这项工作得到了来自 jsp (15K07147、17K07419)、佐野基金会研究补助金 (HI) 和发展医学联合使用/研究中心 (Ishimoto) 的援助资助, 在熊本大学分子胚胎学与遗传学研究所 (HS)。我们感谢梓 Kamikouchi 博士和山田 Daichi 先生在使用成像系统方面的帮助。

1. 幼虫脑外植体的制备
<ol><li>制作一个成像室。 注意: 稳定的记录要求将脑外植体拴在一起。这里, 在 Ca2 +成像系统中使用了一个低成本、手工制作的成像室。<ol>
<li>使用镊子, 划伤塑料培养皿 (35 毫米 x 10 毫米) 的底部中心, 为幼虫大脑的腹神经节创造一个凹痕, 使安装更加容易 (步骤 1.3,图 1)。</li>
		<li>用牙签, 放置一小滴超强力胶水在凹痕的两边, 并附上一个钨棒 (0.125 毫米直径, 6 至7毫米长度) 的胶水。</li>
		<li>使用一小块腻子样的可重用胶粘剂, 使凹痕 (图 1) 周围的圆形壁 (直径10毫米和3毫米高)。 注: 墙内的空间后将填充磷酸盐缓冲生理盐水 (PBS; 0.14 米氯化钠, 0.0027 米氯化钾, 0.01 米 PO43-, pH 7.4 @ 0.05 在25°c)。</li>
	</ol>
</li>
	<li>动物制剂 注: 对于钙成像, 钙指示器, GCaMP6s, 是表达的细胞类型的兴趣, 这是通常实现使用组织特定的 GAL4 和UAS-GCaMP6s的组合。为了确保信号通过候选受体介导, GCaMP6s 也表达在受体缺陷的突变体中。<ol>
<li>当对不同基因型如野生型和突变体进行实验比较时, 年龄匹配试验幼虫避免 GCaMP6 表达水平的变化和生理上的差异, 由于发育阶段。</li>
		<li>保持父母苍蝇 (每性别30苍蝇) 在一个文化小瓶, 其中含有标准的飞行食品8小时, 允许产卵的食物表面。在12小时的光暗循环下, 在25摄氏度和 60-70% 相对湿度下, 培养幼虫的质量。</li>
	</ol>
</li>
	<li>幼虫脑解剖<ol>
<li>在产卵后90-120 小时 (AEL), 收集幼虫, 用蒸馏水冲洗至少3次, 以除去附着的食物碎片。</li>
		<li>将一个幼虫放在一个1.5 寸的方形手表玻璃上, 里面装满了冰冷 PBS。 注: 下两步是在这个手表玻璃下进行解剖显微镜。</li>
		<li>用镊子轻轻抓住幼虫中间部分。使用另一钳抓住嘴钩和拉嘴钩轻轻地把包含大脑的幼虫的前部与身体的其余部分分开。 注: 另外, 可以使用手术剪刀。</li>
		<li>用镊子抓住前尖, 把幼虫翻出来。除去附着在大脑上的多余的组织, 如形象盘、脂肪体和环腺。轻轻地将大脑与口器分开。</li>
		<li>将200µL 的 PBS 应用于在成像室中由腻子状可重用胶粘剂制成的环内。用 PBS 轻轻吮吸被解剖的大脑, 然后把它转移到成像室。</li>
	</ol>
</li>
	<li>将大脑设置成成像室<ol>
<li>使用钳抓取肌肉纤维从腹神经节延伸。将大脑轻轻插入钨线下面的凹痕。</li>
		<li>使用另一个钳, 将钨线稍稍向上拉, 将大脑置于正确的成像位置 (图 1)。</li>
	</ol>
</li>
</ol>2. 获取 Ca2 +荧光图像
