaMP2など遺伝的コード化カルシウム (Ca) 指標 (GECIs) の発明によって可能にしました。GCaMP トランスジェニックゼブラフィッシュ行動動物3Ca イメージングを行うことにより、機能的な神経回路動作を関連付けるために有用であると証明しました。
Ca イメージングは、神経活動と行動間の相関関係を示すことが、一方の抑制神経細胞の活動と行動にその consequence(s) をテストは、因果関係を示す、重要な手順です。これを達成するためにさまざまな方法があります: 特定の神経回路4、特定のニューロン5,6ハロロドプシン7などの光遺伝学的ツールの使用で神経毒の発現を変化させる遺伝子の突然変異の使用と対象となるニューロン8,9のレーザーアブレーションします。レーザーアブレーションは比較的少数の特定のニューロンの活動を排除することに特に適しています。神経細胞を殺すことによって神経細胞の活動の不可逆的な除去を促進する行動の結果を評価します。
ゼブラフィッシュの幼虫の段階で観察することができます 1 つの興味深い現象は、獲物のキャプチャ (図 1A) です。この視覚誘導、目的指向行動視力10、視覚運動変換11,12,13視知覚と認識の研究のため有利な実験システムを提供します。オブジェクト14,15,16,17,18, および意思決定19。どのように獲物は神経行動学20の中心的な質問をされている行動をキャッチ捕食者と獲物の検出が獲物につながる方法によって認識されます。本稿で我々 は、pretectum の核の投射によって形成される pretecto 視床下部の回路の役割に焦点を当てる (核 pretectalis 浅パルス大、以下、単に、pretectum として有名)、または喘鳴症に。獲物捕獲活動を減らす visual 獲物知覚21に関連付けられているまたは喘鳴症で神経活動を廃止し、pretectum のレーザー ・ アブレーションを示した。ここでは、実行するプロトコル レーザー アブレーションと幼虫のとおりゼブラフィッシュの Ca2 +イメージングと行動記録を使用してその効果をテストします。

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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>
		</tr>
		<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

動作における神経細胞の亜集団の役割を識別するためには、生きている動物の活動をブロックの結果をテストするのには不可欠です。レーザーアブレーションのニューロンは、神経細胞が選択的に蛍光プローブ付けこの目的のための効果的な方法です。現在の研究では、2 光子励起顕微鏡を使用して、その機能と行動の結果のテストは神経細胞の亜集団をアブレーション レーザーのプロトコルを説明します。本研究ではゼブラフィッシュ幼虫で獲物のキャプチャ動作は研究モデルとして使用されます。Pretecto 視床下部の回路動作をキャッチこの視覚的に駆動される獲物の根底に知られています。ゼブラフィッシュ pretectum レーザー蒸散、視床下部 (または喘鳴症; pretectal 投影のターゲット) の下の葉の神経活動を調べた。Pretectal アブレーション後獲物キャプチャ動作もテストされました。

特定のニューロンは EGFP と式は Gal4 行で運転された GCaMP6s 遺伝子標識しました。Gal4 ライン gSAIzGFFM119B は、pretectal エリア (大浅 pretectal 核)、および嗅球神経細胞の亜集団の核をラベルに使用されました。別の Gal4 ライン、hspGFFDMC76A、だった、または喘鳴症に使用されます。私たちレーザーアブレーション pretectal ニューロン両側 (図 2...

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

ここでは、ゼブラフィッシュ幼虫からの 2 光子レーザーによる神経細胞の遺伝的標識個体をアブレーションするプロトコルを提案する.

二光子励起レーザーとゼブラフィッシュ幼虫におけるカルシウム イメージング法と行動記録を用いた評価を用いたニューロン集団のアブレーション

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

ゼブラフィッシュ幼生における自動化ハイスループット行動分析

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 ...

神経科学

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

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

二光子<em&gt; in vivoで</em&gt;間引き頭蓋骨の準備を使用して、マウス皮質における樹状突起棘のイメージング

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

電気生理学およびカルシウムイメージングのための急性神経組織の長期のインキュベーション

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

脳スライスにおける神経樹状突起における 2 光子カルシウム イメージング

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

ショウジョウバエ幼虫を用いた熱遺伝学的神経細胞スクリーンにおけるマクロ栄養素摂取量の定量化

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

In vivo Calcium Imaging in Mouse Inferior Olive

インビボ マウス劣オリーブにおけるカルシウムイメージング

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

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

インビボ 大きな頭蓋窓を用いたマウスからの広視野および2光子カルシウムイメージング

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 ...

ゼブラフィッシュの前脳における行動追跡と神経活動パターン

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

著者スポットライト:成体ゼブラフィッシュの脳研究の進歩 

ゼブラフィッシュ成魚の行動性における前脳活動の2光子カルシウムイメージング

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

二光子励起レーザーとゼブラフィッシュ幼虫におけるカルシウム イメージング法と行動記録を用いた評価を用いたニューロン集団のアブレーション

二光子励起レーザーとゼブラフィッシュ幼虫におけるカルシウムイメージング法と行動記録を用いた評価を用いたニューロン集団のアブレーション