Name: multi-fiber photometry to record neural activity in freely-moving animals
Uploaded: 2019-10-20T14:30:00
Description: Watch this Scientific Journal Video about Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals at JoVE.com

这项工作得到了加拿大自然科学和工程研究理事会（NSERC：RGPIN-2017-06131）向C.P.C.P.提供的一笔赠款支持。我们还感谢莫莱库雷病毒（https://www.neurophotonics.ca/fr/pom）的培养，用于本研究中使用的病毒载体。

所有实验均根据加州大学圣地亚哥分校机构动物护理和使用委员会以及《加拿大实验室动物护理和使用指南》进行，并经拉瓦尔动物保护大学批准委员会。
1. CMOS（互补金属氧化物半导体）摄像机与单独或分支配线之间的光路对齐
<ol><li>松开 5 轴转换器上的所有螺钉（11，图 2B）。</li>
	<li>将配线（12，图2B）</str...

<ol>
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纤维光度测量是一种可访问的方法，它允许研究人员记录自由移动动物中从定义的神经元种群中产生的大钙动力学。这种方法可以结合广泛的行为测试，包括"运动重"任务，如强迫游泳测试2，恐惧调节18，社会互动1，4，和其他7，8，19，<su...

将神经反应与动物行为的特定方面联系起来的能力对于理解特定神经元组在指导或响应动作或刺激方面的作用至关重要。鉴于动物行为的复杂性，由于无数的内部状态和外部刺激，甚至会影响最简单的动作，用单次试验分辨率记录信号，为研究人员提供了克服这些困难的必要工具限制。
光纤光度测量技术已成为许多系统神经科学领域研究人员的首选技术，因为它与其他体内记录技术相比相对简单，信噪比高，而且能够记录各种行为范式1，2，3，4，5，6，7，8。与传统的电生理方法不同，光度测量是最常用的光学方法，与基因编码的钙指标（GECIs，GCaMP系列）9。GECIs根据它们是否与钙结合而改变其荧光能力。由于神经元中钙的内部浓度受到非常严格的调节，当神经元激发作用电位时，电压门的钙通道打开，内部钙浓度的瞬时增加，从而导致GECI的荧光能力，可以很好地代理神经元发射9。
使用光纤光度测量，激发光被引导到大脑，将一个细、多模光纤引导到大脑中，并通过同一光纤收集发射信号。由于这些光纤重量轻且可弯曲，动物可以不受阻碍地移动，因此该技术与各种行为测试和条件兼容。某些条件（如光纤配线的快速移动或弯曲超出其可保持总内部反射半径的范围）可能会引入信号伪影。为了消除噪声信号的歧义，我们可以利用GCaMP的特性，称为"异位点"。简而言之，使用 GCaMP 时，当激发光的波长向左移动时，其在钙约束状态下的排放减少，钙未结合状态的发射略有增加。这两个排放的相对强度相等的点称为等值点。此时，当 GCaMP 激发时，其发射不受内部钙浓度变化的影响，信号的方差通常是由于光纤配线过弯或神经组织移动导致信号衰减相对于植入的纤维。
单单元电生理学由于其单单元和单尖峰电平分辨率，仍然是体内自由移动记录的黄金标准。然而，很难确定被记录的细胞的分子特性，而且事后分析可能相当费力。虽然纤维光度测量没有单细胞分辨率，但它确实允许研究人员提出传统技术无法解决的问题。结合病毒策略与转基因动物，GECI的表达可以定向到基因定义的神经元类型，以记录种群或投影定义的神经活动，这可以通过直接监测钙信号在斧头执行端子10，11.此外，通过植入多个光纤导管，可以同时监测来自同一动物12、13中多个大脑区域和通路的神经活动。
在本手稿中，我们描述了单纤维和多纤维光度测量技术，如何校正独立于钙的伪影，并详细介绍了如何执行单色和双色记录。我们还提供了它能够提出的问题类型及其不断增加的复杂性（参见图 1）。该协议中详述的多光纤记录的光纤光度测定可以使用https://sites.google.com/view/multifp/hardware找到的材料列表（图2）。
该系统必须同时配备 410 nm 和 470 nm 激发波长，用于从 GCaMP6 或其变体中发射钙独立和钙依赖荧光。对于自定义设置，或者如果没有可用的软件来运行系统，可以使用免费的开源程序盆景 （http://www.open-ephys.org/bonsai/）。或者，光纤光度测量可以通过MATLAB（例如，https://github.com/deisseroth-lab/multifiber）12或其他编程语言14运行。系统的软件和硬件应允许操作 410 nm 和 470 nm LED 和摄像机、提取图像（图 2），以及计算在光纤周围绘制的感兴趣区域 （ROIs） 的平均荧光强度图像。输出应为使用配线中每根光纤的 470 nm 和 410 nm LED 记录的平均强度值表。执行多纤维实验时，400 μm 捆绑纤维可能会限制小鼠的运动。在这种情况下，我们建议使用 200 μm 的配线，这提供了更大的灵活性。在训练小鼠时，也可以使用较小的虚拟电缆。
在光纤光度采集期间，能够提取感兴趣的事件的时间点至关重要。如果系统不轻易提供集成特定事件的 TTL 的内置系统，则另一种策略是为记录的单个时间点分配时间戳，以便与实验期间的特定时间和事件保持一致。可以使用计算机时钟进行时间戳。