注: 在 PBS 中浸泡的脑外植体采用荧光显微镜, 配备20X 水浸泡物镜 (0.5)。PBS 不会激活大脑中的细胞。如果需要 Z 轴图像, 物镜将与压电激活的透镜移动器一起安装。采用旋转圆盘共焦头, 提高了时间分辨率。对于低光成像, 电子倍增电荷耦合装置相机安装在显微镜上。GCaMP6s 荧光成像 (激发/发射: 488/509 nm) 需要一个 488 nm 激光器的励磁与分色光束分配器和发射过滤器 (e. g, 528, 38 nm 带通滤波器)。
<ol><li>在显微镜下放置包含脑外植体的成像室。</li>
	<li>降低物镜, 直到它触及 PBS。在光明场光照下, 定位大脑并将其聚焦。切换到荧光灯, 并调整 GCaMP6s-labeled 细胞的焦点。 注意: GCaMP6s 的基底绿色荧光应该是可见的。</li>
	<li>使用适当的采集软件 (e. g, µManager), 在水冷模式下以 512 x 512 像素的分辨率开始获取250毫秒/帧。调整曝光时间, 在 CCD 摄像机的动态范围内获得荧光值, 但不低于 1000 (任意单位), 具有16位图像 (动态范围 0-65,535)。 注意: 低曝光时间应用于减少 GCaMP6s 的光漂白。在每个实验中应优化曝光时间, 因为这取决于 GCaMP6 的表达水平和检测系统的灵敏度。这是 100 ms 在我们的实验中使用dilp2 > GCaMP6s.当给定成像深度需要 z 段时, 可以根据曝光时间和帧间隔来获取多个 z 剖面图像。如果相机有足够的空间分辨率, 则应增加 binning 大小 (芯片上的寄存器数, 将作废成一个数字像素), 以减少曝光时间。</li>
	<li>一旦确定了成像参数, 在肽管理之前采取1分钟的图像来检测基线信号强度 (F0)。</li>
	<li>应用试验肽;为此, 在 PBS 中溶解100µL 肽溶液, 并将其直接注入幼虫浴中, 以产生最佳浓度。 注: 肽溶液应缓慢注射 (例如, 流速20µL/秒) 进入浴缸溶液使用吸管。在 PBS 中溶解的不相关合成肽被用作阴性对照。</li>
	<li>记录 GCaMP6s 发射数分钟。</li>
</ol>3. 数据分析
注意: 图像数据与 ImageJ 进行离线分析。在时间推移成像, 吹打导致幼虫大脑移动。因此, 在分析前应纠正序列图像的位置畸变。更正可以使用 ImageJ 插件执行, TurboReg 如下所示。
<ol><li>打开分析软件, 并使用第一帧 (在多肽应用程序之前) 作为参考图像。</li>
	<li>选择插件 |注册 |TurboReg。</li>
	<li>选择串行图像文件作为Source , 并将引用图像作为目标。</li>
	<li>检查刚体(图像的平行位移和旋转) 和精确("快速") 的处理方法和质量分别是粗而快速的分析。</li>
	<li>单击批处理开始图像处理。</li>
	<li>使用分析 | "选择多个感兴趣的区域 (ROIs)" |工具 |ImageJ 中的 ROI 管理器。</li>
	<li>单击更多 |多项措施 |OK在 ROI 管理器窗口中。</li>
	<li>计算ΔF0 = (f f0)/f0, 其中 f 是刺激时的 ROI 强度, 而 f0是在特定实验事件之前 (例如, 30 帧之前) 的时间窗口中的强度。刺激发作)。</li>
	<li>用多个脑样本重复实验, 进行统计处理, 获得平均 (SEM) 在每个时间点的平均值和标准误差。</li>
</ol>

