Name: ablation of a neuronal population using a two-photon laser and its assessment using calcium imaging and behavioral recording in zebrafish larvae
Uploaded: 2018-06-02T13:00:00.000+00:00
Duration: 10 min 29 s
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珍珠珠背景, 以便可以获得珍珠层纯合体。 注意:纯合体的珍珠层不包含体表面上的 melanophores ( 图1 B).在本研究中, Gal4 线 gSAIzGFFM119B (标签 pretectum) 和另一个 Gal4 线 hspGFFDMC76A (标签 ILH) 使用21。</li>
	<li>在交配设置的第二天, 收集斑马鱼的卵, 并把它们提高到幼虫阶段, 此时神经元的兴趣将表达荧光记者。</li>
	<li>将斑马鱼幼虫装入2% 低熔点-琼脂糖 (图 1B)。<ol>
<li>首先准备2% 琼脂糖溶液: 在100毫升系统水中溶解2克低熔点琼脂糖粉 (即, 用于在循环水系统中维持成年斑马鱼的水)22或 E3 水23, 通过将水在微波炉的沸点, 并大力搅拌。</li>
		<li>微波2% 低熔点琼脂糖, 直到它沸腾。将 3.5-4.0 毫升的琼脂糖倒入6厘米培养皿的盖子上。等待, 直到琼脂糖冷却, 以便它是温暖的触摸前继续。</li>
		<li>接下来, 在琼脂糖中放置一个幼虫 (不在这个步骤中被麻醉)。继续调整幼虫的位置和方向, 使其直立, 使用解剖针 (图 1C), 直到琼脂糖固化, 以确保幼虫的正确定位。 注意:当琼脂糖凝固时, 幼虫会继续游动。</li>
		<li>一旦幼虫定位, 允许5分钟的琼脂糖完全凝固。</li>
		<li>在琼脂糖上倒入5毫升的 tricaine 溶液 (0.4% 在1x 工作浓度)。 注意:为了避免幼虫在激光消融过程中的运动, 幼虫必须在消融过程中保持麻醉。</li>
	</ol></li>
	<li>利用双光子激光显微系统中的漂白功能消融幼虫。<ol>
<li>将琼脂糖嵌入幼虫置于双光子激光扫描显微镜下, 启动显微系统。</li>
		<li>启动图像采集软件, 然后单击 "激光" 选项卡上的 "On" 按钮打开激光 (图 1D)。等待激光的 "状态" 从 "忙" 变为 "锁模"。</li>
		<li>在 "通道" 选项卡上, 将激光波长设置为 880 nm (最大功率: 约2300兆瓦) 用于 EGFP 消融, 或在 800 nm (最大功率: 约2600兆瓦) 为 GCaMP 消融。</li>
		<li>通过手动移动镜头左轮手枪, 选择20X 物镜。点击 "定位" 选项卡, 点击 "GFP" 改变光通路, 直接查看荧光的眼睛。</li>
		<li>在视野中心找到斑马鱼幼虫的大脑;然后单击 "获取" 选项卡返回双光子显微镜。</li>
		<li>作为一个记录的 ' 消融前 ' 的条件, 采取 z 堆栈图像与20X 物镜在快速扫描速度 (以避免热损伤)。稍后比较在消融实验之前和之后拍摄的图像 (图 2A)。若要获取 z 堆栈, 请单击 "z 堆栈" 以选择 z 堆栈选项并展开该选项卡. 设置下限 (单击 "设置第一个" 按钮 ") 后, 聚焦在幼虫大脑的腹端, 并设置上限 (单击" 设置最后一个 "按钮) 后, 重点放在大脑的背部最表面。然后, 单击 "开始实验" 按钮以运行 z 堆栈图像获取。</li>
		<li>通过手动移动镜头左轮手枪, 选择63X 物镜。</li>
		<li>点击 "定位" 选项卡切换到荧光显微镜, 找到细胞被消融在视线的中心, 然后点击 "采集" 标签返回激光扫描显微镜。</li>
	</ol></li>
	<li>在 "获取模式" 选项卡上, 将 "帧大小" 设置为 256 x 256。单击 "实时" 并观察感兴趣的神经元。</li>
	<li>从背部最一侧开始, 找到一个焦平面, 其中的细胞被消融的焦点。</li>
	<li>用 "区域" 函数标记一个小的圆形区域, 大约第三个细胞的直径在每个神经元上作为一个感兴趣的区域 (ROI)。</li>
	<li>将扫描速度设置为 13.93 s/256 x 256 像素 (134.42 µm x 134.42 µm), 对应于大约200个µs/像素的激光驻留时间, 或200µs/0.5 µm。另外, 设置4重复 ("迭代周期: 4")。或者, 优化迭代次数和扫描速度, 以便实现足够的消融。</li>
	<li>执行 "漂白" 功能的消融, 结合 "时间序列 (2 周期)", 以图像的样本 (在消融之前和之后) 和 ' 地区 ' (以设置 ROIs) 或等效函数在使用的软件 (图 1D)。</li>
