自动化多模态刺激与同步记录多种小型生物的神经元活动

摘要

我们提出了一种灵活的化学和多模态刺激方法，可同时对多条秀丽隐杆线虫（Caenorhabditis elegans）进行神经活动的刺激与记录。该方法结合微流控技术、开源硬件与软件以及有监督的自动化数据分析，能够测量神经元现象，如适应性、时间抑制和刺激串扰。

摘要

荧光遗传编码钙指示剂极大地促进了我们对从单个神经元到整个脑环路水平神经动态的理解。然而，神经反应可能因先前经验、内部状态或随机因素而发生变化，因此需要能够同时评估大量个体神经功能的方法。尽管大多数记录技术一次仅检测单个动物，本文描述了利用宽场显微镜将神经元记录扩展至数十个秀丽隐杆线虫（Caenorhabditis elegans）或其他亚毫米尺度生物体的技术。开源硬件和软件使得编程完全自动化的实验具有高度灵活性，可精确控制化学、光学、机械、热和电磁等多种刺激的强度与时间。特别是，微流控流动装置能够以亚秒级时间分辨率对化学感受刺激实现精确、可重复且定量的调控。随后，NeuroTracker半自动化数据分析流程可提取个体及群体范围的神经反应，揭示神经兴奋性和动态功能的变化。本文展示了测量神经元适应性、时间抑制以及刺激串扰的应用实例。这些技术提高了刺激的精确性和可重复性，支持对群体变异性的研究，并可推广应用于从小型生物系统（包括细胞、类器官、完整生物体乃至植物）中的其他动态荧光信号检测。

引言

钙成像技术已实现对神经元活动的非侵入性记录 体内 利用荧光显微镜和在靶细胞中表达的基因编码钙指示剂实时监测神经动态^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，以在神经元激活和细胞内钙水平升高时增强荧光强度。钙成像在线虫中尤为强大 秀丽隐杆线虫 用于研究神经元和神经回路在活体动物行为过程中的功能机制^{4,5,6,7,8,9,10}，因为其透明的特性无需手术即可实现光学观察，且细胞特异性基因启动子可将表达靶向至目标细胞。这些技术通常利用微流控装置，在微小物理尺度上提供精确控制的环境，以研究生物学、化学和物理现象^11,12微流控设备广泛用于测量神经活动，新设计不断涌现，且可在研究实验室中轻松制备。然而，许多设计一次仅能固定单个动物，限制了实验通量^7,9,13神经反应在不同动物之间往往存在显著差异，这可能是由于先前经验、压力或饥饿等内部状态，或基因表达水平等随机因素所致。这些差异使得人们需要能够同时刺激并观察多个动物，并从个体中提取信息的方法。⁴.

此外，某些神经调节现象仅在特定刺激条件下才会显现，例如时间抑制¹⁴，即当刺激快速连续发生时，神经反应会出现短暂的抑制。为此，电生理系统能够在广泛的刺激参数空间内驱动神经活动，例如调节电脉冲的电流、电压、频率、波形、占空比以及周期性刺激序列的时间特性。通过自然检测到的刺激或光遗传学系统进行的间接刺激，也将受益于类似范围的控制机制。目前，许多自然刺激通常采用简单的"开关"方式呈现，例如气味的施加与移除，而现有的商用系统在增加灵活性方面进展缓慢。然而，如今低成本的微控制器已能够以可定制化的方式自动递送多种类型的刺激。结合微流控技术，这些系统实现了提高实验通量和灵活性的目标，使得可在多个动物中同时测量神经对多种精确刺激的反应^4,6。多模态刺激可用于进一步探究神经环路，例如在药物暴露等正交干扰前后及期间持续施加刺激，以监测神经兴奋性的变化⁴。低成本、开放式的显微成像系统在推动科学研究方面优势明显，但在实际应用中，元件采购、系统搭建以及性能验证的需求可能阻碍这些技术的广泛采用。

