Name: automated multimodal stimulation and simultaneous neuronal recording from multiple small organisms
Uploaded: 2023-03-03T14:10:00
Description: Watch this Scientific Journal Video about Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms at JoVE.com

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。

1. 神经成像设备
注意：有关构建成像和刺激系统的详细说明，请参阅Lawler和Albrecht15，该系统控制显微镜照明定时，图像采集和刺激传递（图1）。廉价的Arduino Nano激励控制器通过数字信号将流体阀驱动到阀门控制器，并通过模拟电压信号控制光遗传学照明到LED控制器。其他刺激，如振动电机和热加热器，可以使用数字或模拟...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+calcium+sensor+proteins+for+monitoring+neural+activity.">Fast calcium sensor proteins for monitoring neural activity.</a> Neurophotonics. 1, 025008 (2014).</li><li>Tian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nature Methods. 6 (12), 875-881 (2009).</li><li>Larsch, J., Ventimiglia, D., Bargmann, C. I., Albrecht, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+imaging+of+neuronal+activity+in+Caenorhabditis+elegans.">High-throughput imaging of neuronal activity in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (45), 4266-4273 (2013).</li><li>Larsch, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+circuit+for+gradient+climbing+in+C.+elegans+chemotaxis.">A circuit for gradient climbing in C. elegans chemotaxis.</a> Cell Reports. 12 (11), 1748-1760 (2015).</li><li>Lagoy, R. C., Albrecht, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+fluid+delivery+from+multiwell+plates+to+microfluidic+devices+for+high-throughput+experiments+and+microscopy.">Automated fluid delivery from multiwell plates to microfluidic devices for high-throughput experiments and microscopy.</a> Scientific Reports. 8, 6217 (2018).</li><li>Schr&#246;del, T., Prevedel, R., Aumayr, K., Zimmer, M., Vaziri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-wide+3D+imaging+of+neuronal+activity+in+Caenorhabditis+elegans+with+sculpted+light.">Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light.</a> Nature Methods. 10 (10), 1013-1020 (2013).</li><li>Lawler, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+analysis+in+adult+C.+elegans+reveals+state-dependent+alteration+of+neural+and+behavioral+responses.">Sleep analysis in adult C. elegans reveals state-dependent alteration of neural and behavioral responses.</a> The Journal of Neuroscience. 41 (9), 1892-1907 (2021).</li><li>Reilly, D. K., Lawler, D. E., Albrecht, D. R., Srinivasan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+an+adapted+microfluidic+olfactory+chip+for+the+imaging+of+neuronal+activity+in+response+to+pheromones+in+male+C.+elegans+head+neurons.">Using an adapted microfluidic olfactory chip for the imaging of neuronal activity in response to pheromones in male C. elegans head neurons.</a> Journal of Visualized Experiments. (127), e56026 (2017).</li><li>Han, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polymer+index-matched+to+water+enables+diverse+applications+in+fluorescence+microscopy.">A polymer index-matched to water enables diverse applications in fluorescence microscopy.</a> Lab on a Chip. 21 (8), 1549-1562 (2021).</li><li>Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+the+future+of+microfluidics.">The origins and the future of microfluidics.</a> Nature. 442 (7101), 368-373 (2006).</li><li>Albrecht, D. R., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+behavioral+analysis+of+Caenorhabditis+elegans+in+precise+spatiotemporal+chemical+environments.">High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments.</a> Nature Methods. 8 (7), 599-605 (2011).</li><li>Chronis, N., Zimmer, M., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+in+vivo+imaging+of+neuronal+and+behavioral+activity+in+Caenorhabditis+elegans.">Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans.</a> Nature Methods. 4 (9), 727-731 (2007).</li><li>Cohen, R. A., Kreutzer, J. S., DeLuca, J., Caplan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+Inhibition.">Temporal Inhibition.</a> Encyclopedia of Clinical Neuropsychology. , 2480-2481 (2011).</li><li>Lawler, D. E., Albrecht, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+neural+activity+during+sleep/wake+events+in+adult+C.+elegans+by+automated+sleep+detection+and+stimulation.">Monitoring neural activity during sleep/wake events in adult C. elegans by automated sleep detection and stimulation.</a> STAR Protocols. 3 (3), 101532 (2022).</li><li>Edelstein, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+methods+of+microscope+control+using+&#181;Manager+software.">Advanced methods of microscope control using &#181;Manager software.</a> Journal of Biological Methods. 1 (2), 10 (2014).</li><li>Lagoy, R. C., Larsen, E., Lawler, D., White, H., Albrecht, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+behavioral+analysis,+microscopy,+and+neuronal+imaging+in+Caenorhabditis+elegans.">Microfluidic devices for behavioral analysis, microscopy, and neuronal imaging in Caenorhabditis elegans.</a> Methods in Molecular Biology. 2468, 293-318 (2022).</li><li>NeuroTracker. GitHub Available from: <a target="_blank" href="https://github.com/albrechtLab/Neurotracker">https://github.com/albrechtLab/Neurotracker</a> (2022)</li><li>NeuroTracker User Guide. GitHub Available from: <a target="_blank" href="https://github.com/albrechtLab/Neurotracker">https://github.com/albrechtLab/Neurotracker</a> (2022)</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Galotto, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin+triggers+calcium-mediated+immune+response+in+the+plant+model+Physcomitrella+patens.">Chitin triggers calcium-mediated immune response in the plant model Physcomitrella patens.</a> Molecular Plant-Microbe Interactions. 33 (7), 911-920 (2020).</li><li>Qian, X., Song, H., Ming, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+organoids:+Advances,+applications+and+challenges.">Brain organoids: Advances, applications and challenges.</a> Development. 146 (8), 166074 (2019).</li><li>Ventimiglia, D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+synaptic+signaling,+regulation,+and+plasticity+distinguish+two+classes+of+C.+elegans+glutamatergic+neurons.">Diverse modes of synaptic signaling, regulation, and plasticity distinguish two classes of C. elegans glutamatergic neurons.</a> eLife. 6, 31234 (2017).</li><li>Boulin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+gene+fusions.">Reporter gene fusions.</a> WormBook. , (2006).</li></ol>