	<li>通过比较第一个图像 (消融前) 和第二个图像 (消融后) 的时间序列周期, 确保漂白后, 目标细胞中的荧光减少到在背景水平观察 (图 2B)。</li>
	<li>如果在消融后仍然存在荧光, 则增加迭代次数。</li>
	<li>接下来, 将焦点平面稍微更深地移动 (即, 朝向腹侧), 然后选择下一个焦平面, 其中出现未消融的细胞。</li>
	<li>执行以上所述的漂白功能 (步骤 1.9)。重复这些步骤, 直到所有的神经结构的细胞被消融。</li>
	<li>经过所有的焦平面覆盖的神经结构被消融, 检查细胞, 以废除荧光。如果某些细胞因消融不足而被发现仍然是荧光的, 那么激光照射它们就如上文所述。</li>
	<li>如果幼虫需要在激光照射后从琼脂糖中恢复, 不要试图强迫它脱离琼脂糖, 因为这可能会损害幼虫。相反, 小心地将琼脂糖周围的小切口垂直地放到身体表面。然后, 等待幼虫从琼脂糖上游出来时, 麻醉剂就会褪去。</li>
	<li>进入下一个幼虫进行消融或进行控制实验, 使用另一种群的神经元无关的行为正在调查中。 注意:在这里, 作为一个控制, 嗅球神经元的无人参与: EGFP;gSAIzGFFM119B 幼虫使用上面描述的相同协议进行激光消融 (图 2A, 右)。</li>
	<li>在继续进行 Ca 成像 (2 节) 或行为记录 (3 节) 之前, 允许几个小时或1天进行恢复。在此恢复时间, 检查幼虫健康 (即, 正常自发游泳, 消融区周围没有明显的热损伤,等), 并删除显示任何健康不良迹象的幼虫。</li>
</ol>2. 钙成像记录 Pretectum 烧蚀斑马鱼幼虫的猎物诱发神经元活动
<ol><li>要准备一个记录室, 在玻璃滑块上放置一个安全密封的杂交室垫片, 与玻璃上的粘接面, 并卸下顶部密封。 注意:这将创建一个小的记录室, 其直径为9毫米, 深度为0.8 毫米 (图 3a)。</li>
	<li>接下来, 在一个50毫升的管子上放置一个尼龙网 (开口大小:32 µm), 底部切口打开。使用 cap 在管上附加网格 (图 3B)。</li>
	<li>准备草履虫(这是用作幼虫视觉刺激的猎物)。 注意:预先准备草履虫区域性 (步骤2.3.1 和 2.3.2)。<ol>
<li>提前从商业来源或学术生物资源中心获取草履虫种子区域性24 。</li>
		<li>在含蒸压稻草和一粒干啤酒-酵母 (图 3C) 的水中培养草履虫数天。</li>
		<li>依次将草履虫区域性倒入2个筛 (一个具有150µm 光圈, 后跟一个 75-µm 光圈), 以去除该区域性的碎片。</li>
		<li>收集从上一步骤到尼龙网格 (13-µm 光圈) 的流程中的草履虫。</li>
		<li>使用系统水的压缩瓶 (图 3D), 将尼龙网格上的草履虫重新挂载到玻璃烧杯中。</li>
	</ol></li>
	<li>在系统水 2x (图 3E) 中冲洗网格 (图 3B)。 注意:此步骤的目的是删除草履虫培养基中包含的任何可能的气味和 tastants, 因为本研究的目的是观察视觉驱动的神经元活动。</li>
	<li>将斑马鱼幼虫放在 tricaine 溶液中麻醉。</li>
	<li>在2% 低熔点琼脂糖中安装一个幼虫。<ol>
<li>首先, 微波2% 低熔点琼脂糖, 并添加1/10 体积10x 浓缩 tricaine 的琼脂糖。</li>
		<li>将一滴 tricaine 包含的琼脂糖放到录音室 (图 3A)。</li>
		<li>确认琼脂糖不太热后, 将麻醉幼虫 (从步骤 2.5) 放入琼脂糖, 立即去除任何多余的琼脂糖, 使琼脂表面看起来略有凸。</li>
		<li>用解剖针快速将幼虫定向到直立位置 (图 1C)。 注意:这些程序必须在几秒钟内完成琼脂糖固化。</li>
		<li>一旦幼虫正确定位, 允许5分钟琼脂糖完全凝固。然后, 在琼脂糖上倒一滴 1x tricaine 溶液。</li>
	</ol></li>
	<li>除去斑马鱼幼虫头部周围的琼脂糖, 并为草履虫使用外科刀游泳提供空间。为此, 首先, 在琼脂糖中进行一些小切口 (图 3F)。然后, 小心地去除多余的琼脂糖, 轻轻地将系统水倒在房间里。 注意:在这一步, tricaine 将被淘汰。</li>
	<li>接下来, 在录音室中放入一个草履虫作为视觉刺激。 注意:当草履虫在斑马鱼幼虫的头部附近游动时, 它会唤起神经元的响应 (图 3G)。</li>
	<li>将录音室置于装有科学 CMOS 照相机的荧光显微镜下 (图 3H), 然后选择2.5X 物镜 (图 3I)。</li>
	<li>收集 GCaMP 荧光信号的定时记录。<ol>
<li>启动已装备相机的图像采集应用程序。</li>
		<li>单击 "序列窗格", 然后在窗格上选择 "硬盘记录"。在 "扫描设置" 部分, 将 "帧计数" 设置为900或所需的任何其他总帧数。 注意:如果可用, 请使用带有模块 (此处为 "硬盘记录") 的定时录制软件, 以便将数据有效地保存到硬盘上。</li>