本方案旨在缓解其中一些技术难题。以往的方案主要关注微流控装置的使用和基本刺激^9,15,17我们在此描述一种用于神经成像研究的灵活、自动化、多模式刺激递送系统的构建与使用方法 秀丽隐杆线虫 或利用先前描述的微流控装置的其他小型生物体⁴. 该开源系统已进行编程 通过 使用简单的文本文件定义实验，NeuroTracker 数据分析程序可从显微镜视频中半自动提取神经活动数据。我们以化学感受神经元 AWA 为例，展示该系统的应用，用于评估时间抑制、去抑制以及刺激串扰；AWA 神经元在响应不同食物气味时会发生去极化，或在表达光遗传学光敏离子通道时响应光照而发生去极化。^5,6.

方案

1. Neural imaging equipment

NOTE: See Lawler and Albrecht¹⁵ for detailed instructions on building the imaging and stimulation system, which controls the microscope illumination timing, image acquisition, and stimulus delivery (Figure 1). An inexpensive Arduino Nano stimulus controller actuates the fluidic valves through digital signals to a valve controller and controls the optogenetic illumination through analog voltage signals to an LED controller. Other stimuli, such as vibration motors and thermal heaters, can be controlled using digital or analog signals. The stimulus controller synchronizes the stimulation and image recording via camera signals, as specified by the open-source Micro-Manager microscope control software (µManager)¹⁶. See the Table of Materials for details related to all the materials, reagents, equipment, and organisms used in this protocol.

1. Set up the neural imaging equipment, including an epifluorescence microscope with GFP optics, an sCMOS camera with a digital exposure output signal, and a stimulus controller¹⁵.
2. Connect the stimulus controller to the desired systems via digital or analog signals. For the examples presented below, the following systems are used:
   1. Use a valve controller for chemical stimulation. Connect the valve controller to fluidic solenoid or pinch valves (Figure 1)¹⁵.
   2. Use a 615 nm red LED and controller for optogenetic stimulation, mounted above the microscope stage.
3. Set up the computer with the microscope control software, create a Configuration Preset with equipment settings, and ensure proper operation of the stimulus control¹⁵.

2. Microfluidic device fabrication

NOTE: See Lagoy et al.¹⁷ for detailed information about obtaining or fabricating the master molds and the production, use, and cleaning of the microfluidic devices. These steps are summarized below.

1. Obtain or fabricate a master mold using the microfluidic design file provided (albrechtlab.github.io/microfluidics)¹⁷. For young adult C. elegans, ensure that the channel height is 55-70 µm.
2. Combine the PDMS base and curing agent at a 10:1 ratio by weight and mix thoroughly with transfer pipettes.
3. Degas for 30-60 min in a vacuum desiccator until the bubbles disappear.
4. Place the master mold into a large (150 mm diameter) Petri dish, and pour the degassed PDMS up to a depth of 4-5 mm (~100 g). Inspect and remove any dust or bubbles with a transfer pipette.
5. Bake at 65 °C on a level oven shelf for 3 h to overnight.
6. Once cured, use a scalpel to cut the PDMS from the mold and a straight razor blade to separate the devices.
7. Punch inlet and outlet holes using a 1 mm dermal punch, and clean them with dH₂O, ethanol, and again with dH₂O. Dry the device in an airstream (see Figure 2A).
8. Clean both sides of the PDMS device with adhesive tape, removing any dust or debris.
9. Prepare the glass slides to complete the microfluidic device as described¹⁷. Drill inlet holes in the top slide using a diamond bit, and render the bottom slide hydrophobic by exposure to TFOCS vapors or by applying water-repellant glass treatment (Figure 2B).
10. Assemble the glass-PDMS device sandwich into a clamp (Figure 2C).

