Name: automated rat single-pellet reaching with 3-dimensional reconstruction of paw and digit trajectories
Uploaded: 2019-07-10T15:00:00
Description: Watch this Scientific Journal Video about Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories at JoVE.com

作者要感谢卡鲁内什·甘古利和他的实验室对熟练完成任务的建议,并感谢亚历山大和麦肯齐·马希斯帮助改编DeepLabCut。这项工作得到了美国国家神经疾病和中风研究所(授予编号K08-NS072183)和密歇根大学的支持。

此处描述的所有涉及动物使用的方法均已获得密歇根大学机构动物护理和使用委员会 (IACUC) 的批准。
1. 设置接触室
注:有关仪器的详细信息和图表,请参阅 Ellens 等人14。零件号参见图1。
<ol><li>用丙烯酸水泥粘结透明聚碳酸酯面板,以构建到达室(宽15厘米,长40厘米,高40厘米)(部分#1)。一侧面板(部分#2)有一...

<ol>
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R., 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=Medial+premotor+cortex+shows+a+reduction+in+inhibitory+markers+and+mediates+recovery+in+a+mouse+model+of+focal+stroke.">Medial premotor cortex shows a reduction in inhibitory markers and mediates recovery in a mouse model of focal stroke.</a> Stroke. 44, 483-489 (2013).</li><li>Fenrich, K. K., 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=Improved+single+pellet+grasping+using+automated+ad+libitum+full-time+training+robot.">Improved single pellet grasping using automated ad libitum full-time training robot.</a> Behavioural Brain Research. 281, 137-148 (2015).</li><li>Azim, E., Jiang, J., Alstermark, B., Jessell, T. 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啮齿动物熟练达到已成为研究运动系统生理学和病理生理学的标准工具。我们已经描述了如何实现自动大鼠熟练到达任务,允许:训练和测试与最小的监督,三维爪和数字轨迹重建(在达到,抓握,和爪子缩回),实时识别爪子在到达,并与外部电子同步。它非常适合将前肢运动学与生理学联系起来,或在到达运动过程中进行精确时间的介入。
自从我们最初报告这个设计14</sup...

人类严重依赖灵巧的技能,即需要精确协调的多关节和数字运动的运动。这些技能受一系列常见的中枢神经系统疾病的影响,包括结构病变(如中风、肿瘤、脱骨髓性病变)、神经退行性疾病(如帕金森病)和运动功能异常电路(例如肌张力障碍)。因此,了解中央电机电路如何学习并实施灵巧的技能,有可能提高大量人口的生活质量。此外,这种理解有可能通过优化训练和康复策略来改善健康人的运动性能。
解剖人类灵巧技能背后的神经回路受到技术和伦理因素的限制,因此有必要使用动物模型。非人类灵长类动物通常用于研究灵巧的肢体运动,因为它们的运动系统和行为与人类相似1。然而,非人类灵长类动物由于生成时间长而昂贵,限制了研究对象和基因干预的数量。此外,虽然适用于非人类灵长类动物的神经科学工具箱比人类大,但许多最近的技术进步在灵长类动物中要么不可用,要么被大大限制。
啮齿熟练达到是研究灵巧电机控制的一种互补方法。大鼠和小鼠可以训练达到,抓住,并检索糖颗粒在一个定型的动作序列,与人类到达模式2一样。由于其相对较短的生成时间和较低的住房成本,以及他们获得技能的能力,在几天到数周内达到,有可能在学习和技能巩固阶段学习大量的科目。使用啮齿动物,特别是小鼠,也有助于使用强大的现代神经科学工具(如光遗传学,钙成像,疾病遗传模型)来研究灵巧的技能。
几十年来,啮齿熟练接触已经被用来研究正常的运动控制,以及它是如何受到中风和帕金森病等特定疾病的影响。然而,大多数版本的这个任务是劳动密集型和时间密集型的,减轻了研究啮齿动物的好处。典型的实现包括将啮齿动物放在一个接触室中,在狭窄的槽前有一个搁板,啮齿动物必须通过这个槽到达。研究人员手动将糖粒放在架子上,等待动物到达,然后放置另一个。实时或视频评论4将到达作为成功或失败进行评分。然而,只要成功或失败就达到,就忽略丰富的运动学数据,这些数据可以洞察(而不是简单地说是否)到达是如何受损的。此问题已通过实施详细的审查达到视频,以确定和半定量得分达到子运动5。虽然这增加了一些关于到达运动学的数据,但它也大大增加了实验者的时间和工作量。此外,高水平实验者参与可能导致方法和数据分析的不一致,即使在同一实验室内也是如此。
最近,已经开发出了几个自动版本的熟练接触。有的附着在家里的笼子里6、7号,省去了转移动物的需要。这既减轻了动物的压力,也无需将它们适应专门的接触室。其他版本允许爪子跟踪,以便运动学的变化在特定干预下可以研究8,9,10,或有机制自动确定颗粒是否从货架上敲下来11。自动熟练的到达任务是特别有用的高强度训练,可能需要在受伤后的康复12。自动化系统允许动物长时间执行大量到达,而无需研究人员的密集参与。此外,允许爪子跟踪和自动结果评分的系统减少了研究人员执行数据分析的时间。
我们开发了一个自动化的大鼠熟练到达系统,具有几个专业功能。首先,通过使用可移动的底座将颗粒从下方置于"到达位置",我们获得了前肢几乎一览无余的视图。其次,镜子系统允许使用单个摄像机同时查看多个覆盖视图,允许使用高分辨率、高速 (300 fps) 摄像机对到达轨迹进行三维 (3-D) 重建。随着最近开发强大的机器学习算法的无标记运动跟踪13,我们现在不仅跟踪爪子,但个别关节提取详细的伸手和掌握运动学。第三,执行简单视频处理的帧抓取器允许实时识别不同的到达阶段。此信息用于触发视频采集(由于文件大小,连续视频采集不可行),还可用于在精确时刻触发干预(例如,光遗传学)。最后,单个视频帧由晶体管-晶体管逻辑 (TTL) 脉冲触发,使视频能够与神经记录(例如电生理学或光度测量)精确同步。在这里,我们描述了如何构建这个系统,训练大鼠执行任务,将设备与外部系统同步,以及重建三维到达轨迹。