		<li>在 "捕获" 窗格中, 将曝光时间设置为30毫秒 (即、33.3 fps) 和 Binning 2 x 2。</li>
		<li>在显微镜控制触摸屏上, 选择一个用于 gfp 的过滤器, 因为 GCaMP 有类似于 gfp 的激发/发射光谱。</li>
		<li>单击 "捕获" 窗格上的 "实时", 在照相机视图和焦点 (图 4A) 中找到幼虫, 然后单击 "停止实时", 然后单击 "开始" 按钮。将影片保存在. cxd 或任何其他格式中, 以保留有关获取条件的信息。</li>
	</ol></li>
	<li>录音后, 通过使用自由软件斐济25, 通过获取在时间推移影片中设置的 ROI 的平均像素值, 分析 GCaMP6s 荧光信号 (Ca 信号)。 注意:斐济是一个版本的 ImageJ, 有许多有用的插件预装, 可以读取. cxd 使用生物格式插件格式化图像文件。</li>
	<li>如果幼虫在录制过程中稍有移动, 请在斐济运行 TurboReg 插件26执行图像注册。从菜单中, 选择 "插件"、"注册" 和 "TurboReg"。</li>
	<li>考虑如何根据研究的目的分析数据。下面是一个示例。<ol>
<li>计算 F/F0 (每一次的荧光强度, 除以静止时的荧光强度或发生任何 Ca 瞬态之前), 如下所示:</li>
		<li>使用 ROI 管理器在 pretectum 或 ILH 上设置 ROIs: 在菜单中, 选择 "分析"、"工具"、"ROI 管理器" 和 "添加" 到 ROIs 列表 (图 4A)。</li>
		<li>通过在 ROI 管理器面板上选择 ROIs (i. e) 的平均像素值 (GCaMP6s 的荧光强度): "更多"、"多度量" 和 "确定"。将结果另存为电子表格文件。</li>
		<li>由于信号很嘈杂, 因此使用可以处理电子表格数据的应用程序, 平均11个时间点 (移动平均值) 的信号。</li>
		<li>通过 F0 (基底层的荧光强度) 将 F (荧光强度随时间) 除以, 以计算归一化荧光强度。作为数据可视化的示例, pretectum 和 ILH 中规范化的 GCaMP6s 荧光强度的变化是彩色编码的, 并映射在草履虫轨迹上 (图 4B D)。</li>
	</ol></li>
</ol>3. 激光消融后行为后果的评估
<ol><li>建立一个由装有摄像机的立体镜组成的行为记录系统。使用具有适当图像采集软件的照相机, 商业或定制 (图 5a)。</li>
	<li>优化光照条件, 以便通过图像处理可以检测到草履虫。例如, 使用带分隔符的环形白色 LED 灯 (图 5B)。 注意:LED 的距离和角度影响示例的外观 (即, 在深色背景下亮起或在明亮背景下为深色), 因此对成功的图像处理至关重要。确定间隔的最佳高度 (图 5C)。</li>
	<li>将斑马鱼幼虫 (从1节中描述的激光消融实验中恢复) 放在一个行为记录室 (连接到玻璃幻灯片上), 并卸下原来的顶部封条 (图 5D)。</li>
	<li>使用微从区域性 (步骤 2.3.5) 收集 50草履虫, 并将它们放在记录室中。</li>
	<li>在录音室的顶部放置一个盖子。把一小滴水放在盖子上, 放在记录室的水面上, 以免在室内引入空气。 注意:在此步骤中, 某些带有草履虫的水将从记录室溢出。记录室中的草履虫的剩余数目将大约为30-40。</li>
	<li>将录音室 (包含幼虫和草履虫) 放在照明系统中 (图 5D)。</li>
	<li>以 10 fps 为11分钟 (6600 帧) 开始定时录制。</li>
	<li>选对于每一个为行为进行测试的幼虫, 通过荧光显微镜观察激光消融区域的荧光, 以确认激光烧蚀的有效性 (即、缺席或显著减少的荧光细胞数)。激光照射的区域)。 注意:即使辐照后, 一些荧光细胞仍然可以观察, 由于后期 Gal4 基因表达的细胞, 在消融后分化27。</li>
	<li>记录了幼虫的行为后为了弥补背景中的不均匀性, 在斐济的平均图像上执行每个帧的逐像素划分: 单击 "进程"、"图像计算器"、"操作: 除法"、"32 位 (浮点)" 结果和 "好的。若要制作普通图像, 请单击 "图像"、"堆栈"、"Z 项目"、"平均强度" 和 "确定" (图 5E、F)。</li>
	<li>通过单击 "分析", "分析粒子", 设置覆盖所有草履虫的区域范围, 检查 "显示: 蒙版" 复选框, 然后单击 "确定", 计算分庭中剩余的草履虫的数目。"显示: 掩码" 选项将提供提取的草履虫图像 (图 5G), 并列出每个帧中的粒子数 (草履虫) 的列表。</li>
	<li>绘制在记录室中随时间推移而保留的草履虫的数目。由于草履虫的数量估计是嘈杂的, 因此建议在电子表格上对600帧 (1 分钟) 的每个点执行移动平均值。</li>
</ol>

