Name: targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging
Uploaded: 2012-12-12T11:00:00
Description: Watch this Scientific Journal Video about Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging at JoVE.com

这项工作是支持的国家眼科研究所R01EY017925和R21EY020985的资金从达纳白厅基础PK我们也感谢马修·彼得雷拉外科手术的帮助，格雷斯·迪翁示于图5A追踪树突补助; PRATIK Chhatbar为的手稿上的意见。

1。手术准备<ol><li>诱导麻醉，并持续监测心跳率，结束潮汐CO 2，脑电图，及温度。南卡罗来纳大学医疗机构动物管理和使用委员会批准了所有的程序，是基于我们以前出版9,15。 </li><li>公开的背用手术刀切割皮肤表面的头骨。解剖的结缔组织，覆盖骨使用一个Brudon的刮匙。用棉签涂抹和棉花纱布清洁骨。应用骨蜡（如需要）的头骨，偶尔通过小使者静脉的出血停止。 </li><li>钛?...

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我们提出一个方法来定位标签在预先确定的功能性微域的新皮层的神经元细胞体（或树突和轴突）。合并内在信号光学成像与双光子显微镜提供的可能性确定哪些突触和细胞有助于微域的任何功能的地图，是否选择性神经元的神经元中的功能图的位置，以及神经元电路相关组件这种变化的视觉体验7或应用临床治疗的药物18。 据报道，在视觉皮层的成年野生型?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 1。生命支持/实验准备 </td></tr><tr><td>异氟醚</td><td>韦伯斯特兽医</td><td> NDC 57319-474-05 </td><td></td></tr><tr><td>异氟醚蒸发器</td><td> MIDMARK </td><td> VIP 3000 </td><td></td></tr><tr><td>反馈调节加热毯</td><td>哈佛设备</td><td> 50-7079F </td><td></td></tr><tr><td>心电监护仪</td><td> Digicare生物医学</td><td> LifeWindow精简版</td><td></td></tr><tr><td>脑电放大器</td><td> AM系统</td><td> 1800 </td><td></td></tr><tr><td>脑显示器</TD&gt; <td>惠普</td><td> 78304A </td><td></td></tr><tr><td>呼气末CO 2显示器</td><td>伟</td><td> Novametrix呼气Capnoguard 1265 </td><td>优化通风</td></tr><tr><td>硬质合金钻头钻骨毛刺</td><td>亨利施恩</td><td>罚款（0.5毫米头），粗（1.25毫米头） </td><td></td></tr><tr><td>水泥磁头板/室</td><td>登士柏</td><td> 675571，675572 </td><td></td></tr><tr><td>黑火药滕佩拉漆</td><td>萨金特艺术公司</td><td> 22-7185 </td><td>巩固提高遮光，减少反射</td></tr><tr><td>琼脂糖 - Ⅲ型-A </td><td>西格玛</td><td> A9793 </td><td>用于最小化内在信号和双光子成像过程中的脉动</td></tr><tr><td>盖玻片：5或8毫米，直径0.17毫米的厚度</td><td>世界精密剂量率仪</td><td> 502040，502041 </td><td>为了最大限度地减少脉动在成像过程中，根据需要可能会被切除玻璃罩</td></tr><tr><td> Brudon刮匙</td><td>乔治·蒂曼</td><td> 105-715-0，105-715-3 </td><td>清洁颅骨表面</td></tr><tr><td>骨蜡</td><td>爱惜</td><td> W31G </td><td>快速止血</td></tr><tr><td>棉签</td><td>电子显微镜科学</td><td> 72308-05 </td><td>清洁，干燥，骨面</td></tr><tr><td>杜蒙5CO钳</td><td>精细科学工具</td><td> 11295-20 </td><td>抓住各个层硬脑膜或软</td></tr><tr><td> Vannas春风似剪刀</td><td>精细科学工具</td><td> 15000-03 </td><td>切硬膜</td></tr><tr><td>明胶海绵</td><td>辉瑞公司</td><td> 09-0396-05 </td><td>要停止硬脑膜上出血</td></tr><tr><td>吸收矛</td><td>精细科学工具</td><td> 18105-01 </td><td>超快速和无绒排汗CSF </td></tr><tr><td>停电材料</td><td> Thorlabs公司</td><td> BK5 </td><td>盾开颅手术</td></tr><tr><td></td><td></td><td></td><td> 2。染料制备/注射 </td></tr><tr><td>二甲基亚砜（DMSO）的</td><td>西格玛</td><td> D2650 </td><td></td></tr><tr><td>普朗尼克</td><td>西格玛</td><td> P2443 </td><td></td></tr><tr><td>俄勒冈绿488 BAPTA-AM </td><td> Invitrogen公司</td><td> O6807 </td><td>钙指示剂</td></tr><tr><td>的Alexa Fluor 594 </td><td> Invitrogen公司</td><td> A10438 </td><td></td></tr><tr><td>离心式过滤器（0.45μm孔径） </td><td>密理博</td><td> UFC30HV00 </td><td>注射前要除去杂质</td></tr><tr><td>玻璃吸管拉马</td><td>萨特仪器</td><td> P97 </td><td></td></tr><tr><td>高硼硅玻璃filamented毛细管（1.5毫米外径） </td><td>世界精密仪器</td><td> 1B150F-4 </td><td>染料弹射移液管</td></tr><tr><td> Microloader </td><td> Eppendorf公司</td><td> 5242 956 003 </td><td>加载染料吸管</td></tr><tr><td>微操作机器人</td><td>萨特仪器</td><td> MP-285 </td><td>要定位移液管</td></tr><tr><td>压力脉冲控制器</td><td>派克汉尼汾</td><td> PicoSpritzer III </td><td>对于染料的压力注入</td></tr><tr><td>单细胞电穿孔</td><td> Molecular Devices公司</td><td> Axoporator 800A </td><td>有关染料的电穿孔</td></tr><tr><td></td><td></td><td></td><td> 3。本质IMA晋</td></tr><tr><td> 4倍的目标（0.13 NA，17毫米WD） </td><td>奥林巴斯</td><td> UPLFLN4X </td><td></td></tr><tr><td>内在的硬件/软件</td><td>光学成像公司</td><td>成像仪3001 / VDAQ </td><td>情节成像软件是用于VDAQ </td></tr><tr><td> CCD相机</td><td> Adimec公司</td><td> Adimec公司-1000 </td><td></td></tr><tr><td>光源电源</td><td> KEPCO </td><td>吃了15-15M </td><td></td></tr><tr><td>光源</td><td>光学成像公司</td><td> HAL 100 </td><td>在皮层表面的光照强度为3-5毫瓦</td></tr><tr><td>绿色过滤器（用于血管图像） </td><td>光学成像公司</td><td> λ= 546 nm处（带通30纳米） </td><td>对于参考图像的表面脉管</td></tr><tr><td>红色过滤器（内在信号） </td><td>光学意马纳克公司</td><td> λ= 630 nm处（带通30纳米） </td><td> ，要收集内在的信号</td></tr><tr><td>热过滤器</td><td>光学成像公司</td><td> KG-1 </td><td></td></tr><tr><td></td><td></td><td></td><td> 4。双光子台/影像 </td></tr><tr><td>双光子显微镜和软件</td><td>草原技术</td><td></td><td> Shen 等人 2012年的光路，过滤器和激光功率</td></tr><tr><td>掺钛蓝宝石激光器</td><td>光谱物理公司</td><td>麦态XF </td><td></td></tr><tr><td> 20倍（0.5 NA，3.5毫米WD） </td><td>奥林巴斯</td><td> UMPLFLN20X </td><td>仅用于对准移液器超过开颅0.5 NA物镜（未用于两个光子成像） </td></tr><tr><td> 20X（1.0 NA，2.0毫米WD） </td><td>奥林巴斯</td><td> XLUMPLFLN20X </td><td></td></tr><tr><td> 40X（0.8 NA; WD3.3毫米） </td><td>奥林巴斯</td><td> LUMPLFLN40X/IR </td><td></td></tr><tr><td>航空表</td><td>新港</td><td> ST-200 </td><td>从外部振动隔离准备</td></tr><tr><td> XY阶段</td><td>迈克机械有限公司（Attleboro，麻萨诸塞州） </td><td></td><td>放置在 xy阶段的实验对象和Sutter微操作机器人</td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td colspan="2"> 食谱 </td></tr><tr><td>人工脑脊髓液</td><td>氯化钠（135毫摩尔），氯化钾（5.4毫摩尔），的MgCl 2（1.0毫摩尔），的CaCl 2（1.8毫摩尔），HEPES（5毫摩尔），pH为7.4 </td></tr><tr><td>液14 </td><td>氯化钠（150毫摩尔），氯化钾（2.5毫摩尔），HEPES（10mM的），pH值7.4 </td></tr></tbody>
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targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging

在非啮齿类哺乳动物初级视觉皮层的神经元聚集根据自己的喜好刺激的功能，如方向1-4，5-7方向，眼优势8,9和双眼视差9。方位选择性是最被广泛研究的功能，并具有准周期的布局为择优取向的连续映射在整个初级视觉皮层10,11是本。整合的突触，手机和网络的贡献，在这些功能的地图，以的刺激选择性的反应，需要杂交成像技术，跨越亚微米到毫米的空间尺度。与传统的内源信号光学成像，整体布局功能地图的整个表面上的视觉皮层可确定12。 在体内双光子显微镜使用钙敏感染料的发展使一个确定SYNAPT到达13或记录个人的树突棘IC输入端的活动，同时从数以百计的单个神经元细胞体6,14。因此，结合特性的信号与亚微米的空间分辨率的双光子显微镜成像提供准确地确定树突状段和细胞有助于在新皮质的任何功能的地图的微域的可能性。在这里，我们展示了一个高收益的方法快速获得皮质的方向图，针对一个特定的微域在这个功能的地图，用荧光染料标记细胞在非啮齿类哺乳动物。用于双光子成像具有相同的显微镜中，我们首先生成一个定向地图使用固有的信号的光学成像。然后，我们如何定位的利益，用微装有染料为标签的微域人口的神经元细胞体或标签的单个神经元的树突棘和轴突中是可见的体内。我们比以前的方法的改进，促进神经元的结构与功能的关系与亚细胞分辨率的新皮层的功能体系结构的框架中的一个考试。

为了说明我们的染料标记方法的精度，我们针对最小的微域非啮齿类动物的新皮质中的任何已知的功能图。在初级视觉皮层的方向图是稀疏穿插在整个奇点。这些发生在所有优选方位点收敛等，在优选的方向，周围的区域“风车”（ 图2A-B）的奇异性的样子的假彩色地图。每一个风车开颅手术选择染料标记（绿色圆圈在图2C），一定要避免大血管和小动脉，特别是15。<...

Watch this Scientific Journal Video about Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging at JoVE.com

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

用荧光染料标记细胞在预定的新皮层的功能性微域的一种方法。首先，本征信号光学成像用来获得一个功能图。双光子显微镜被用于标签和图像的神经元内微域的地图。

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

Research

JoVE Journal

Neuroscience

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

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

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

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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

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

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 ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Two-photon Calcium Imaging in Neuronal Dendrites in 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+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

轻度创伤性脑损伤大鼠海马学的高级扩散成像

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...