在神经生理学实验中利用神经元刺激活性触发闭环刺激
Summary
该协议演示如何使用电生理系统进行由神经元活动模式触发的闭环刺激。还提供可轻松针对不同刺激设备修改的示例 Matlab 代码。
Abstract
闭环神经生理学系统使用神经元活动模式来触发刺激，进而影响大脑活动。这种闭环系统已经在临床应用中找到，是基础脑研究的重要工具。最近一个特别有趣的发展是闭环方法与光遗传学的整合，这样神经元活动的特定模式可以触发选定神经元群的光学刺激。然而，为闭环实验建立电生理系统是很困难的。在这里，提供了一个随时应用的Matlab代码，用于根据单神经元或多个神经元的活动触发刺激。此示例代码可根据个人需求轻松修改。例如，它显示了如何触发声音刺激以及如何更改声音以触发连接到 PC 串行端口的外部设备。提出的协议设计用于动物研究（Neuralynx）的流行神经元记录系统。闭环刺激的实现在醒着的大鼠中得到证明。
Introduction
该协议的目的是演示如何在神经生理学实验中实现闭环刺激。神经科学中闭环实验的典型设置涉及基于神经元活动的在线读出触发刺激。这反过来又导致大脑活动的变化，从而关闭反馈循环1，2。这种闭环实验比标准开环设置具有多种优势，特别是与光遗传学结合时，这允许研究人员针对特定的神经元子集。例如，Siegle和Wilson使用闭环操作来研究ta振荡在信息处理中的作用3。他们证明，刺激海马神经元在塔振荡的下降阶段对行为的影响与在上升阶段应用相同的刺激不同。闭环实验在临床前研究也越来越重要。例如，多种癫痫研究表明，癫痫发作时触发的神经元刺激是降低癫痫发作严重程度的一种有效方法。此外，自动癫痫检测系统和或送治疗7、8的系统对癫痫患者9、10、11、12显示出显著益处。闭环方法迅速发展的另一个应用领域是使用皮质脑机接口控制神经假体。这是因为向假肢装置的用户提供即时反馈可显著提高准确性和能力13。
近年来，一些实验室已经开发出定制系统，用于同步电子记录神经元活动和在闭环系统14，15，16，17，18的刺激。尽管其中许多设置具有令人印象深刻的特性，但在其他实验室中实现它们并不总是那么容易。这是因为系统通常需要经验丰富的技术人员来组装所需的电子元件和其他必要的硬件和软件组件。
因此，为了便于在神经科学研究中采用闭环实验，本文提供了一个协议和Matlab代码，将开环电生理记录设置19、20、21、22转换为闭环系统2、6、23。该协议旨在与数字 Lynx 记录硬件配合使用，这是一种用于神经元群记录的常用实验室系统。典型的实验包括：1） 记录 5-20 分钟的尖峰数据;2） 尖峰排序创建神经元模板;3） 使用这些模板对神经活动模式进行在线检测;4） 检测到用户指定的模式时触发刺激或实验事件。
Protocol
此处描述的所有程序均根据莱特布里奇大学动物福利委员会批准的动物研究协议进行。 1. 外科 注：用于植入神经生理学记录的探针的手术程序已在其他出版物24、25、26中发表。闭环刺激手术的确切细节取决于所使用的记录探头类型和目标的大脑区域。然而，在大多数情况下，典型的手?…
Representative Results
Fisher-Brown挪威大鼠在现场出生和长大，在实验前两周习惯处理。一个记录驱动器被手术植入，类似于前面描述的方法28，29，30，31，32，33，34。使用数字采集系统在32 kHz下记录神经元信号。神经元信号首先被放大，使用统?…
Discussion
此处描述的协议演示如何使用标准神经生理记录系统执行闭环刺激。该协议允许在计算机科学方面拥有有限专业知识的神经科学家以很少的成本快速实施各种闭环实验。这种实验经常是必要的，以研究在大脑的因果关系。
在准备动物并安装软件（步骤 1 和 2）后，闭环实验由两个单独的阶段组成。首先，初始数据采集（步骤3）收集数据，以定义与单个神经元活动对应的模板?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NSERC发现公司对AL和AG的资助。
Materials
| Baytril | Bayer, Mississauga, CA | DIN 02169428 | antibiotic; 50 mg/mL |
| Cheetah 6.4 | NeuraLynx, Tucson, AZ | 6.4.0.beta | Software interfaces for data acquisition |
| Digital Lynx 4SX | NeuraLynx, Tucson, AZ | 4SX | recording equipment |
| Headstage transmitter | TBSI | B10-3163-GK | transmits the neural signal to the receiver |
| Isoflurane | Fresenius Kabi, Toronto, CA | DIN 02237518 | inhalation anesthetic |
| Jet Denture Powder & Liqud | Lang Dental, Wheeling, US | 1230 | dental acrylic |
| Lacri-Lube | Allergan, Markham, CA | DIN 00210889 | eye ointment |
| Lido-2 | Rafter 8, Calgary | DIN 00654639 | local anesthetic; 20 mg/mL |
| Matlab | Mathworks | R2018b | software for signal processing and triggering external events |
| Metacam | Boehringer, Ingelheim, DE | DIN 02240463 | analgesic; 5 mg/mL |
| Netcom | NeuraLynx | v1 | Application Programming Interface (API) that communicates with Cheetah |
| Silicone probe | Cambridge Neurotech | ASSY-156-DBC2 | implanted device |
| SpikeSort 3D | NeuraLynx, Tucson, AZ | SS3D | spike waveform-to-cell classification tools |
| Wireless Radio Receiver | TBSI | 911-1062-00 | transmits the neural signal to the Digital Lynx |
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Tags
Neuron Spiking ActivityClosed-loop StimuliNeurophysiological ExperimentsClosed-loop StimulationCausal InteractionsBrainEpilepsySeizure OnsetNeuroscience ResearchCheetah 6.4 Data Acquisition SystemSpikeSort-3DNetCom Development PackageMATLABData AcquisitionSpike SortingSpikeSort3-DKlustaKwikClosed-loop Experiment
Cite This Article
Molina, L. A., Ivan, V. E., Gruber, A. J., Luczak, A. Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments. J. Vis. Exp. (153), e59812, doi:10.3791/59812 (2019).