JoVE Journal > Neuroscience
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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신경과학

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

Published: August 26, 2020
doi:
10.3791/61659
,
1Department of Biological Sciences,Korea Advanced Institute of Science and Technology (KAIST), 2KAIST Institute for BioCentury (KIB),Korea Advanced Institute of Science and Technology (KAIST)

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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…

Representative Results

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…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

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

Keywords: In Vivo Calcium ImagingMiniaturized MicroscopyAmygdalaAAV InjectionStereotaxic FrameCraniotomyViral InjectionSkull ScrewsMotorized Surgery ArmStereotactic FrameAmygdala Coordinates
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Lee, H., Han, J. Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse. J. Vis. Exp. (162), e61659, doi:10.3791/61659 (2020).
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