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作者没有什么可透露的。

此处描述的 Ca2 +成像方法是一种用于测试大脑中荷尔蒙功能的有用系统。在体内,大脑接收来自各种内分泌器官的荷尔蒙。此外, 大脑不断感知上升的感官信息, 这会导致自发的神经元活化。此前体系统消除了此类噪音, 并产生了比体内成像更好的信号/噪声比。在这个系统中, 分子可以单独或组合测试。除了肽荷尔蒙, 任何水溶性分子, 包括天然或合成化合物, 都可以应用?...

器官对器官的沟通是一种进化保守的策略, 用于维持动态平衡以应对环境变化。在人类中, 各种激素 arereleased 从内分泌腺体进入循环。许多这些荷尔蒙的目标是大脑的下丘脑, 它调节新陈代谢过程和基本行为, 如喂养1,2。许多荷尔蒙是用哺乳动物模型发现的。然而, 它们的行动机制, 特别是他们参与的 interorgan 网络, 在很大程度上仍然不清楚。
果蝇已成为研究器官与器官沟通的有用模型。在昆虫中, 许多生理过程是由荷尔蒙控制的。早期研究侧重于生长和变形使用大昆虫。在这些研究中, 特定器官的移除或移植预测了器官间信号分子的存在;后来, 青少年激素 (JH), Prothoracicotropic 激素 (PTTH) 和昆虫蜕皮激素生化纯化3, 4, 5.在昆虫生命周期的6、7中, 大量的细胞间信号肽被认为涉及各种生理事件。大多数这些肽作用于 G 蛋白偶联受体 (受体), 虽然特定的受体最初难以识别使用常规方法。果蝇全基因组序列8的发布是一个突破, 它使果蝇生物活性肽的鉴定与其他昆虫的同源性有关。此外, 用细胞培养为基础的 GPCR 配体结合试验, 从基因组预测的受体中发现了几种多肽受体。其次, 表达分析预测这些肽和受体诱发的器官对器官通路。值得注意的是, 许多假定的肽受体在大脑中表达, 这表明大脑是肽类激素的主要目标9。此外, 在果蝇中, 先进的基因工具有助于鉴定肽 GPCR 组合的生理作用。例如, 基于 GAL4 和莱克萨的二进制转录系统可以在空间和世俗控制的方式下使基因击倒或过度表达。GAL4 是酵母中识别的转录因子, 它与一种称为上游活化序列的特定顺规序列结合在一起。在 GAL4/UAS 系统中, 驱动程序行提供了组织特定的或特定于阶段的 GAL4 表达式, 而应答器线携带感兴趣的基因或构造来驱动 shRNA 表达式的无人机上游。莱克萨/LexAop 系统基于类似的机制。表型分析的组织特异性肽击倒和组织特异性 GPCR 的动物可以揭示的信息, 肽 GPCR 信号的模式和行动地点。然而, 仅仅通过遗传数据就可以得出的结论是有限的。另一方面, 一旦特定肽类激素的假定目标被缩小到组织或细胞类型, 在器官外植体中的活体钙成像可以用来阐明由肽 GPCR 信号介导的器官对器官的传播。在 Gq 耦合的 GPCR 激活后, 由于从 ER10释放 ca2 + , 胞内 ca2 +浓度增加。在大脑中, 神经激活还提升了胞内 Ca2 +级。此类 Ca2 +的增加可以通过钙传感器检测, GCaMP 在 Ca2 +的存在下发生构象变化, 导致荧光发射11。
本文介绍了一种使用果蝇脑外植体的钙成像方法。为了测试多肽激活特定神经元的能力, 将测试肽应用于 GCaMP 表达的脑外植体, 并通过共聚焦显微镜对荧光变化进行监测。GPCR 的参与, 然后确认通过执行相同的化验, 使用突变的大脑缺乏 GPCR。这种成像和遗传学的结合提供了关于多肽受体的器官与器官通讯的精确信息. 还讨论了此协议的可能修改和应用。

