Name: successful in vivo calcium imaging with a head-mount miniaturized microscope in the amygdala of freely behaving mouse
Uploaded: 2020-08-26T14:00:00
Description: Watch this Scientific Journal Video about Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse at JoVE.com

这项工作得到了三星科技基金会（项目编号SSTF-BA1801-10）的资助。

所有程序均得到韩国高等科学技术研究所动物伦理委员会的批准。所有实验均按照机构动物护理和使用委员会的准则进行。
注：此协议包括六个主要步骤：病毒注射手术、GRIN 镜头植入手术、GRIN 镜头植入验证、底板附件、行为测试期间 GCaMP 信号的光学记录以及数据处理（图1A）。除手术外，还使用商业软件包（Inscopix）。
1. 立...

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熟练的手术技术对于在更深的大脑区域（如杏仁核）中成功实现体内光学钙成像至关重要，正如我们在这里描述的那样。因此，尽管此协议为基板附件和 GRIN 镜头植入的优化手术过程提供了指导，但关键步骤可能需要额外的优化过程。如协议部分所述，手术中的杏仁核坐标、基板附件步骤中的气流速度、钙记录中的图像采集设置（帧速率、LED功率等）和数据处理中的变量（ICA时间重量、事?...

钙是无处不在的第二信使，在几乎每一个细胞功能中都起着至关重要的作用。在神经元中，动作电位激发和突触输入导致细胞内自由[Ca2]9，10的快速变化。因此，跟踪钙瞬态提供了一个监测神经元活动的机会。GECIs 是一种强大的工具，可以监测 [Ca2+] 在定义的细胞群和细胞内隔间11，12。在许多不同类型的基于蛋白质的钙指标中，基于单个GFP分子13的Ca2+探针GCaMP是最优化的，因此被广泛使用GECI。通过多轮工程，已开发出12、14、15、16等多种GCAMP变型。我们使用最近开发的GCaMP之一，GCaMP7b，在此协议16。GCaMP传感器为研究一些模型生物体的神经回路功能做出了巨大贡献，如17日开发过程中的Ca2+瞬态成像、特定皮质层18的体内成像、运动任务学习19中的电路动力学测量以及与海马和杏仁核20、21中关联恐惧记忆相关的细胞合奏活性的成像。
GECI的光学成像有几个优点。基因编码使 GECIs 能够在由遗传特征或解剖连接特定模式定义的特定细胞子集中长期稳定地表达。光学成像使活体动物中数百到数千个神经元的体内慢性同步监测。一些光学成像系统已经开发出来，用于对大脑内的GECI进行体内的活体成像和分析，这些小鼠的大脑中带有头部安装的微型荧光显微镜21、23、24、25。尽管基于GECIs、GRIN镜头和头坐式微型显微镜的体内光学成像技术是研究神经回路活动和行为之间联系的有力工具，但由于将GLIN透镜瞄准杏仁核中表达GECIs的细胞而未引起运动伪影，从而严重降低了图像采集质量和寻找表达GECIs的细胞，因此将这项技术应用于杏仁核一直存在一些技术问题。该协议旨在为基板附件和 GRIN 镜头植入的外科手术提供一个有用的指南，这是成功在杏仁核体内进行光学钙成像的关键步骤。虽然此协议针对杏仁核，但此处描述的大多数程序通常适用于其他更深的大脑区域。虽然此协议基于特定系统（例如 Inscopix），但与其他替代系统很容易实现相同的目的。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">26G needle</td>
			<td style="width:121px;">BD</td>
			<td align="right" style="width:161px;">302002</td>
			<td style="width:167px;">Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AAV1-Syn-GCaMP7b-WPRE</td>
			<td style="width:121px;">Addgene</td>
			<td style="width:161px;">104493-AAV1</td>
			<td>Surgery</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">AAV2/1-CaMKii&#945;-GFP</td>
			<td style="width:121px;">custom made</td>
			<td style="width:161px;"></td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acrylic-Dental cement (Ortho-jet Acrylic Pink)</td>
			<td style="width:121px;">Lang</td>
			<td style="width:161px;">1334-pink</td>
			<td>Surgery &#38; Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Air flow manipulator</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">NTR000253-04</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amoxicillin</td>
			<td style="width:121px;">SIGMA</td>
			<td style="width:161px;">A8523-5G</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Baseplate</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002192</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Baseplate cover</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002193</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Behavioral apparatus (chamber)</td>
			<td style="width:121px;">Coulbourn Instrument</td>
			<td style="width:161px;">Testcage</td>
			<td>Behavior test</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Behavioral apparatus (software)</td>
			<td style="width:121px;">Coulbourn Instrument</td>
			<td style="width:161px;">Freeze Frame</td>
			<td>Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbon cage</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">180mm x 70mm</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofen</td>
			<td style="width:121px;">SIGMA</td>
			<td style="width:161px;">PHR1452-1G</td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Data processing software</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">INSCOPIX Data Processing Software</td>
			<td>Baseplate Attachment &#38; Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dexamethasone</td>
			<td style="width:121px;">SIGMA</td>
			<td style="width:161px;">D1756-500MG</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Drill</td>
			<td style="width:121px;">Seyang</td>
			<td style="width:161px;">marathon-4</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Drill bur</td>
			<td style="width:121px;">ELA</td>
			<td style="width:161px;">US1/2, Shank104</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass needle</td>
			<td style="width:121px;">WPI</td>
			<td style="width:161px;">PG10165-4</td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GRIN lens (INSCOPIX Proview Lens Probe)</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002208</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hamilton Syringe</td>
			<td style="width:121px;">Hamilton</td>
			<td align="right" style="width:161px;">84875</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Head plate</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">Model 5</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hex-key</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-004195</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laptop computer</td>
			<td style="width:121px;">Samsung</td>
			<td style="width:161px;">NT950XBV</td>
			<td>Surgery &#38; Baseplate Attachment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lens holder, Stereotaxic rod (INSCOPIX proview implant kit)</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-004223</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope gripper</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002199</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope, DAQ software, hardware</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">nVista 3.0</td>
			<td>Baseplate Attachment &#38; Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mobile homecage</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">MHC V5</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moterized arm</td>
			<td style="width:121px;">Neurostar</td>
			<td style="width:161px;">Customized</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moterized arm software</td>
			<td style="width:121px;">Neurostar</td>
			<td style="width:161px;">Customized</td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NI board</td>
			<td style="width:121px;">National instrument</td>
			<td style="width:161px;"></td>
			<td>Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Removable epoxy bond</td>
			<td style="width:121px;">WPI</td>
			<td style="width:161px;">Kwik-Cast</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resin cement (Super-bond)</td>
			<td style="width:121px;">Sun medical</td>
			<td style="width:161px;">Super bond C&#38;B</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Skull screw</td>
			<td style="width:121px;">Stoelting</td>
			<td align="right" style="width:161px;">51457</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereotaxic electrode holder</td>
			<td style="width:121px;">ASI</td>
			<td style="width:161px;">EH-600</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereotaxic frame</td>
			<td style="width:121px;">Stoelting</td>
			<td align="right" style="width:161px;">51600</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereotaxic manipulator</td>
			<td style="width:121px;">Stoelting</td>
			<td align="right" style="width:161px;">51600</td>
			<td>Baseplate Attachment</td>
		</tr>
	</tbody>
</table>