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E.&#160;coli (OP50)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>Cat# OP50</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental models: Organisms/strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C.&#160;elegans strains expressing GCaMP (and optionally, Chrimson) in desired neurons</td>
			<td>Caenorhabditis Genetics Center (CGC) or corresponding authors of published work</td>
			<td>NZ1091, for example</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals, Treatments, and Worm Preparation Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# B85307</td>
			<td>diacetyl, example chemical stimulus</td>
		</tr>
		<tr>
			<td>Calcium chloride, CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein, Sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# F6377</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass water repellant</td>
			<td>Rain-X</td>
			<td>Cat #800002250</td>
			<td>glass hydrophobic treatment (single-use)</td>
		</tr>
		<tr>
			<td>Magnesium chloride, MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Medium (NGM) agar</td>
			<td>Genesee</td>
			<td>Cat #: 20-273NGM</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (60&#160;mm)</td>
			<td>Tritech</td>
			<td>Cat #T3305</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(dimethyl siloxane) (PDMS): Sylgard 184</td>
			<td>Dow Chemical</td>
			<td>Cat# 1673921</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (TFOCS)</td>
			<td>Gelest</td>
			<td>CAS# 78560-45-9</td>
			<td>glass hydrophobic treatment (durable)</td>
		</tr>
		<tr>
			<td>Software and algorithms</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino IDE</td>
			<td>Arduino</td>
			<td>https://www.arduino.cc/en/software</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>https://www.mathworks.com/products/matlab.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-manager</td>
			<td>Micro-manager</td>
			<td>https://micro-manager.org/</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope control software</td>
			<td>Albrecht Lab</td>
			<td>https://github.com/albrechtLab/MicroscopeControl</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurotracker data analysis software</td>
			<td>Albrecht Lab</td>
			<td>https://github.com/albrechtLab/Neurotracker</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Microscope and Stimulation System</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Observer.A1 inverted microscope set up for epifluorescence (GFP filter cubes, 5&#215; objective or similar)</td>
			<td>Zeiss</td>
			<td>Cat #491237-0012-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Excelitas X-cite XYLIS LED illuminator</td>
			<td>Excelitas</td>
			<td>Cat #XYLIS</td>
			<td></td>
		</tr>
		<tr>
			<td>Orca Flash 4.0 Digital sCMOS camera</td>
			<td>Hamamatsu</td>
			<td>Cat #C11440-22CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino nano</td>
			<td>Arduino</td>
			<td>Cat #A000005</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way Miniature Diapragm Isolation Valve (LQX12)</td>
			<td>Parker</td>
			<td>Cat #LQX12-3W24FF48-000</td>
			<td>Valve 1: Control</td>
		</tr>
		<tr>
			<td>2-way normally-closed (NC) Pinch Valve</td>
			<td>Bio-Chem Valve Inc</td>
			<td>Cat #075P2-S432</td>
			<td>Valve 2: Outflow</td>
		</tr>
		<tr>
			<td>3-way Pinch Valve</td>
			<td>NResearch</td>
			<td>Cat #161P091</td>
			<td>Valve 3: Stimulus selection</td>
		</tr>
		<tr>
			<td>Optogenetic stimulation LED and controller (615 nm)</td>
			<td>Mightex</td>
			<td>Cat #PLS-0625-030-S and #SLA-1200-2</td>
			<td></td>
		</tr>
		<tr>
			<td>ValveLink 8.2 digital/manual valve controller</td>
			<td>AutoMate Scientific</td>
			<td>Cat #01-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Wires and connectors</td>
			<td>various</td>
			<td>See Fig. 2 of Cell STARS Protocol (Lawler, 2021)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic Device Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dremel variable speed rotary cutter 4000&#160;</td>
			<td>Dremel</td>
			<td>Cat #F0134000AB</td>
			<td>Set speed to 5k RPM for cutting glass</td>
		</tr>
		<tr>
			<td>Dremel drill press rotary tool workstation</td>
			<td>Dremel</td>
			<td>Cat #220-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond drill bit</td>
			<td>Dremel</td>
			<td>Cat #7134</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide, 1&#160;mm thick</td>
			<td>VWR</td>
			<td>Cat #75799-268</td>
			<td></td>
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			<td>Glass scribe (Diamond scriber)</td>
			<td>Ted Pella</td>
			<td>Cat #54468</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer 3-way stopcock</td>
			<td>Cole-Parmer</td>
			<td>Cat #EW-30600-07</td>
			<td></td>
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		<tr>
			<td>Luer 23 G blunt needle</td>
			<td>VWR</td>
			<td>Cat #89134-100</td>
			<td></td>
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			<td>Microfluidic device</td>
			<td>Corresponing author or fabricate from CAD files associated with this article</td>
			<td>N/A</td>
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			<td>Microfluidic device clamp</td>
			<td>Warner Instruments (or machine shop)</td>
			<td>P-2</td>
			<td></td>
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			<td>Microfluidic tubing, 0.02&#8243; ID</td>
			<td>Cole-Parmer</td>
			<td>Cat #EW-06419-01</td>
			<td></td>
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			<td>Tube 19 G, 0.5&#8243;</td>
			<td>New England Small Tube</td>
			<td>Cat #NE-1027-12</td>
			<td></td>
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automated multimodal stimulation and simultaneous neuronal recording from multiple small organisms

