这项工作得到了中央研究院分子生物学研究所和台湾国家科学技术委员会的支持。中央研究院物理研究所的机械车间帮助制造定制设计的零件。我们还要感谢 P. Argast（瑞士巴塞尔弗里德里希·米歇尔生物医学研究所）设计了头部载物台的快速锁定机制。

所有动物程序均按照中央研究院动物护理和使用机构委员会的指导方针批准和执行。研究工具的详细信息可以在 材料表中找到。
1、录音室的准备
<ol><li>准备一个半六角形罐、一个底板和一个头部阶段（图1A;补充文件1-3）。头部平台由连接到圆形板上的两个金属柱组成。圆形板包含可以锁定在底板上的凹槽。锁定在一起后，头级和底板铺设在半六角形罐的底部。</li>
	<li>将半六角形槽放在显微镜的实验平台上。平台应该能够在 X 和 Y 方向上移动，而不会达到平移阶段的极限。</li>
	<li>将 810 nm 红外 （IR） 光和相机对准头部tage 进行行为记录。确保相机的视野足够大，可以容纳成年斑马鱼。<ol><li>为防止双光子激光和视觉刺激干扰行为记录，请在相机前分别设置875nm短通滤光片和700nm长通滤光片。</li>
	</ol></li>
	<li>为了呈现视觉刺激，将背投薄膜贴在半六边形罐的三个壁的内侧（图1A）。</li>
	<li>将三个投影仪对准水箱的三个表面。三个图像应对齐以形成一个连续的视觉场景。为防止投影机灯污染钙荧光信号，请在每台投影机前面放置一个中性密度滤光片和一个红色滤光片（600 nm，长通），并在光电倍增管 （PMT） 前面放置一个带通滤光片 （510/80 nm）。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65526/65526fig1v8.jpg"> 图 1：头部约束手术所需的器械。 （A）半六角罐内头部阶段的圆板与底板之间的快速锁定机构。定制零件的计算机辅助设计 （CAD） 文件可在补充文件 1-4 中找到。（二）用于头枕的Ω形头杆。（C） 用于将头杆定位到连接部位的三轴显微操纵器。插图：头杆在粘土中的方向。（D） 在手术过程中用大炮固定鱼。插图：鱼在大炮内的方向。（E） 鱼装载模块和用于将鱼装载到头部阶段的显微操纵器。插图：模块内鱼的方向。<a href="https://www.jove.com/files/ftp_upload/65526/65526fig1v8large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
2. 头枕手术
<ol><li>准备一个Ω形的顶杆（图1B; 补充文件 4）用于斑马鱼头枕。为此，将一块油基建模粘土连接到三轴显微操纵器上。确保粘土足够坚固以固定头杆，但又足够柔软，以便在头枕后从头杆上分离。</li>
	<li>将头杆的臂插入粘土中，并使头杆水平定向（图1C）。这将确保稍后将头杆连接到斑马鱼上是水平的。 注意：头杆可以由钛（24毫克）或不锈钢（43毫克）制成，可以重复用于多个实验。头杆材料不会影响成年斑马鱼（体重范围为 300-1,000 毫克）在头枕下的行为。</li>
	<li>准备大炮，一个用于在手术过程中将鱼体固定到位的空心管（图1D）。</li>
	<li>在鱼缸水中制备0.03%和0.01%甲磺酸三卡因（TMS;见 材料表）。这将用于在手术期间麻醉和保持鱼处于镇静状态。</li>
	<li>准备四根组织拭子，以去除颅骨附着部位多余的皮肤和水分。准备每根拭子时，将纸巾切成 3 厘米见方，并沿其对角线折叠以产生管状结构。将管子的末端拧成一个细点。附着部位非常小，因此细小的点可以更精细地控制拭子。</li>
	<li>为显微操纵器准备鱼加载模块（图1E）。 注意： 该模块由两个钢柱组成，钢柱由直角柱固定在一起amp.一根柱子连接到显微操纵器上，而另一根柱子则装有携带鱼的旋转柱夹。该模块用于通过精细控制将鱼定位到头部舞台上。旋转的立柱夹用作螺距调节器，以改变鱼的螺距角。</li>
	<li>在鱼缸水中用0.03%TMS麻醉鱼。在以下步骤中，使用注射器每 90 秒以短脉冲将鱼缸水中的 0.01% TMS 输送到口腔中。灌注中的水流应引起鳃运动。这将使鱼在手术过程中存活超过 40 分钟。 注：Tg[neuroD：GCaMP6f] 因其在幼虫和成虫阶段10 的前脑中广泛表达钙指示剂而使用。 注意：或者，在双手被占用的手术期间，使用重力灌注为口腔提供恒定的水流。</li>
	<li>将鱼包裹在胶囊中（图2A），将鱼固定在大炮内。<ol><li>将麻醉的鱼包裹在一块湿纸巾中，从尾尖到尾部约1毫米到鳃。确保包裹牢固，以防止鱼从组织中滑出。</li>
		<li>在纸巾周围缠绕一根带有纵向狭缝的软塑料管（例如，切开的中等大小的热缩管），以覆盖从尾巴末端到尾部约 2 毫米到鳃的鱼。管子确保鱼的身体在整个手术过程中保持笔直。</li>
		<li>将纸巾缠绕在管道周围，其直径与大炮的直径大致相似。</li>
		<li>将一根从塑料移液管的灯泡上切下的软塑料管缠绕在纸巾上。这确保了胶囊可以顺利地装入大炮中。</li>
		<li>将鱼装入大炮。</li>
	</ol></li>
