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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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, 使用不同的荧光报告蛋白, 需要对每个记者进行消融条件的优化。GCaMP 表达在无细胞核的细胞质中观察到。相比之下, EGFP 荧光出现在整个细胞内, 这可能有利于有效的和特定的细胞消融, 比 GCaMP。GCaMP 的使用也很不利, 因为激光消融 GCaMP 表达的细胞可以表现出增加的荧光强度, 由于立即大量的钙流入, 与降低荧光强度时, EGFP 被用于消融。因此, 荧光的变化不能成为 GCaMP 消融程度的可靠指标。在这种情况下, 可能需要通过观察特定时期后荧光细胞的丢失来验证消融。消融区的 Ca 信号的缺失或显著减少, 可能是 GCaMP 成功烧蚀的另一个标准。
其次, 成功消融所需的激光照射的量 (即、扫描速度、扫描次数) 可能因神经元从脑表面的位置的深度而异。与位于表面附近的细胞相比, 位于大脑深处的人需要更多的激光照射, 从而使消融更加困难。激光照射量的优化应具体确定为每个目标的神经元亚群。
第三, 据经验观察, 在一个扫描中设置一些 ROIs 在一个小区域内经常会对细胞周围的组织造成热损伤 (由激光照射区域内气泡的出现而判断), 而仅针对一个细胞或疏生在同一条件下, 在扫描中分布的 ROIs 没有有害的影响。在本研究中, 漂白仅适用于小区域 (大约是细胞体大小的第三个), 以避免对靶细胞外的组织造成损伤。通常, 5-10 个单元是针对每个焦点平面 (4-8 个平面覆盖整个 pretectal 区域)。
除了优化激光照射量外, 通过执行控制实验对激光消融进行广泛的验证是至关重要的。一个控制实验可以包括激光消融另一个亚群的细胞, 不太可能涉及的行为正在研究。标记在同一 Gal4 线上的嗅球神经元在本研究中充当控制项 (图 2A, 右,图 5I)。然而, 由于上述原因, 没有一组神经元可以作为理想的控制。神经元的每个亚群不同于其在大脑中的位置、细胞密度、荧光报告基因表达水平、增殖率和报告人转基因表达的时机。这些差异使得在控制实验中使用完全相同的消融 (漂白功能) 是不可能的。因此, 还应考虑不同类型的控制实验, 例如测量对视响应、视觉行为28和运动21中的基本活动, 以确保消融的具体效果。
根据我们的经验, 在双边的 pretectal 细胞消融, 大约需要1小时 (从嵌入琼脂糖, 消融使用双光子显微镜, 以从琼脂糖消融后检索)。可将每天多达8只幼虫 (例如, 4 只幼虫用于实验和4用于控制) 消融;因此, 可能需要进行几组实验, 以确保实验组 (例如、n = 12) 和对照组 (例如、n = 12) 有足够的幼虫。此外, 最好允许半天或一天的幼虫恢复从消融 (例如, 激光消融在 4-dpf 随后的行为记录在 5-dpf, 或消融在早上后, Ca 成像在下午)。在这个恢复时间, 任何看起来不健康的幼虫都应该被排除在研究之外。
用于分析所得数据的协议完全取决于研究的目的。本研究的重点是草履虫在视觉领域的位置与 pretectum 和 ILH 的神经元活动之间的关系。考虑到 GCaMP6s 信号29的慢衰减 (~ 秒), GCaMP6s 信号的增量 (而不是减少) 的时间间隔与草履虫的位置相关。即使草履虫移开, Ca 信号也可能很高。因此, 我们只包括增加荧光强度的 GCaMP6s 在数据呈现。这种类型的分析通常需要使用特定编程语言或宏语言编写的定制脚本。本文中使用的脚本可根据要求提供。
抑制条件 (即, 琼脂糖嵌入) 在一定程度上可能对斑马鱼幼虫有压力;然而, 当与自由游泳条件21、22进行比较时, 我们没有观察到明显的应力诱发的神经元活动或行为。琼脂糖嵌入斑马鱼幼虫至少能存活24小时, (可能更多), 没有任何明显的问题。在 pretectum 中的草履虫驱动的 Ca 信号和受约束条件下的 ILH 类似于在自由游动幼虫21中观察到的。斑马鱼的视觉行为可以由昼夜节律控制。因此, 我们进行了行为实验从早晚到深夜。在录制期间, 野生类型和控制幼虫消耗了大量的草履虫。
这里描述的功能和行为分析利用了在许多实验室中可以找到的最常用的设备, 或者可以很容易地建立起来, 因此它应该在行为科学的不同领域有广泛的应用。