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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>. Fiji Available from: <a target="_blank" href="https://fiji.sc">https://fiji.sc</a> (2017)</li><li>Thevenaz, P., Ruttimann, U. E., Unser, 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+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7 (1), 27-41 (1998).</li><li>Mueller, T., Wullimann, M. 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提交人没有报告的利益冲突。

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。

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

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 ...

ablation-neuronal-population-using-two-photon-laser-its-assessment

Research

JoVE Journal

Neuroscience

8.7K 次观看.  SOKENDAI (The Graduate University for Advanced Studies). 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。

视频: Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

发育生物学

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns.

Embryonic development requires large-scale coordination of cell motion. Two-photon excitation mediated laser ablation allows the spatially controlled 3-dimensional ablation of large groups of deep cells. In addition, this technique can probe the reaction of collectively migrating cells in vivo to perturbations in their mechanical environment.

在斑马鱼胃中使用双光子显微镜进行深度和空间控制的体积消融

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly ...

斑马鱼前脑的行为跟踪和神经活动模式

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

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

作者聚焦:成年斑马鱼大脑研究的进展 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

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 ...

神经科学

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

Prey Capture Assay: A Method to Study the Prey Capture Behavior of Zebrafish Larva

Source: Muto, A., et al. <a href="https://www.jove.com/v/57485/" target="_blank">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae</a>. &#160;J. Vis. Exp.&#160;(2018)
This video describes the prey capture assay. The video discusses the method to study prey capture behavior of zebrafish larvae after the laser ablation of specific neurons.

猎物捕获测定:一种研究斑马鱼幼虫猎物捕获行为的方法

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 ...

JoVE Encyclopedia of Experiments

蓝斑马（斑马鱼）

幼虫

Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Source: Muto, A. et al.,&#160; <a href="https://app.jove.com/v/57485">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.</a> J. Vis. Exp. (2018)
This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

分析转基因斑马鱼幼虫中猎物诱发的神经元活动

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope
NOTE: If users plan on performing Ca imaging following ablation, use the ...

Neurophysiology

Stimulation and Modulation Techniques

Two-Photon Laser Axotomy: A Method to Injure Axons in Zebrafish Embryos and Observe Axonal Recovery

Source: O&#39;Brien G. S., et al. <a href="https://www.jove.com/v/1129/" target="_blank">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos</a>. J. Vis. Exp. (2009).
This video describes the method to injure axons in zebrafish embryos using two photon laser axotomy and observing axonal recovery from injury.

双光子激光轴索切开术:一种损伤斑马鱼胚胎轴突并观察轴突恢复的方法

Source: O&#39;Brien G. S., et al. Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. J. Vis. Exp. (2009).
This video describes ...

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

摘要

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此视频中的章节

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

在斑马鱼胃中使用双光子显微镜进行深度和空间控制的体积消融

成年斑马鱼前脑活动的双光子钙成像

成年斑马鱼前脑活动的双光子钙成像

成年斑马鱼前脑活动的双光子钙成像

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

猎物捕获测定:一种研究斑马鱼幼虫猎物捕获行为的方法

分析转基因斑马鱼幼虫中猎物诱发的神经元活动

双光子激光轴索切开术:一种损伤斑马鱼胚胎轴突并观察轴突恢复的方法