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

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提交人没有什么可透露的

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。

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

successful-vivo-calcium-imaging-with-head-mount-miniaturized

Universidad de Cartagena UDEC

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Neuroscience

12.2K 次观看.  Korea Advanced Institute of Science and Technology (KAIST). 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。

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

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

科研

生物学

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 ...

神经科学

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

立体定向病毒注射和梯度指数晶状体植入术用于深部脑 体内 钙成像

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

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

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

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

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

体内 使用大颅窗的小鼠宽场和双光子钙成像

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

上丘的双光子和宽场钙成像

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

作者聚焦:揭示上丘的神经编码和视觉处理机制 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

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 ...

小鼠齿状回细胞神经元活动的实时成像 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

作者聚焦:用微型显微镜研究齿状回状颗粒细胞的神经活动

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

小鼠海马齿状回管颗粒细胞的体内钙成像 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

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

摘要

探索更多视频

在体内2光子层钙成像对小鼠的2 / 3

在体内小鼠劣质橄榄中的钙成像

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

立体定向病毒注射和梯度指数晶状体植入术用于深部脑体内钙成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

体内使用大颅窗的小鼠宽场和双光子钙成像

小鼠上丘的钙成像

小鼠上丘的钙成像

小鼠上丘的钙成像

小鼠海马齿状回管颗粒细胞的体内钙成像

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

摘要

探索更多视频

在体内2光子层钙成像对小鼠的2 / 3

在体内 小鼠劣质橄榄中的钙成像

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

立体定向病毒注射和梯度指数晶状体植入术用于深部脑 体内 钙成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

体内 使用大颅窗的小鼠宽场和双光子钙成像

小鼠上丘的钙成像

小鼠上丘的钙成像

小鼠上丘的钙成像

小鼠海马齿状回管颗粒细胞的体内钙成像

在体内小鼠劣质橄榄中的钙成像

立体定向病毒注射和梯度指数晶状体植入术用于深部脑体内钙成像

体内使用大颅窗的小鼠宽场和双光子钙成像