3. Animal preparation

1. Obtain or create animals with genetically encoded calcium indicators expressed in the neurons of interest.
   NOTE: For example, line NZ1091 (kyIs587 [gpa-6p::GCaMP2.2b; unc-122p::dsRed]; kyIs5662 [odr-7p::Chrimson:SL2:mCherry; elt-2p::mCherry]) expresses GCaMP and the red light-sensitive cation channel Chrimson in the AWA sensory neuron pair⁶. Both transgenes are integrated into the genome for stable expression in every animal.
2. One day before experimentation, place at least 20 L4 larval stage C. elegans per experiment onto a nematode growth medium (NGM) agar plate seeded with an OP50 E. coli lawn. When maintained at 20 °C, this will synchronize the wild-type animals at the young adult stage the next day.
   NOTE: For array transgenes, pick animals using a fluorescence stereoscope to ensure transgene expression in the chosen animals.

4. Solution preparation

1. Prepare 1x S Basal buffer (100 mM NaCl and 0.05 M KPO₄, pH 6.0) from a 10x stock solution.
2. Prepare 1 mM tetramisole buffer by diluting 1 M stock in 1x S Basal. Use this paralytic buffer to prepare all the stimuli. An experiment typically uses about 150 mL.
3. Add 0.1-1 µg/mL fluorescein to the "control" buffer reservoirs to visualize the flow.
4. Create stimulus solutions by serial dilution to the desired final concentration. For example, create a 10⁻⁷ dilution of diacetyl attractant by first creating a 10⁻³ stock.
   NOTE: A small amount of fluorescein (0.1–1 µg/mL) may be added to the stimulus or buffer solution to verify stimulus timing, but concentration should be minimal to avoid artifacts in neural fluorescence.

5. Microfluidic device preparation

NOTE: See Reilly et al.⁹ for a video protocol showing the reservoir generation, device setup, and the loading of the animals. See also Lagoy et al.¹⁷ for a written protocol including many helpful tips.

1. Prepare three or more fluid reservoirs. For each, attach a 30 mL or 60 mL syringe reservoir, a 3 mL priming syringe, and a needle stub to a three-way Luer valve (Figure 2D). Connect the needle to microbore tubing fitted with a metal tube at the end. Label the reservoirs, mount them onto a rack attached to a ring stand, and fill them with the corresponding buffer or stimulus fluids (Figure 2E).
2. Degas the assembled microfluidic device in a vacuum desiccator for ~1 h.
3. Fill and remove the air bubbles from the reservoir tubing using the priming syringe. Fill the outflow tubing with buffer.
4. Remove the microfluidic device from the vacuum, and quickly insert the outlet tubing and inject the fluid through the device until a droplet emerges from one inlet.
5. At this inlet, use a "drop-to-drop" connection¹⁷ to insert the corresponding fluidic inlet tube (Figure 2F). Ensure that liquid drops are present on both the inlet tubing and the device port hole to avoid introducing a bubble.
6. Inject more fluid from the outlet, connect the next inlet tube, and repeat until all the inlets are filled. Insert a solid blocking pin at any unused inlets and the worm loading port.
7. Initiate flow by opening the inlet and outlet Luer valves. Inspect the device for leaks at the inlets and glass base. Inspect the device for any bubbles within the flow channels or inlets using the microscope image capture software in live mode.
   ​NOTE: If bubbles are present, wait for them to absorb into the PDMS material.

6. Animal loading

NOTE: See Lagoy et al.¹⁷.

1. Transfer young adult animals onto an unseeded NGM agar plate using a wire-tipped "pick".
2. Flood the plate with approximately 5 mL of 1x S Basal buffer such that the animals are swimming.
3. Draw the worms into a loading syringe (1 mL or 3 mL syringe with attached tubing that has been prefilled with 1x S Basal).
   1. Using a stereoscope, move the tubing end below the liquid surface with one hand to each desired animal, and draw it into the tubing using the syringe held in the other hand.
   2. Draw the worms only into the tubing, not into the syringe.
      NOTE: The tubing typically holds only about 100 µL. The animals can be expelled into a local area and then drawn again into the tubing with a small volume.
4. Close the outlet line, remove the worm loading pin, and connect the worm loading syringe to the device using a drop-to-drop connection.
5. Gently flow the animals into the arena, establish buffer flow, and allow up to 1 h for immobilization by tetramisole.
   ​NOTE: During the immobilization period, ensure that only buffer fluid enters the arena. The stimulus and control reservoirs can be turned off during this period but open them and verify the correct flow before running the stimulation trials.