<table>
	<tbody>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 1&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S100</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 1/2&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S050</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 3/8&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S038</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;m, 0.50 NA, SMA-SMA Fiber Patch Cable</td>
			<td>Thorlabs</td>
			<td>M59L01</td>
			<td></td>
		</tr>
		<tr>
			<td>12.7 mm Optical Post</td>
			<td>Thorlabs</td>
			<td>TR30/M</td>
			<td></td>
		</tr>
		<tr>
			<td>12.7 mm Pedestal Post Holder</td>
			<td>Thorlabs</td>
			<td>PH20EM</td>
			<td></td>
		</tr>
		<tr>
			<td>15 V, 2.4 A Power Supply Unit with 3.5 mm Jack Connector for T-Cube</td>
			<td>Thorlabs</td>
			<td>KPS101</td>
			<td></td>
		</tr>
		<tr>
			<td>20x objective</td>
			<td>Thorlabs</td>
			<td>RMS20X</td>
			<td>#10 in Figure 2, #11 in Figure 5</td>
		</tr>
		<tr>
			<td>30 mm Cage Cube with Dichroic Filter Mount</td>
			<td>Thorlabs</td>
			<td>CM1-DCH/M</td>
			<td>#8-9 in Figure 2, #8-10 in Figure 5</td>
		</tr>
		<tr>
			<td>405 nm LED</td>
			<td>Doric Lenses</td>
			<td>CLED_405</td>
			<td>#2 in Figure 2</td>
		</tr>
		<tr>
			<td>410 nm bandpass filter</td>
			<td>Thorlabs</td>
			<td>FB410-10</td>
			<td>#5 in Figure 2; #7 in Figure 5</td>
		</tr>
		<tr>
			<td>465 nm. LED</td>
			<td>Doric Lenses</td>
			<td>CLED_465</td>
			<td>#1 in Figure 2</td>
		</tr>
		<tr>
			<td>470 nm bandpass filter</td>
			<td>Thorlabs</td>
			<td>FB470-10</td>
			<td>#4 in Figure 2; #6 in Figure 5</td>
		</tr>
		<tr>
			<td>560 nm bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-560/14-25</td>
			<td>#5 in Figure 5</td>
		</tr>
		<tr>
			<td>560 nm LED</td>
			<td>Doric Lenses</td>
			<td>CLED_560</td>
			<td>#1 in Figure 3</td>
		</tr>
		<tr>
			<td>5-axis kinematic Mount</td>
			<td>Thorlabs</td>
			<td>K5X1</td>
			<td>#11 in Figure 2, #12 in Figure 5</td>
		</tr>
		<tr>
			<td>Achromatic Doublet</td>
			<td>Thorlabs</td>
			<td>AC254-035-A-ML</td>
			<td>#7 in Figure 2</td>
		</tr>
		<tr>
			<td>Adaptor for 405 collimator</td>
			<td>Thorlabs</td>
			<td>AD11F</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Adaptor for ajustable collimator</td>
			<td>Thorlabs</td>
			<td>AD127-F</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Aluminum Breadboard</td>
			<td>Thorlabs</td>
			<td>MB1824</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping Fork</td>
			<td>Thorlabs</td>
			<td>CF125</td>
			<td></td>
		</tr>
		<tr>
			<td>Cube connector</td>
			<td>Thorlabs</td>
			<td>CM1-CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual 493/574 dichroic</td>
			<td>Semrock</td>
			<td>FF493/574-Di01-25x36</td>
			<td>#10 in Figure 5</td>
		</tr>
		<tr>
			<td>Emission filter for GCaMP6</td>
			<td>Semrock</td>
			<td>FF01-535/22-25</td>
			<td>#6 in Figure 2</td>
		</tr>
		<tr>
			<td>Enclosure with Black Hardboard Panels</td>
			<td>Thorlabs</td>
			<td>XE25C9</td>
			<td></td>
		</tr>
		<tr>
			<td>Externally SM1-Threaded End Cap for Machining</td>
			<td>Thorlabs</td>
			<td>SM1CP2M</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast-change SM1 Lens Tube Filter Holder</td>
			<td>Thorlabs</td>
			<td>SM1QP</td>
			<td>#4-6 in Figure 2, #5-7 in Figure 5</td>
		</tr>
		<tr>
			<td>Fixed Collimator for 405 nm light</td>
			<td>Thorlabs</td>
			<td>F671SMA-405</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Fixed collimator for 470 and 560 nm light</td>
			<td>Thorlabs</td>
			<td>F240SMA-532</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Green emission filter</td>
			<td>Semrock</td>
			<td>FF01-520/35-25</td>
			<td>In light beam splitter</td>
		</tr>
		<tr>
			<td>High-Resolution USB 3.0 CMOS Camera</td>
			<td>Thorlabs</td>
			<td>DCC3260M</td>
			<td>#13 in Figure 2, #15 in Figure 5</td>
		</tr>
		<tr>
			<td>Light beam splitter</td>
			<td>Neurophotometrics</td>
			<td>SPLIT</td>
			<td>#14 in Figure 5</td>
		</tr>
		<tr>
			<td>Longpass Dichroic Mirror, 425 nm Cutoff</td>
			<td>Thorlabs</td>
			<td>DMLP425R</td>
			<td>#8 in Figure 2, #9 in Figure 5</td>
		</tr>
		<tr>
			<td>Longpass Dichroic Mirror, 495 nm Cutoff</td>
			<td>Semrock</td>
			<td>FF495-Di03</td>
			<td>#9 in Figure 2, #8 in Figure 5</td>
		</tr>
		<tr>
			<td>Metabond dental cement</td>
			<td>C&#38;B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M8 - M8 cable</td>
			<td>Doric Lenses</td>
			<td>Cable_M8-M8</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic fiber cannulas</td>
			<td>Doric Lenses</td>
			<td></td>
			<td>Need to specify that these will be used to photometry experiments requiring low autofluorescence</td>
		</tr>
		<tr>
			<td>Optic fiber Patchcords</td>
			<td>Doric Lenses</td>
			<td></td>
			<td>Need to specify that these will be used to photometry experiments requiring low autofluorescence</td>
		</tr>
		<tr>
			<td>Red emission filter</td>
			<td>Semrock</td>
			<td>FF01-600/37-25</td>
			<td>In light beam splitter</td>
		</tr>
		<tr>
			<td>T7 LabJack</td>
			<td>LabJack</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-cube LED Driver</td>
			<td>Thorlabs</td>
			<td>LEDD1B</td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 I/O Cable, Hirose 25, for DCC3240</td>
			<td>Thorlabs</td>
			<td>CAB-DCU-T3</td>
			<td></td>
		</tr>
	</tbody>
</table>

