来自多个小生物体的自动多模态刺激和同时神经元记录
Summary
我们提出了一种灵活的化学和多模式刺激和记录许多 秀丽隐杆线虫 同时神经活动的方法。该方法使用微流体、开源硬件和软件以及有监督的自动数据分析来测量神经元现象，例如适应、时间抑制和刺激串扰。
Abstract
荧光基因编码的钙指示剂极大地促进了我们对神经动力学的理解，从单个神经元的水平到整个大脑回路。然而，神经反应可能因先前的经验、内部状态或随机因素而异，因此需要能够同时评估多个个体神经功能的方法。虽然大多数记录技术一次检查一只动物，但我们描述了使用宽场显微镜将神经元记录放大到数十种 秀丽隐杆线虫 或其他亚毫米级生物。开源硬件和软件在编程全自动实验方面具有极大的灵活性，这些实验控制各种刺激类型的强度和时间，包括化学、光学、机械、热和电磁刺激。特别是，微流体流动装置以亚秒级时间分辨率提供化学感觉刺激的精确、可重复和定量控制。然后，NeuroTracker半自动数据分析管道提取个体和人群范围的神经反应，以揭示神经兴奋性和动力学的功能变化。本文介绍了测量神经元适应、时间抑制和刺激串扰的示例。这些技术提高了刺激的精度和可重复性，允许探索群体变异性，并且可以推广到小型生物系统中从细胞和类器官到整个生物体和植物的其他动态荧光信号。
Introduction
钙成像技术允许使用荧光显微镜和靶细胞中表达的遗传编码钙指示剂实时无创记录体内神经动力学1，2，3。这些传感器通常使用绿色荧光蛋白（GFP），例如GFP-钙调蛋白-M13肽（GCaMP）家族，以增加神经元激活时的荧光强度和细胞内钙水平升高。钙成像在线虫秀丽隐杆线虫中特别强大，用于检查神经元和神经回路在活的，行为动物中的功能4，5，6，7，8，9，10，因为它们的透明性质意味着光学访问不需要手术过程，并且细胞特异性基因启动子将表达靶向感兴趣的细胞。这些技术通常利用微流体装置，其提供精确控制的环境，以在小物理尺度上研究生物，化学和物理现象11，12。用于测量神经活动的微流体装置比比皆是，新设计正在不断开发中，并且它们很容易在研究实验室中制造。然而，许多设计一次捕获一只动物，限制了实验吞吐量7，9，13。由于先前经验的差异，压力或饥饿等内部状态或基因表达水平等随机因素，动物的神经反应通常差异很大。这些差异确立了对能够同时刺激和观察许多动物并从个体中提取信息的方法的需求4.
此外，某些神经调节现象仅在特定的刺激条件下变得明显，例如时间抑制14，它指的是当刺激快速连续发生时对反应的短暂抑制。为此，电生理系统可以在广阔的刺激空间中驱动神经活动，例如，调节电脉冲电流、电压、频率、波形、占空比和周期性刺激序列的时间。自然检测到的刺激或光遗传学系统的间接刺激将受益于类似的控制机制。目前，许多自然刺激以简单的“开关”方式呈现，例如气味呈现和去除，使用商业系统在增加灵活性方面进展缓慢。然而，廉价的微控制器现在可以根据研究人员的需求定制的方式自动传递几种类型的刺激。结合微流体，这些系统已经实现了提高实验通量和灵活性的目标，允许在许多动物中同时测量对各种精确刺激的神经反应4，6。多模态刺激可用于进一步询问神经元回路，例如在正交扰动（如药物暴露）之前、期间和之后持续刺激时监测神经兴奋性的变化4。廉价、开放的显微镜系统对于推进科学研究的好处是显而易见的，但在实践中，对零件采购、构造和性能验证的需求可能会阻碍这些技术的采用。
该协议旨在缓解其中一些技术挑战。虽然以前的协议侧重于微流体装置的使用和基本刺激9，15，17，但我们在这里描述了一个灵活的，自动化的，多模态刺激传递系统的构建和使用，用于秀丽隐杆线虫或其他小生物体的神经成像利用先前描述的微流体装置4。开源系统通过简单的文本文件进行编程以定义实验，NeuroTracker数据分析程序半自动地从显微镜视频中提取神经活动数据。我们通过使用化学感觉神经元AWA评估时间抑制，去抑制和刺激串扰的示例来演示该系统，该神经元在表达光遗传学光敏离子通道5，6时响应不同的食物气味或响应光而去极化。
Protocol
Representative Results
Discussion
在该协议中，我们描述了一种开放式显微镜系统，用于使用不同刺激模式的时间精确传递来评估神经活动现象。微流体平台提供可重复的刺激，同时将数十只动物保持在显微镜视野中。很少有商业显微镜软件包允许对各种刺激定时模式进行轻松编程，而那些通常需要手动输入每种模式或专有文件格式的软件包。相比之下，实验在该系统中由文本文件定义，文本文件可以是计算机生成的，并且可以?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Fox Avery测试这些协议并审阅手稿，并感谢Eric Hall的编程帮助。本文介绍的方法的资金部分由美国国家科学基金会1724026（D.R.A.）提供。
Materials
| Bacterial strains | |||
| E. coli (OP50) | Caenorhabditis Genetics Center (CGC) | Cat# OP50 | |
| Experimental models: Organisms/strains | |||
| C. elegans strains expressing GCaMP (and optionally, Chrimson) in desired neurons | Caenorhabditis Genetics Center (CGC) or corresponding authors of published work | NZ1091, for example | |
| Chemicals, Treatments, and Worm Preparation Supplies | |||
| 2,3-Butanedione | Sigma-Aldrich | Cat# B85307 | diacetyl, example chemical stimulus |
| Calcium chloride, CaCl2 | Sigma-Aldrich | Cat# C3881 | |
| Fluorescein, Sodium salt | Sigma-Aldrich | Cat# F6377 | |
| Glass water repellant | Rain-X | Cat #800002250 | glass hydrophobic treatment (single-use) |
| Magnesium chloride, MgCl2 | Sigma-Aldrich | Cat# M2393 | |