7. Automated stimulation and neuronal recording

1. Create a stimulus definition text file called "User Defined Acquisition Settings.txt" with a text editor (e.g., Notepad) containing the stimulation settings for the automated image acquisition (see examples in Figure 3). The settings are divided into two sections:
   1. Microscope acquisition settings: Define the experiment type (Single-Stimulus or Multi-Pattern), exposure and excitation timing, trial duration and intervals, and save directory.
   2. Stimulation settings: Define the stimulus control parameters. A "stimulation command" specifies the actions occurring at certain video frames with the syntax <letter code><frame number>, where the letter codes are A = valve1, B = valve2, C = valve3, L = LED light; additionally, uppercase = on and lowercase = off. The LED intensity is set with the letter code "i" and a value of 0 (off) to 255 (maximum brightness).
      NOTE: The LED intensity value from 0 to 255 sets the output analog voltage from 0 V to 5 V. The current controller used here has a linear intensity scaling, and others should be calibrated with a light power meter.
      1. For a Single-Stimulus experiment with only one repeated stimulation command, use the format in Figure 3A.
      2. For a Multi-Pattern experiment with multiple stimulation commands, use the format in Figure 3B. Include a "Pattern Sequence" of digits that represents the order of the stimulus patterns. For each pattern, enter a stimulation command on a separate line.
         NOTE: A pseudorandom sequence or m-sequence can be useful to investigate stimulus history dependence.
2. Run the microscope control software. Verify that all the fluidic inlets are open, the flow is as desired within the arena (see Figure 2H), and the neurons of interest are in focus within the live window.
3. Close the live window and run the script "MultiPattern_RunScript.bsh" within the software.
   NOTE: It is useful to run a test experiment without animals to verify proper flow and stimulation. Substituting a different fluorescein concentration for each stimulus fluid can help visualize and document new stimulus patterns.
4. After experimentation, disassemble the microfluidic device, and rinse all the surfaces, tubing, and reservoirs with water.
   ​NOTE: All the components can be reused dozens of times if kept clean. Ensure that reservoirs, tubing, and microfluidic devices are maintained wet or fully dried in an air stream to avoid salt crystallization, which can clog and is difficult to remove. The devices can be kept in ethanol to sterilize but should be fully dried before reuse (>1 h at 65 °C)¹⁷.

8. Data analysis using NeuroTracker

NOTE: NeuroTracker^4,18,19 is an ImageJ/FIJI²⁰ software plugin for tracking the fluorescence intensity of multiple neurons and animals, even as they move during trials. This plugin saves data as text files with each neuron's position and background-corrected fluorescence intensity (F). The fluorescence data are normalized to the baseline fluorescence (F₀), for example, the average of several seconds prior to stimulation, as ΔF/F₀ = (F -F₀)/F₀, which can be averaged across populations.