multi-fiber photometry to record neural activity in freely-moving animals

记录一组神经元在自由移动的动物中的活动是一项具有挑战性的任务。此外，当大脑被解剖成越来越小的功能亚群时，从神经元的投影和/或遗传定义的亚群中记录变得至关重要。纤维光度测量是一种可访问且功能强大的方法，可以克服这些挑战。通过结合光学和遗传方法，神经活动可以通过表达基因编码的钙指标在深脑结构中测量，将神经活动转化为易于测量的光学信号。目前的协议详细说明了多光纤光度测定系统的组件，如何访问深脑结构来传递和收集光，一种考虑运动伪影的方法，以及如何处理和分析荧光信号。该协议详细介绍了从单一或多植入光纤执行单色和双色成像时的实验注意事项。

行为反应的神经相关性可能因多种因素而异。在此示例中，我们在体内纤维光度测量中用于测量从侧下丘脑区域 （LHA） 端接在侧下丘脑 （LHb） 中的斧子端子的活性。野生型小鼠在LHA中注射了编码GCaMP6s（AAV-hSyn-GCaMP6s）的腺相关病毒（AAV），并植入了一条光纤，尖端紧接在LHb上方（图4A）。GCaMP6s表达在LHA的细胞体及其投影到LHb的斧子端子中被发现，在那里可以?...