	<li>使用手术刀去除覆盖附着部位的皮肤，即小脑外侧和鳃上方颅骨喙外侧的两个三角形区域（图2B），然后去除覆盖附着部位之间区域的皮肤。使用组织棉签擦干附着部位并清除任何残留的皮肤。<ol><li>在去除皮肤时使用可调节的平台支撑头部，但平台不得接触眼睛，以免在手术过程中受伤。 注意： 彻底去除附着部位的皮肤对于减少成像过程中的运动伪影至关重要。</li>
	</ol></li>
	<li>使用 10 μL 移液器吸头在每个附着部位的中心涂抹一滴组织胶（参见 材料表）。 注意： 纸巾胶覆盖附着部位并迅速变干。组织胶在颅骨和用于粘合头杆的牙胶之间提供粘合界面，并防止鳃中的水到达附着部位。</li>
	<li>将鱼体直立放置，将头杆略高于附着部位，并尾部贴合，为头杆胶合做准备。</li>
	<li>准备牙科水泥（见 材料表）并立即将其用于将头杆粘在颅骨上（图2C）。该过程对时间敏感，必须在 45 秒内完成。<ol><li>将一小勺聚合物与四滴快速单体和一滴催化剂V混合，将混合物均匀搅拌15秒。</li>
		<li>使用微量移液器和 10 μL 移液器吸头将混合物涂抹在附着部位和部位之间的区域。避免遮盖鳃和眼睛。</li>
		<li>使用显微操纵器将头杆轻轻压在水泥上。用剩余的水泥盖住头杆。</li>
		<li>等待 12 分钟，让牙水泥固化。避免水与水泥接触。</li>
	</ol></li>
	<li>为了改善前脑的光学通路进行钙成像，请去除端脑上方的皮肤。皮肤去除可以在3分钟内完成，使用手术刀进行五次切割（图2D）。确保去除粘附在颅骨表面的脂滴。</li>
	<li>固化后，从头杆上取下粘土。</li>
	<li>将整个胶囊从大炮转移到显微操纵器的鱼加载模块中的螺距调节器。</li>
	<li>使用显微操纵器定位鱼。头杆应位于头台金属柱的顶部tage.稍微增加鱼的俯仰角，使前脑表面在双光子成像时能更好地对准光学平面。</li>
	<li>用紫外线 （UV） 固化胶水将头杆粘在金属柱上。15 秒的紫外线照射足以固化。</li>
	<li>从胶囊中拉出鱼，并将头部tage锁定在半六角形水箱内的底板上。</li>
	<li>将动物浸入鱼缸水中。鱼应在 1-2 分钟内开始呼吸并从麻醉中恢复。 注意：或者，使用注射器灌注将淡水输送到口腔。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65526/65526fig02.jpg"> 图 2：头部约束手术中的关键步骤 。 （A）大炮内部胶囊的组成。（B） 颅骨上的附着部位（红色）。红色箭头表示血管部位。（C） 顶部：头杆附着在鱼头骨上。底部：鱼装载到头部阶段。（D） 切除前脑上方皮肤所需的切口。数字表示切割顺序。避免在标记部位（箭头）去除皮肤，以防止动物出血。 <a href="https://www.jove.com/files/ftp_upload/65526/65526fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
3. 双光子成像
<ol><li>打开激光器并等待 30 分钟，让电源稳定下来。将样品的波长设置为920 nm，功率设置为50 mW左右。 注意： 由谐振扫描仪控制的激光束在扫描路径的转弯点缓慢移动。为了防止组织损伤，使用普克尔斯池来降低转折点的激光功率。</li>
	<li>将包含头部束缚斑马鱼的记录室放在实验平台上（图3A）。平台应该能够在 X 和 Y 方向上移动，而不会达到平移阶段的极限。</li>
	<li>将物镜放置在尽可能靠近前脑表面的位置。物镜应对准瞳孔的前缘（图3B）。</li>
	<li>打开激光快门并启用PMT。 逐渐抬高物镜（~1mm），直到荧光图像中显示背侧前脑（图3C）。</li>
	<li>为了增加记录的神经元数量，使用压电陶瓷促动器获取多个深度（六个图像平面，相距15μm）的图像。这将以牺牲帧速率为代价来增加数据产量。启用快速 Z 轴扫描以控制压电陶瓷促动器（均匀模式，切片数 = 6，步长 = 15 μm，波形 = 锯齿波，反激式时间 = 4 ms，促动器滞后 = 8 ms）。</li>
	<li>要进行行为记录，请打开水箱两侧的 810 nm 红外 （IR） 灯。调整角度以照亮整个身体，这在相机中应该清晰可见。</li>
	<li>打开投影仪。</li>
	<li>开始记录数据。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65526/65526fig3v2.jpg"> 图 3：用于执行钙成像、行为记录和 视觉刺激显示的设置。 （A） 三台投影仪将视觉刺激投射到半六角形水箱的壁上。侧面的红外灯用于照亮斑马鱼的身体。（B） 物镜的定位。左：前视图。右：侧视图。16倍物镜与目标大脑区域之间的距离约为2.5毫米。 （C）双光子图像示例。左图：Tg[neuroD：GCaMP6f]中整个背前脑的最大投影。右图：放大图像，显示多个大脑区域的神经元。插图：来自不同大脑区域的更高放大倍率。图像是以 5Hz 记录的 10 秒数据的平均值。 <a href="https://www.jove.com/files/ftp_upload/65526/65526fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