1. Install NeuroTracker scripts as instructed (github.com/albrechtLab/Neurotracker).
2. Run NeuroTracker by clicking on Plugins | Tracking | NeuroTracker.
3. Select the folder containing the .TIF video files to be tracked, and select the desired settings.
   NOTE: If only a subset of the video files are to be tracked, select the numerical range of the files to analyze at the prompt. Default tracking settings are appropriate for nonmotile animals at 250 pix/mm resolution. Adjust the parameters proportionally for other resolutions. See the User Guide¹⁹ for further settings information.
4. Set the intensity threshold such that the neurons are visible for selection (Figure 4).
5. Open the Brightness/Contrast (B/C) and Threshold control windows using the Image | Adjust menu.
6. In the B/C window, adjust the Minimum and Maximum sliders until the neurons are clearly distinguishable (Figure 4A).
7. In the Threshold window, check Dark background and Don't Reset Range.
8. Slide the frame slider to observe the neuron movement and intensity changes, noting any animals to exclude from tracking, such as due to overlap with other animals.
9. Identify the neurons for tracking.
10. For each animal, adjust the threshold level such that the red threshold area above the neuron is visible in every frame before, during, and after stimulation.
11. Click on the neuron to record its position and threshold level.
12. Repeat the threshold adjustment and selection for each animal and neuron to track. See Figure 4C for a good threshold level example and Figure 4D,E for over-threshold and under-threshold examples. Previously selected neurons are indicated by a small box.
13. When all the neurons are selected, press the spacebar to begin tracking.
    NOTE: For additional NeuroTracker examples, see previous references^4,19.
14. Monitor the tracking process for each animal, and make any necessary corrections.
15. If NeuroTracker pauses, it has lost the neuron. Reclick on the neuron, adjusting the threshold level as needed.
16. If the integration box jumps to another nearby animal or non-neuronal structure, press the spacebar to pause, move the slider back to the first erroneous frame, and reclick on the correct neuron location.

9. Data exploration and visualization

NOTE: The MATLAB analysis script is used for data processing and visualization and to generate summary PDFs for each analysis.

1. Run the "NeuroTrackerSummary_pdf.m" file in MATLAB, and select the folder containing the NeuroTracker data text files.
2. Wait for a summary PDF to be produced, allowing the verification of the tracking process (Figure 5). Animals are identified by a number (Figure 5A), and the neural responses from each animal and trial can be viewed to assess the population variability (Figure 5B).
3. Use the function "databrowse.m" to explore the neural data, for example, grouping by trial number (Figure 5C), by animal number (Figure 5D), by stimulation pattern, or by another category.

结果

我们展示了多种用于评估不同神经现象的刺激模式示例，包括时间抑制、适应和去抑制。时间抑制是指当第二次刺激在初次刺激后短时间内呈现时，神经反应出现的短暂性抑制¹⁴。为检验这一现象，在双脉冲实验中，呈现了八种由两个相隔0 s至20 s的1 s气味脉冲组成的刺激模式（图6B；对应的刺激控制文件见图3B）。实验装置设置为单个刺激液（1.1 µM 二乙酰）、缓冲液和对照流路管，未使用的进样口用实心针封闭（图6A）。当阀1开启时，对照液进入ctrl1进样口，使流体流路发生偏移，从而使刺激液进入实验区域；当阀1关闭时，对照液进入ctrl2进样口，缓冲液则流经实验区域（见图2G、H）。在检测二乙酰的AWA神经元中，第一个1 s气味脉冲引发的反应幅度与第二个脉冲的反应幅度...

讨论

在本实验方案中，我们描述了一种开放获取的显微成像系统，用于通过精确的时间控制递送不同刺激模式，以评估神经活动现象。该微流控平台能够在显微镜视野中同时维持数十个生物样本，并可重复递送刺激。目前市面上的显微成像软件中，仅有少数支持便捷地编程设置多种刺激时序模式，而具备此功能的软件通常需要手动输入每种模式或依赖专有文件格式。相比之下，本系统通过文本文件定义实验，这些文件可由计算机生成，并能被分析软件直接读取。本文展示了利用可变刺激模式评估神经元现象（如时间依赖性抑制、适应性以及多模态刺激下的信号串扰）的有效性。数据分析流程可将庞大的显微视频文件转化为神经活动数据，进而用于可视化和分析群体间的变异性（图5B）、随时间的变化（图7C 和 图8C、D），以及扰动后的变化（图7C）。