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。

我们提出了几个刺激模式的例子，这些刺激模式评估了不同的神经现象，包括时间抑制、适应和去抑制。 时间抑制 是在初始呈现后不久发生的对第二次刺激呈现的神经反应的瞬时抑制14。为了测试这种现象，在配对脉冲实验中，提出了由两个1 s气味脉冲组成的八种模式，间隔范围为0 s至20 s（图6B;相应的刺激控制文件见 图3B</stro...

Watch this Scientific Journal Video about Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms at JoVE.com

Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms

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。

来自多个小生物体的自动多模态刺激和同时神经元记录

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons ...

神经反应可以洞察大脑活动，但可能因实验和个人而异。该协议有助于自动化刺激记录和分析，并简化不同类型和模式的刺激编程。自动化技术允许以相同的微流体格式并行记录多个神经元和生物体。这提高了记录的可重复性，并允许探索跨群体的神经元变异性。需要不同的刺激模式来揭示适应和感觉统合等神经现象。该方法可推广到从细胞和类器官到生物体和植物的其他动态信号。首先，打开显微镜，使用灌注注射器填充并去除储液管中的气泡，然后用缓冲液填充流出管。从真空干燥器中取出微流体装置。将设备放在显微镜上并快速插入出口管。轻轻推动出口注射器，将液体注入设备，直到液滴从入口中流出。在这个液滴出现的入口处，使用液滴到液滴的连接插入相应的流体入口管，确保液滴同时存在于入口管和设备端口孔上，以避免引入气泡。将下一个入口管连接到下一个液滴。如果需要，从出口注入更多液体，然后重复直到所有入口都充满。在未使用的入口和蜗杆装载口插入实心阻挡销。检查屏幕上的流量，以确保流量沿所需方向流动。翻转阀门控制器上的一个，观察流入微流体领域的刺激流。如果流量不平衡，请调整水库高度。使用钢丝尖镐将年轻的成年动物转移到未播种的线虫生长培养基或NGM琼脂平板上，然后用大约5毫升的一个XS基础缓冲液淹没平板，使动物可以游泳。使用预先填充了一个XS基础缓冲液的附加管将蠕虫吸入一到三毫升的加载注射器中。使用立体镜，用一只手将管子移到每个所需的动物的液体表面以下，并使用另一只手握住的注射器将其拉入管中。关闭出口轮廓，取下蠕虫装载销，并使用滴对滴连接将蠕虫装载注射器连接到设备。然后轻轻地将动物带入竞技场。建立缓冲流，并允许长达一小时的四米唑固定。使用文本编辑器，创建一个名为 userdefinedacquisition settings 的刺激定义文本文件。TXT包含自动图像采集的刺激设置。该文件定义显微镜采集参数，如曝光和激发时序、试验持续时间、间隔和保存目录。它还定义了实验类型和刺激时间设置。对于单个刺激实验，请使用只有一个重复刺激命令的格式。对于多模式实验，请使用具有多个刺激命令的格式，方法是包含表示刺激模式顺序的模式数字序列。对于每个模式，在单独的行上输入刺激命令。