斑马鱼前脑的行为跟踪和神经活动模式

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作者声明他们没有相互竞争的经济利益。

在这里，我们描述了一种详细的方案，以限制成年斑马鱼的头部进行双光子钙成像。要实现足够稳定的头枕，有两个关键步骤可以进行激光扫描成像。首先，头杆必须粘在头骨的特定附着部位。颅骨的其他部位通常太薄，无法提供机械稳定性，甚至可能在剧烈的身体运动中骨折。其次，必须彻底去除附着部位上方的皮肤。残留的水也应彻底干燥。这使得头骨能够与组织胶紧密结合。提供了一个表?...

使用基因编码指示剂或合成染料的钙荧光成像一直是测量行为动物（包括非人灵长类动物、啮齿动物、鸟类和昆虫）神经元活动的有效方法1。使用多光子成像可以同时测量数百个细胞的活动，这些细胞在脑表面以下约800μm处2,3。特定细胞类型的活性也可以通过在遗传定义的神经元群中表达钙指示剂来测量。成像方法在小型脊椎动物模型中的应用为跨大脑区域的神经元计算领域开辟了新的可能性。
斑马鱼是神经科学研究中广泛使用的模型系统。受精后 6 天左右的斑马鱼幼虫由于其微型大脑和透明的身体而被用于钙成像4.幼年斑马鱼（3-4周龄）也用于研究感觉运动通路的神经机制5,6。然而，复杂行为（包括联想学习和社会行为）的最大表现水平是在年龄较大的 7,8 岁时达到的。因此，需要一个可靠的方案来使用成像方法研究成年斑马鱼大脑中的多种认知功能。虽然斑马鱼幼虫和幼年斑马鱼可以嵌入琼脂糖中进行活体成像，但 2 个月或以上的成年斑马鱼在这种情况下会缺氧，并且身体太强壮而无法被琼脂糖限制。因此，需要外科手术来稳定大脑并使动物能够通过鳃自由呼吸。
在这里，我们描述了一种头枕协议，该协议涉及单个头杆的新颖设计。缩短的手术时间缩短了 25 分钟，是以前方法9 的两倍。我们还描述了记录室（半六角形罐）、头部级和将两个部分组合在一起的快速锁定机构的设计9.最后，还描述了呈现沉浸式视觉刺激以研究视觉触发的大脑活动和行为的设置。总体而言，此处描述的程序可用于在头部束缚的成年斑马鱼的遗传定义的细胞群中进行双光子钙成像，从而能够研究各种行为范式中的大脑活动。