successful in vivo calcium imaging with a head-mount miniaturized microscope in the amygdala of freely behaving mouse

在体内实时监测自由移动动物的神经元活动是将神经元活动与行为联系起来的关键方法之一。为此，利用基因编码的钙指标（GECIs）、微型荧光显微镜和梯度折射指数（GRIN）透镜，开发并成功应用于许多大脑结构1、2、3、4、5、6，检测神经元中的钙瞬态。这种成像技术特别强大，因为它能够长期同时对基因定义的细胞群进行长达几周的成像。虽然有用，这种成像技术不容易应用于大脑结构，位于大脑深处，如杏仁核，情感处理和关联恐惧记忆7的基本大脑结构。有几个因素使得很难将成像技术应用于杏仁核。例如，在更深的大脑区域进行成像时，运动伪影物通常更频繁地发生，因为植入大脑深处的头部安装显微镜相对不稳定。另一个问题是，横向心室位于靠近植入的 GRIN 透镜的位置，其呼吸过程中的运动可能会导致无法轻松校正的高度不规则运动伪影，从而难以形成稳定的成像视图。此外，由于杏仁核中的细胞通常处于静止或麻醉状态，因此很难在碱基板过程中找到并聚焦在杏仁核中表达GECI的目标细胞，以备日后成像之用。该协议为如何用头安装微型显微镜有效定位杏仁核中表达GECI的细胞提供了一个有用的指南，从而成功地在如此深的大脑区域进行体内钙成像。需要注意的是，此协议基于特定系统（例如 Inscopix），但不限于此系统。

格林 镜头植入验证 在通过粘固将底板长期连接到大脑之前，GRIN 镜头植入需要经过验证。在成功植入镜片的动物中，GCaMP表达细胞和血管都清晰地观察到由显微镜客观透镜与植入的G GRIN透镜（图2A和B）之间的距离决定的焦点平面范围内。相比之下，在进行脱靶植入的动物中，在焦点平面范围内（无论是失焦的还是失视的）没有观察到G...

Watch this Scientific Journal Video about Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse at JoVE.com

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

体内显微内窥镜钙成像是一种宝贵的工具，能够实时监测自由行为动物的神经元活动。然而，将这种技术应用于杏仁核是很困难的。该协议旨在为成功瞄准小鼠的杏仁核细胞提供有用的指南。

在自由行为鼠标的杏仁核中，使用头山小型显微镜成功进行体内钙成像

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

Research

JoVE Journal

Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

科研

神经科学

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

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 ...

Long-term Sensory Conflict in Freely Behaving Mice

自由生育小鼠的长期感觉冲突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse 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 of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

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

In vivo Calcium Imaging in Mouse Inferior Olive

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 ...

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

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

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Subcellular Imaging of Neuronal Calcium Handling In Vivo

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus (Video) | JoVE

小鼠上丘的钙成像

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment (Video) | JoVE

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...