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 border="0" cellpadding="0" cellspacing="0" style="width:1283px;" width="1282">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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>
</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右窗格) gSAIzGFFM119B与无人使用者交配: EGFP 记者鱼。结果表明, 双光子激光器可以消融靶细胞, 而不影响相邻细胞或突起 (图 2B)。
pretectal 神经元投射它们的轴突朝向 ILH ipsilaterally。在这里, 我们研究了他们的功能连接使用 Ca 成像。该区域的神经元活动可以被观察为 ca 信号与 ca 探针 GCaMP6s (图 4a), 其表达式由 Gal4 线 gSAIzGFFM119B (图 4B) 或另一个 Gal4 行 hspGFFDMC76A (图 4C). 彩色箭头 (图 4) 显示了草履虫的游泳方向和轨迹, 以及在指定脑区的颜色编码的 Ca 信号变化, 这是由游泳的视线诱发的草履虫(图 4B-D)。pretectum (图 4B) 和 ILH (图 4C) 都显示了在猎物的近端存在神经元活动, 这表明两种神经元活动都是视觉驱动的。
采用双 Gal4 GCaMP6s 幼虫 (gSAIzGFFM119B; hspGFFDMC76A;UAShspzGCaMP6s), 我们对 pretectal 神经元进行单边消融, 进一步进行 Ca 成像, 观察 pretectum 和 ILH 在消融幼虫中的神经细胞活性。被激光消融的左 pretectum 显示无神经元活动的残余 (图 4D, 左上), 表明激光消融是成功的。然而, 同侧 ILH 显著减少了神经元活动 (图 4D左下角), 这表明对 ILH 的主要输入来自同侧 pretectum。相比之下, 激光消融 pretectum 的另一侧的 ILH (图 4D右下角) 显示了与同侧 pretectum (图 4D, 右上角) 神经元活动相媲美的神经元活动, 这建议正确的 ILH 从右 pretectum 接收输入。
在实验中使用的行为分析的选择取决于所调查的神经元的可能作用。在这里, 我们展示了一个在 pretectal 核消融后被确定为猎物探测器21的猎物捕获试验的例子。使用图 5A-d中显示的照明系统, 可以通过图像处理 (图 5草履虫) 来计算记录室中的数。自动提取眼睛和计算眼睛位置 (即, 眼睛角度的变化) 也是可能的, 因为斑马鱼幼虫的眼睛比这个光照条件下的背景看起来更暗 (图 5H).
实验结果表明, pretectum 的双侧消融消除了猎物捕获活动 (图 5I, PT), 而嗅球中神经元亚群的消融没有 (图 5I, OB)。这些结果连同图 4中所示的实验表明, pretecto 下丘脑电路对捕获猎物行为至关重要。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57485/57485fig1.jpg"> 图 1.斑马鱼幼虫(A) 五天高龄 (5 天后受精 (5-dpf)) 斑马鱼幼虫及其猎物, 一个草履虫。(B) 4-dpf珍珠珠幼虫嵌入2% 低熔点琼脂糖。注意, 当视网膜色素上皮完好无损时,珍珠层菌株在人体上缺乏黑色色素。刻度条: 0.5 毫米 (C) 用于在琼脂糖中定向斑马鱼幼虫的解剖针。刻度条: 1 厘米 (D) 双光子显微术中使用的软件截图, 显示 "漂白"、"时间序列" 和 "区域" 面板, 用于消融。<a href="/files/ftp_upload/57485/57485fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57485/57485fig2.jpg"> 图 2.用双光子激光显微镜消融神经元的亚群。(A) EGFP 在神经元消融之前和之后的 4-dpf 斑马鱼幼虫的荧光图像。使用图像处理软件对3D 重建的 z 栈图像进行了顶部视图和侧面视图。z 叠加图像采用20X 物镜。消融区 (pretectum 或嗅球) 呈黄色圆圈。刻度条: 100 µm (B) 在单个焦平面上使用 "漂白" 功能进行激光消融的例子。激光辐照区域 (设置为 ROIs) 显示在每个单元格的颜色中。刻度条:10 µm.<a href="/files/ftp_upload/57485/57485fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57485/57485fig3.jpg"> 图 3.制备斑马鱼幼虫和用于 Ca 成像的草履虫。() 记录室.采用商用9毫米直径 x 0.8 毫米深度杂交垫片, 8 室作为记录室。垫片被放在玻璃滑梯上并粘附在一起。垫片顶部的密封件被剥离。(B) 用于冲洗草履虫的尼龙网 (32 µm)。网格被放在一个50毫升的管子上。(C)草履虫养殖, 含有稻草和干酵母丸。(D)草履虫由 C 中所示的区域性编写的库存解决方案 (E)草履虫库存解决方案用系统水冲洗两次, 以去除培养基中可能的嗅觉和味觉提示。