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			<td>Vidrio vDAQ</td>
			<td>Microscope</td>
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			<td>Back-projection film</td>
			<td>Kimoto</td>
			<td>Diland screen - GSK</td>
			<td>present visual stimulus</td>
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			<td>Band-pass filter (510/80 nm)</td>
			<td>Chroma</td>
			<td>ET510/80m</td>
			<td>Microscope</td>
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		<tr>
			<td>Base plate for the semi-hexagonal tank</td>
			<td>custom made</td>
			<td>see supplemental files</td>
			<td>recording chamber</td>
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			<td>Camera filter (&#60;875 nm)</td>
			<td>Edmund optics</td>
			<td>#86-106</td>
			<td>Behavior recording</td>
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			<td>Camera filter (&#62;700 nm)</td>
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			<td>#43-949</td>
			<td>Behavior recording</td>
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			<td>Camera lens</td>
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			<td>MVL50M23</td>
			<td>Behavior recording</td>
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			<td>Chameleon Vision-S</td>
			<td>Coherent</td>
			<td>Vision-S</td>
			<td>Laser</td>
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		<tr>
			<td>Circular plate for the head stage</td>
			<td>custom made</td>
			<td>see supplemental files</td>
			<td>recording chamber</td>
		</tr>
		<tr>
			<td>Controller for piezo actuator</td>
			<td>Physik Instrumente&#160;</td>
			<td>E-665. CR</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Current amplifier</td>
			<td>Thorlabs</td>
			<td>TIA60</td>
			<td>Microscope</td>
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		<tr>
			<td>Elitedent Q-6</td>
			<td>Rolence Enterprise</td>
			<td>Q-6</td>
			<td>Surgery: UV lamp</td>
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		<tr>
			<td>Emission Filter 510/80 nm</td>
			<td>Chroma</td>
			<td>ET510/80m</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Head bar</td>
			<td>custom made</td>
			<td>see supplemental files</td>
			<td>recording chamber</td>
		</tr>
		<tr>
			<td>Infrared light</td>
			<td>Thorlabs</td>
			<td>M810L3</td>
			<td>Behavior recording</td>
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		<tr>
			<td>LED projector</td>
			<td>AAXA</td>
			<td>P2B LED Pico Projector</td>
			<td>present visual stimulus</td>
		</tr>
		<tr>
			<td>Moist paper tissue (Kimwipe)</td>
			<td>Kimtech Science</td>
			<td>34155</td>
			<td>Surgery: moist paper tissue</td>
		</tr>
		<tr>
			<td>Motorized XY sample stage</td>
			<td>Zaber</td>
			<td>X-LRM050</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Neutral Density Filters (50% Transmission)</td>
			<td>Thorlabs</td>
			<td>NE203B</td>
			<td>present visual stimulus</td>
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		<tr>
			<td>&#216;1/2&#34; Post Holder</td>
			<td>ThorLabs</td>
			<td>PH1.5V</td>
			<td>Surgery: hollow tube for cannon</td>
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			<td>&#216;1/2&#34; Stainless Steel Optical Post</td>
			<td>ThorLabs</td>
			<td>TR150/M</td>
			<td>Surgery: fish loading module</td>
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		<tr>
			<td>Objective lens 16x, 0.8NA</td>
			<td>Nikon</td>
			<td>CF175</td>
			<td>Microscope</td>
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		<tr>
			<td>Oil-based modeling clay</td>
			<td>Ly Hsin Clay</td>
			<td>C4086</td>
			<td>Surgery: head bar holder</td>
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		<tr>
			<td>Optical adhesive</td>
			<td>Norland Products</td>
			<td>NOA68</td>
			<td>Surgery: UV curable glue</td>
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			<td>Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H11706P-40</td>
			<td>Microscope</td>
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		<tr>
			<td>Piezo actuator</td>
			<td>Physik Instrumente&#160;</td>
			<td>P-725.4CA PIFOC</td>
			<td>Microscope</td>
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		<tr>
			<td>Pockels Cell</td>
			<td>Conoptics</td>
			<td>M350-80-LA-BK-02</td>
			<td>Microscope</td>
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		<tr>
			<td>Red Wratten filter (&#62; 600 nm)</td>
			<td>Edmund optics</td>
			<td>#53-699</td>
			<td>present visual stimulus</td>
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		<tr>
			<td>Resonant-Galvo Scan System</td>
			<td>INSS</td>
			<td>RGE-02</td>
			<td>Microscope</td>
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		<tr>
			<td>Right-Angle Clamp for &#216;1/2&#34; Post</td>
			<td>ThorLabs</td>
			<td>RA90/M</td>
			<td>Surgery: fish loading module</td>
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			<td>Rotating Clamp for &#216;1/2&#34; Post</td>
			<td>ThorLabs</td>
			<td>SWC/M</td>
			<td>Surgery: fish loading module</td>
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		<tr>
			<td>ScanImage</td>
			<td>MBF Bioscience</td>
			<td>Basic version</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Semi-hexagonal tank</td>
			<td>custom made</td>
			<td>see supplemental files</td>
			<td>recording chamber</td>
		</tr>
		<tr>
			<td>Super-Bond C&#38;B Kit</td>
			<td>Sun Medical Co.</td>
			<td>Super-Bond C&#38;B</td>
			<td>Surgery: dental cement</td>
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			<td>Tricaine methanesulfonate</td>
			<td>Sigma Aldrich</td>
			<td>E10521</td>
			<td>Surgery: anesthetic</td>
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			<td>USB Camera</td>
			<td>FLIR</td>
			<td>BFS-U3-13Y3M-C</td>
			<td>Behavior recording</td>
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			<td>Vetbond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>Surgery: tissue glue</td>
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two-photon calcium imaging of forebrain activity in behaving adult zebrafish