| Nematode Growth Medium (NGM) agar | Genesee | Cat #: 20-273NGM | |
| Petri dishes (60 mm) | Tritech | Cat #T3305 | |
| Poly(dimethyl siloxane) (PDMS): Sylgard 184 | Dow Chemical | Cat# 1673921 | |
| Potassium phosphate monobasic | Sigma-Aldrich | Cat# P5655 | |
| Potassium phosphate dibasic | Sigma-Aldrich | Cat# P8281 | |
| Sodium chloride, NaCl | Sigma-Aldrich | Cat# S7653 | |
| (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (TFOCS) | Gelest | CAS# 78560-45-9 | glass hydrophobic treatment (durable) |
| Software and algorithms | |||
| Arduino IDE | Arduino | https://www.arduino.cc/en/software | |
| ImageJ | NIH | https://imagej.nih.gov/ij/ | |
| MATLAB | MathWorks | https://www.mathworks.com/products/matlab.html | |
| Micro-manager | Micro-manager | https://micro-manager.org/ | |
| Microscope control software | Albrecht Lab | https://github.com/albrechtLab/MicroscopeControl | |
| Neurotracker data analysis software | Albrecht Lab | https://github.com/albrechtLab/Neurotracker | |
| Automated Microscope and Stimulation System | |||
| Axio Observer.A1 inverted microscope set up for epifluorescence (GFP filter cubes, 5× objective or similar) | Zeiss | Cat #491237-0012-000 | |
| Excelitas X-cite XYLIS LED illuminator | Excelitas | Cat #XYLIS | |
| Orca Flash 4.0 Digital sCMOS camera | Hamamatsu | Cat #C11440-22CU | |
| Arduino nano | Arduino | Cat #A000005 | |
| 3-way Miniature Diapragm Isolation Valve (LQX12) | Parker | Cat #LQX12-3W24FF48-000 | Valve 1: Control |
| 2-way normally-closed (NC) Pinch Valve | Bio-Chem Valve Inc | Cat #075P2-S432 | Valve 2: Outflow |
| 3-way Pinch Valve | NResearch | Cat #161P091 | Valve 3: Stimulus selection |
| Optogenetic stimulation LED and controller (615 nm) | Mightex | Cat #PLS-0625-030-S and #SLA-1200-2 | |
| ValveLink 8.2 digital/manual valve controller | AutoMate Scientific | Cat #01-18 | |
| Wires and connectors | various | See Fig. 2 of Cell STARS Protocol (Lawler, 2021) | |
| Microfluidic Device Preparation | |||
| Dremel variable speed rotary cutter 4000 | Dremel | Cat #F0134000AB | Set speed to 5k RPM for cutting glass |
| Dremel drill press rotary tool workstation | Dremel | Cat #220-01 | |
| Diamond drill bit | Dremel | Cat #7134 | |
| Glass slide, 1 mm thick | VWR | Cat #75799-268 | |
| Glass scribe (Diamond scriber) | Ted Pella | Cat #54468 | |
| Luer 3-way stopcock | Cole-Parmer | Cat #EW-30600-07 | |
| Luer 23 G blunt needle | VWR | Cat #89134-100 | |
| Microfluidic device | Corresponing author or fabricate from CAD files associated with this article | N/A | |
| Microfluidic device clamp | Warner Instruments (or machine shop) | P-2 | |
| Microfluidic tubing, 0.02″ ID | Cole-Parmer | Cat #EW-06419-01 | |
| Tube 19 G, 0.5″ | New England Small Tube | Cat #NE-1027-12 |
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