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			<td height="40" style="height:40px;width:295px;">Tungsten rod</td>
			<td style="width:327px;">A-M systems, Sequim, WA, USA</td>
			<td style="width:165px;">717000</td>
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			<td height="40" style="height:40px;">Blu Tack</td>
			<td>Bostik, Paris, France</td>
			<td>3049100</td>
			<td>putty-like reusable adhesives</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Watch glass, square, 1 5/8 in</td>
			<td>Carolina Biological Supply Company, Burlington, NC, USA</td>
			<td>742300</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>TAKARA Bio Inc., Kusatsu, Shiga, Japan</td>
			<td>T900</td>
			<td>Phosphated buffered salts</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CCHa2</td>
			<td>SCRUM Inc., Tokyo, Japan</td>
			<td></td>
			<td>Custum-synthesized peptide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ghrerin</td>
			<td>Peptide institute Inc., Osaka, Japan</td>
			<td>4372-s</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nociceptin</td>
			<td>Peptide institute Inc., Osaka, Japan</td>
			<td>4313-v</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Axio Imager A2</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td>Axio Imager A2</td>
			<td>fluorescence microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective W N-Achroplan 20x/0.5 M27</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td>420957-9900-000</td>
			<td>water-immersion objective lens</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">P-725 PIFOC objective scanner with long travel range</td>
			<td>Physik Instrumente GmbH &#38; Co. KG, Karlsruhe, Germany</td>
			<td>N/A</td>
			<td>piezoelectric-activated lens mover</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Confocal Scanner Unit CSU-W1</td>
			<td>Yokogawa Electric Corporation, Tokyo, Japan</td>
			<td>CSU-W1</td>
			<td>spinning disc confocal head</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ImagEM C9100-13</td>
			<td>Hamamatsu Photonics, Sizuoka, Japan</td>
			<td>C9100-13</td>
			<td>EM-CCD camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OBIS 488 nm LS 60 mW</td>
			<td>Coherent, Santa Clara, CA, USA</td>
			<td>1178770</td>
			<td>488-nm laser</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">488/568/647 nm Yokogawa dichroic beamsplitter</td>
			<td>Semrock, Rochester, NY, USA</td>
			<td>Di01-T405/488/568/647-13x15x0.5</td>
			<td>dichroic beam splitter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">528/38 nm BrightLine single-band bandpass filter</td>
			<td>Semrock, Rochester, NY, USA</td>
			<td>FF01-528/38-25</td>
			<td>emission filter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#181;Manager</td>
			<td>Open Imaging, Inc.</td>
			<td>N/A</td>
			<td>https://micro-manager.org</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ImageJ</td>
			<td>U. S. National Institutes of Health</td>
			<td>N/A</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TurboReg</td>
			<td>Philippe Th&#233;venaz, Biomediccal Imaging Group, Swiss Federal Institute of Technology Lausanne</td>
			<td>N/A</td>
			<td>http://bigwww.epfl.ch/thevenaz/turboreg/</td>
		</tr>
	</tbody>
</table>

ex vivo calcium imaging for visualizing brain responses to endocrine signaling in drosophila

通过内分泌信号, 例如从外围到大脑, 器官对器官的传播对于维持稳态是必不可少的。作为内分泌研究的动物模型, 具有成熟基因工具和基因组信息的果蝇正在越来越多地被使用。本文介绍了一种用于果蝇脑外植体的钙成像方法。这种方法能够检测到大脑中荷尔蒙的直接信号。众所周知, 许多肽荷尔蒙通过 G 蛋白偶联受体 (受体) 作用, 其活化导致胞内 Ca2 +浓度增加。神经激活还提升了胞内 ca2 +级别, 从 ca2 +的流入和释放 ca2 +存储在内质网 (ER)。钙传感器 GCaMP 可以监视这些 Ca2 +的更改。在该方法中, GCaMP 表达在感兴趣的神经元中, GCaMP 表达的幼虫脑被解剖和培养为前体.然后将试验肽应用于脑外植体, 用装有 CCD 摄像机的旋转圆盘共焦显微镜检测 GCaMP 的荧光变化。使用这种方法, 任何水溶性分子都可以进行测试, 与神经活化相关的各种细胞事件可以使用适当的荧光指示器进行成像。此外, 通过修改成像室, 该方法可用于图像其他果蝇器官或其他动物的器官。

利用这一协议, 研究了肽类激素 CCHa2 对脑胰岛素生成细胞的活化作用。野生型脑的化学品方案使用dilp2GAL412 (来自 Rulifson 博士的礼物) 和UAS-GCaMP6s13 (金博士的礼物) 的组合标记了 GCaMP6s。采用合成 CCHa2 肽对幼虫脑外植体进行治疗, 并实时记录 GCaMP6s 的荧光。在这个实验中, 图像是从一个单一的焦平面获得的。每个 IPC 群集包含?...

Watch this Scientific Journal Video about Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila at JoVE.com

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

本文介绍了 果蝇脑的体外钙成像协议。在这种方法中, 自然或合成化合物可以应用到缓冲, 以测试他们的能力, 激活特定神经元的大脑。

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

ex-vivo-calcium-imaging-for-visualizing-brain-responses-to-endocrine

Research

JoVE Journal

Neuroscience

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

9.1K 次观看.  Nagoya University. 本文介绍了 果蝇脑的体外钙成像协议。在这种方法中, 自然或合成化合物可以应用到缓冲, 以测试他们的能力, 激活特定神经元的大脑。

视频: Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

在行为和刺激反应期间为整个大脑成像准备成人德罗索菲拉褪黑麦片加斯特

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

在活体钙成像小鼠致病性甘里昂神经元对味觉刺激的反应

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

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 ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...