成年斑马鱼（Danio rerio）表现出丰富的行为，用于研究认知功能。它们还有一个微型大脑，可用于通过光学成像方法测量跨大脑区域的活动。然而，关于记录成年斑马鱼大脑活动的报道很少。本研究描述了在成年斑马鱼背侧前脑进行双光子钙成像的程序。我们专注于限制成年斑马鱼移动头部的步骤，这提供了稳定性，可以对大脑活动进行激光扫描成像。头部束缚的动物可以在没有辅助工具的情况下自由移动身体部位和呼吸。该手术旨在缩短头部约束手术的时间，最大限度地减少大脑运动，并最大限度地增加记录的神经元数量。本文还描述了在钙成像过程中呈现沉浸式视觉环境的设置，可用于研究潜在的视觉触发行为的神经相关性。

该协议由两部分组成：头部约束手术和前脑神经元活动的双光子钙成像。手术的成功取决于动物的存活率和头枕的稳定性。手术时经口频繁灌注0.01%TMS溶液可大大提高存活率。鱼应从麻醉中恢复，并在浸入鱼缸水后1-2分钟内积极呼吸。双光子钙成像能够通过完整的颅骨（~40 μm）在距脑表面200μm深度的背侧前脑中记录单个神经元的活动。该成像范围覆盖端脑背侧 （D） 的多个区域，包括内侧区 （Dm?...

Watch this Scientific Journal Video about Advancements in Adult Zebrafish Brain Research at JoVE.com

作者聚焦：成年斑马鱼大脑研究的进展 

在这里，我们提出了一种在成年斑马鱼的背前脑中进行双光子钙成像的方案。

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 ...

鱼类前脑所在的区域被怀疑与参与复杂行为的哺乳动物大脑区域同源。这些包括海马体、皮层和杏仁核。我们目前正在试图了解感觉信息对这些大脑区域活动的影响。目前斑马鱼神经科学的研究使用固定幼虫的双光子钙成像。结合基于虚拟现实的行为论文，研究大脑中的感觉运动处理。大多数关于斑马鱼大脑的研究都是在幼虫上进行的，但是鱼类的一些复杂行为直到成年才完全发育。我们的协议允许研究成人在行为过程中的大脑活动。与以前的方法相比，该方案将手术时间缩短了 50%。这是一种更有效的方法，可以限制成年斑马鱼的头部以获得更好的图像。这种头部约束方案为在成年斑马鱼大脑上进行体内实验提供了机会。除了双光子钙图像外，头部约束还可能允许对活鱼进行光遗传学操作、电生理学甚至超声成像。

two-photon-calcium-imaging-forebrain-activity-behaving-adult

Research

JoVE Journal

Neuroscience

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

2.2K 次观看.  Academia Sinica. 鱼类前脑所在的区域被怀疑与参与复杂行为的哺乳动物大脑区域同源。这些包括海马体、皮层和杏仁核。我们目前正在试图了解感觉信息对这些大脑区域活动的影响。目前斑马鱼神经科学的研究使用固定幼虫的双光子钙成像。结合基于虚拟现实的行为论文，研究大脑中的感觉运动处理。大多数关于斑马鱼大脑的研究都是在幼虫上进行的，但是鱼类的一些复杂行为直到成?...

视频: 作者聚焦：成年斑马鱼大脑研究的进展 

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute 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+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

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

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

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 ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

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

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

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

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 other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

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

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

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 ...