(F)草履虫嵌入到记录室中。为了去除琼脂糖的一部分, 做了几次切割。(G) 带有斑马鱼幼虫和草履虫的记录室。大多数琼脂糖被移除, 以允许草履虫游泳。斑马鱼幼虫的头被暴露。(H) 用于 Ca 成像的直立荧光显微镜。显微镜上装有一个科学的 CMOS 照相机。(I) 斑马鱼幼虫在录音室在显微镜下与励磁灯。与2.5X 物镜, 照明面积略大于房间的大小。<a href="/files/ftp_upload/57485/57485fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57485/57485fig4.jpg"> 图 4.代表性 Ca 映像数据.(A) pretectum 和下丘脑 (ILH) 下叶的位置, 以黄色圆圈, 在双 Gal4 hspGFFDMC76A;gSAIzGFFM119B;UAShspzGCaMP6s 斑马鱼幼虫。(B) pretectum Ca 信号 (平均双边) 的变化, 颜色映射到 gSAIzGFFM119B 中草履虫的轨迹上;UAShspzGCaMP6s 幼虫。缩放条: 1 毫米 (C) ILH Ca 信号 (双边平均) 的变化, 在 hspGFFDMC76A 中草履虫的轨迹上的颜色映射;UAShspzGCaMP6s 幼虫。缩放条: 1 毫米 (D) pretectum 中的更改和 ILH Ca 的信号在 hspGFFDMC76A 中的草履虫的轨迹上进行颜色映射;gSAIzGFFM119B;UAShspzGCaMP6s 幼虫, 受到双光子激光消融的左 pretectum。刻度条: 1 毫米。此图像是从以前发布的21中的相同数据复制的。<a href="/files/ftp_upload/57485/57485fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57485/57485fig5.jpg"> 图 5.激光烧蚀斑马鱼幼虫的捕食捕获试验和典型数据.(A) 行为记录系统。显微镜装有 CMOS 相机。图像是使用定制的脚本获取的。(B) 照明系统的组件。一张灰色的纸, 一个玻璃扩散器, 白色的 LED 环形灯, 扩散器, 一个定制的间隔 (纸板), 并在中心孔的扩散。(C) 从 B. (D) 记录室中所示的部件组装的照明系统, 用于捕捉猎物的化验。一个腔 (直径:20 毫米; 深度: 2.5 毫米) 放在玻璃滑梯上。房间的最初的顶部封印被剥离了。在录音室上放了一个玻璃罩。(E) 从录制影片的框架中原始图像。(F) 由影片中的平均图像划分 (逐像素像素) 的帧。注意, 这种图像处理可以补偿照明中的非均匀背景。(G)草履虫使用斐济的 "粒子分析" 功能从框架中提取。(H) 在斐济具有应用阈值的图像示例。草履虫看起来很亮, 而斑马鱼幼虫的眼睛看起来很黑, 背景是灰色的。如果需要, 可以计算眼睛位置的变化 (即、与身体轴有关的角度)。(I) 在嗅球消融控制幼虫 (OB) 和 pretectum 消融幼虫 (PT) 中的草履虫消耗量的典型实验数据。Pretectum 消融显著降低了猎物的狩猎能力, 而嗅球消融并没有影响它。<a href="/files/ftp_upload/57485/57485fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

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 measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

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 ...

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 regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

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

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

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

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+ ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

脑磁图在儿童脑功能研究中的应用

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

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 cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

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

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.