接下来，运行显微镜控制软件。验证所有流体入口是否打开，竞技场内需要流动，并且感兴趣的神经元在实时窗口中聚焦。然后关闭实时窗口并运行脚本多模式运行脚本。软件中的BSH。要分析数据，请依次单击插件，然后跟踪，然后运行NeuroTracker。选择包含要跟踪的 tiff 视频文件的文件夹，然后选择要处理的文件范围。接下来，使用图像和调整菜单，打开亮度对比度和阈值控制窗口。检查深色背景，不要在阈值窗口中重置范围，将阈值方法设置为默认值，将可视化效果设置为红色。然后在亮度对比度窗口中单击自动以获得神经元可见性。在亮度对比度窗口中，调整最小和最大滑块，直到神经元清晰可辨。然后调整阈值水平，直到所有神经元都显示为与其他物体分开的小红点。调整帧滑块以观察神经元运动和强度变化，注意要从跟踪中排除的任何动物，例如由于与其他动物重叠。在确定要跟踪的神经元后，根据需要调整每只动物的阈值水平，使得神经元上方的红色阈值区域在每一帧中都可见。单击神经元以记录其位置和阈值水平。选择所有神经元后，按空格键开始跟踪。监控每只动物的跟踪过程并进行任何必要的更正。如果NeuroTracker暂停，它就会失去神经元。根据需要调整阈值水平并重新单击神经元。如果积分框跳转到附近的另一个动物或非神经元结构，请按空格键暂停。将滑块移回第一个错误帧，然后重新单击正确的神经元位置。运行神经追踪器摘要pdf。m 文件，然后选择包含神经追踪器数据文本文件的文件夹。等待生成摘要 PDF，以便验证跟踪过程。可以通过每只动物和试验的数量和神经反应来识别动物，并观察以评估种群变异性。使用函数数据浏览。m 探索按试验编号、动物数量、刺激模式或其他类别分组的神经数据。在配对脉冲实验中测试了时间抑制现象，该实验产生了八种模式，由两个一秒的气味脉冲组成，间隔从0到20秒不等。第一个一秒气味脉冲在双乙酰检测AWA神经元中引起相等的响应幅度，并且第二个脉冲中的响应随刺激间隔而变化。通过捕获试验实验评估去抑制，其中观察到对双乙酰刺激的适应，但新型2-甲基吡嗪刺激的呈现没有引起强烈的去抑制效果。测试的多模态刺激表明，单独的化学刺激会引起适应，而单独的光刺激则不会。然而，组合的多模态刺激模式表明，光生反应容易受到化学诱导的适应。通过修改微流体设计以包括两个领域，我们可以同时比较遗传或其他扰动以及匹配的参考。设置完成后，可以通过多种方式修改系统。例如，我们已经自动测量了化学剂量反应和神经活动的状态依赖性变化，例如睡眠和清醒状态之间的变化。

Research

JoVE Journal

Neuroscience

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

同时神经刺激，并记录在水迷宫的设计和构建一个具有成本效益探头

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

科研

神经科学

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

金纳米棒辅助神经细胞的刺激光

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

电图同时记录和麻醉大鼠视觉诱发电位

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...