多纤维光测量记录自由移动动物的神经活动
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1/4"-20 Stainless Steel Cap Screw, 1" Long | Thorlabs | SH25S100 | |
| 1/4"-20 Stainless Steel Cap Screw, 1/2" Long | Thorlabs | SH25S050 | |
| 1/4"-20 Stainless Steel Cap Screw, 3/8" Long | Thorlabs | SH25S038 | |
| 1000 µm, 0.50 NA, SMA-SMA Fiber Patch Cable | Thorlabs | M59L01 | |
| 12.7 mm Optical Post | Thorlabs | TR30/M | |
| 12.7 mm Pedestal Post Holder | Thorlabs | PH20EM | |
| 15 V, 2.4 A Power Supply Unit with 3.5 mm Jack Connector for T-Cube | Thorlabs | KPS101 | |
| 20x objective | Thorlabs | RMS20X | #10 in Figure 2, #11 in Figure 5 |
| 30 mm Cage Cube with Dichroic Filter Mount | Thorlabs | CM1-DCH/M | #8-9 in Figure 2, #8-10 in Figure 5 |
| 405 nm LED | Doric Lenses | CLED_405 | #2 in Figure 2 |
| 410 nm bandpass filter | Thorlabs | FB410-10 | #5 in Figure 2; #7 in Figure 5 |
| 465 nm. LED | Doric Lenses | CLED_465 | #1 in Figure 2 |
| 470 nm bandpass filter | Thorlabs | FB470-10 | #4 in Figure 2; #6 in Figure 5 |
| 560 nm bandpass filter | Semrock | FF01-560/14-25 | #5 in Figure 5 |
| 560 nm LED | Doric Lenses | CLED_560 | #1 in Figure 3 |
| 5-axis kinematic Mount | Thorlabs | K5X1 | #11 in Figure 2, #12 in Figure 5 |
| Achromatic Doublet | Thorlabs | AC254-035-A-ML | #7 in Figure 2 |
| Adaptor for 405 collimator | Thorlabs | AD11F | #3 in Figure 2; #4 in Figure 5 |
| Adaptor for ajustable collimator | Thorlabs | AD127-F | #3 in Figure 2; #4 in Figure 5 |
| Aluminum Breadboard | Thorlabs | MB1824 | |
| Clamping Fork | Thorlabs | CF125 | |
| Cube connector | Thorlabs | CM1-CC | |
| Dual 493/574 dichroic | Semrock | FF493/574-Di01-25×36 | #10 in Figure 5 |
| Emission filter for GCaMP6 | Semrock | FF01-535/22-25 | #6 in Figure 2 |
| Enclosure with Black Hardboard Panels | Thorlabs | XE25C9 | |
| Externally SM1-Threaded End Cap for Machining | Thorlabs | SM1CP2M | |
| Fast-change SM1 Lens Tube Filter Holder | Thorlabs | SM1QP | #4-6 in Figure 2, #5-7 in Figure 5 |
| Fixed Collimator for 405 nm light | Thorlabs | F671SMA-405 | #3 in Figure 2; #4 in Figure 5 |
| Fixed collimator for 470 and 560 nm light | Thorlabs | F240SMA-532 | #3 in Figure 2; #4 in Figure 5 |
| Green emission filter | Semrock | FF01-520/35-25 | In light beam splitter |
| High-Resolution USB 3.0 CMOS Camera | Thorlabs | DCC3260M | #13 in Figure 2, #15 in Figure 5 |
| Light beam splitter | Neurophotometrics | SPLIT | #14 in Figure 5 |
| Longpass Dichroic Mirror, 425 nm Cutoff | Thorlabs | DMLP425R | #8 in Figure 2, #9 in Figure 5 |
| Longpass Dichroic Mirror, 495 nm Cutoff | Semrock | FF495-Di03 | #9 in Figure 2, #8 in Figure 5 |
| Metabond dental cement | C&B | ||
| M8 – M8 cable | Doric Lenses | Cable_M8-M8 | |
| Optic fiber cannulas | Doric Lenses | Need to specify that these will be used to photometry experiments requiring low autofluorescence | |
| Optic fiber Patchcords | Doric Lenses | Need to specify that these will be used to photometry experiments requiring low autofluorescence | |
| Red emission filter | Semrock | FF01-600/37-25 | In light beam splitter |
| T7 LabJack | LabJack | ||
| T-cube LED Driver | Thorlabs | LEDD1B | |
| USB 3.0 I/O Cable, Hirose 25, for DCC3240 | Thorlabs | CAB-DCU-T3 |
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