Watch this Scientific Journal Video about Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals at JoVE.com

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

该协议详细介绍了如何实现和执行多光纤光度测定记录，如何校正与钙无关的伪影，以及双色光度成像的重要注意事项。

多纤维光测量记录自由移动动物的神经活动

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

光纤光度测量 CSOM 成像结合了基因编码的自定义指示器和光纤，以监控自由流动动物的神经活动。这对于理解一组特定的神经元在指挥或响应动作或刺激时如何发挥作用至关重要。这种技术是一种从特定大脑区域记录由其连接或基因特征定义的可访问方法，内置控制功能可将信号与噪声分离。证明处理器将是，叶卡捷琳娜·马泰诺娃，一个研究生在我的实验室。首先松开五轴转换器上的所有螺钉。将零线拧到贴在五轴转换器上的适配器上。然后在低功耗下打开 470 纳米激发灯，将光纤尖端指向自动荧光塑料幻灯片。下一个记录从免费金属氧化物半导体或CMOS相机在实时模式。增加增益或调整查找表，直到图像在目标的焦点可见。将五轴转换器推进到目标方向，确保 470 纳米光以配线 SMA 或 FC 端的光纤为中心，直到在相机上解析图像。最后，调整 X 轴和 Y 轴，直到图像居中并解析良好。可视化从配线变种端发出的光，因为它应显示为等向性圆。首先打开所有激励灯，以更好地可视化光纤。并调整摄像机增益，使像素不饱和，并存在光纤的清晰图像。然后拍摄初步图像。在光纤周围绘制感兴趣的区域或 ROIS。并保留它们以测量录制过程中的均值。对于多个光纤录制，通过从所有光纤进行实时录制来测试独立光纤。将一根光纤指向光源，然后用手指轻点。观察通道中的波动。然后将彩色胶带应用到光纤的末尾，以标注哪个 ROI 对应于哪个光纤。最后，使用支架、夹子和支架将配线悬挂在竞技场上方，设置录制区域。最后，确保动物不会受到纤维长度的移动限制，并可以在整个竞技场上自由移动。首先，用眼睛检查配线纤维的端端，并用迷你光纤显微镜检查。如果纤维表面被划伤，使用纤维抛光膜用细砂砾重新抛光纤维。然后用 70% 乙醇和棉片施用器清洁配线的端端。使用 70% 乙醇和棉尖施用器清洁光纤管。使用覆盖着黑色收缩管的陶瓷分套将配线变频端连接到植入的纤维。在连接过程中，确保套筒紧固。让动物恢复几分钟。然后开始记录光信号，然后运行实验。在录制时，请仔细观察实时跟踪，以确保录制的质量。并注意信号的任何跳跃，表示套筒不够紧。对于双色记录，在光度测量系统中添加 516 纳米 LED 以激发红色荧光钙传感器，以及适当的双色镜和滤镜。然后在目标和 CMOS 摄像机之间添加图像拆分器，以分隔绿色和红色发射波长度。最后，在红色和绿色两种颜色的所有光纤周围绘制 ROIS。确保用相应的光纤和通道清楚地识别每个 ROI。使用 470 纳米和 560 纳米 LED 同时触发激发，并用 410 纳米 LED 交替。结果表明，测量的荧光与LHA-LHB通路激活小鼠的空气泡芙同时显著增加。然而，在表达绿色荧光蛋白或GP的小鼠中，在空气泡芙的给法过程中未检测到信号的变化。这项技术使研究人员能够从不同大脑区域的特定类型的细胞中合理记录。当动物自由行为时。这进一步剖析这些大脑区域的功能的能力，因为它们与行为有关。

Research

JoVE Journal

Neuroscience

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

高密度的自由移动，使用基于聚酰亚胺的微电极的小鼠的脑电图记录

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

科研

神经科学

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

自由移动鸽子穿越三个阶段学习记录单个神经元&#39;动作电位

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（<em&gt;欧洲熊蜂</em&gt;）

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Noninvasive EEG Recordings from Freely Moving Piglets

自由移动仔猪的无创脑电图记录

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...