自动化的关键优势在于能够在多次试验重复以及不同实验之间实现刺激条件的...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
细菌菌株
E. coli (OP50)	Caenorhabditis 遗传中心 (CGC)	Cat# OP50
实验模型：生物体/菌株
C. elegans 菌株，在目标神经元中表达 GCaMP（可选地，同时表达 Chrimson）	Caenorhabditis 遗传中心 (CGC) 或已发表文献的通讯作者	例如 NZ1091
化学品、处理剂及线虫制备耗材
2,3-丁二酮	Sigma-Aldrich	Cat# B85307	二乙酰，示例化学刺激物
氯化钙，CaCl2	Sigma-Aldrich	Cat# C3881
荧光素钠盐	Sigma-Aldrich	Cat# F6377
玻璃防水涂层剂	Rain-X	Cat #800002250	玻璃疏水处理（一次性使用）
氯化镁，MgCl2	Sigma-Aldrich	Cat# M2393
线虫生长培养基（NGM）琼脂	Genesee	Cat #: 20-273NGM
培养皿（60 mm）	Tritech	Cat #T3305
聚二甲基硅氧烷（PDMS）：Sylgard 184	Dow Chemical	Cat# 1673921
磷酸二氢钾	Sigma-Aldrich	Cat# P5655
磷酸氢二钾	Sigma-Aldrich	Cat# P8281
氯化钠，NaCl	Sigma-Aldrich	Cat# S7653
（十三氟-1,1,2,2-四氢辛基）三氯硅烷（TFOCS）	Gelest	CAS# 78560-45-9	玻璃疏水处理（耐用型）
软件与算法
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/
显微镜控制软件	Albrecht 实验室	https://github.com/albrechtLab/MicroscopeControl
Neurotracker 数据分析软件	Albrecht 实验室	https://github.com/albrechtLab/Neurotracker
自动化显微镜与刺激系统
Axio Observer.A1 倒置显微镜，配置落射荧光系统（GFP 滤光片组，5× 物镜或类似规格）	Zeiss	Cat #491237-0012-000
Excelitas X-cite XYLIS LED 照明器	Excelitas	Cat #XYLIS
Orca Flash 4.0 数字 sCMOS 相机	Hamamatsu	Cat #C11440-22CU
Arduino nano	Arduino	Cat #A000005
三通微型隔膜隔离阀（LQX12）	Parker	Cat #LQX12-3W24FF48-000	阀门 1：控制
二通常闭（NC）夹管阀	Bio-Chem Valve Inc	Cat #075P2-S432	阀门 2：流出
三通夹管阀	NResearch	Cat #161P091	阀门 3：刺激选择
光遗传学刺激 LED 及控制器（615 nm）	Mightex	Cat #PLS-0625-030-S 和 #SLA-1200-2
ValveLink 8.2 数字/手动阀门控制器	AutoMate Scientific	Cat #01-18
导线与连接器	多种来源	参见 Cell STARS 方案（Lawler, 2021）中的图 2
微流控装置制备
Dremel 可调速旋转切割器 4000	Dremel	Cat #F0134000AB	切割玻璃时设定转速为 5,000 RPM
Dremel 钻台旋转工具工作站	Dremel	Cat #220-01
金刚石钻头	Dremel	Cat #7134
载玻片，厚度 1 mm	VWR	Cat #75799-268
玻璃刻划笔（金刚石刻划器）	Ted Pella	Cat #54468
鲁尔三通旋塞阀	Cole-Parmer	Cat #EW-30600-07
鲁尔 23G 钝头针	VWR	Cat #89134-100
微流控装置	通讯作者提供，或根据本文附带的 CAD 文件自行制备	N/A
微流控装置夹具	Warner Instruments（或机械加工车间）	P-2
微流控管路，内径 0.02″	Cole-Parmer	Cat #EW-06419-01
管路 19G，0.5″	New England Small Tube	Cat #NE-1027-12