<table>
	<tbody>
		<tr>
			<td>clear polycarbonate panels</td>
			<td>TAP Plastics</td>
			<td></td>
			<td>cut to order (see box design)</td>
		</tr>
		<tr>
			<td>infrared source/detector</td>
			<td>Med Associates</td>
			<td>ENV-253SD</td>
			<td>30&#34; range</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Basler</td>
			<td>acA2000-340kc</td>
			<td>2046 x 1086 CMV2000 340 fps Color Camera Link</td>
		</tr>
		<tr>
			<td>camera lens</td>
			<td>Megapixel (computar)</td>
			<td>M0814-MP2</td>
			<td>2/3&#34; 8mm f1.4 w/ locking Iris &#38; Focus</td>
		</tr>
		<tr>
			<td>camera cables</td>
			<td>Basler</td>
			<td>#2000031083</td>
			<td>Cable PoCL Camera Link SDR/MDR Full, 5 m - Data Cables</td>
		</tr>
		<tr>
			<td>mirrors</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>linear actuator</td>
			<td>Concentrics</td>
			<td>LACT6P</td>
			<td>Linear Actuator 6&#34; Stroke (nominal), 110 Lb Force, 12 VDC, with Potentiometer</td>
		</tr>
		<tr>
			<td>pellet reservoir/funnel</td>
			<td>Amico (Amazon)</td>
			<td>a12073000ux0890</td>
			<td>6&#34; funnel</td>
		</tr>
		<tr>
			<td>guide tube</td>
			<td>ePlastics</td>
			<td>ACREXT.500X.250</td>
			<td>1/2&#34; OD x 1/4&#34; ID Clear. Extruded Plexiglass Acrylic Tube x 6ft long</td>
		</tr>
		<tr>
			<td>pellet delivery rod</td>
			<td>ePlastics</td>
			<td>ACRCAR.250</td>
			<td>0.250&#34; DIA. Cast Acrylic Rod (2&#39; length)</td>
		</tr>
		<tr>
			<td>plastic T connector</td>
			<td>United States Plastic Corp</td>
			<td>#62065</td>
			<td>3/8&#34; x 3/8&#34; x 3/8&#34; Hose ID Black HDPE Tee</td>
		</tr>
		<tr>
			<td>LED lights</td>
			<td>Lighting EVER</td>
			<td>4100066-DW-F</td>
			<td>12V Flexible Waterproof LED Light Strip, LED Tape, Daylight White, Super Bright 300 Units 5050 LEDS, 16.4Ft 5 M Spool</td>
		</tr>
		<tr>
			<td>Light backing</td>
			<td>ePlastics</td>
			<td>ACTLNAT0.125X12X36</td>
			<td>0.125&#34; x 12&#34; x 36&#34; Natural Acetal Sheet</td>
		</tr>
		<tr>
			<td>Light diffuser films</td>
			<td>inventables</td>
			<td>23114-01</td>
			<td>.007x8.5x11&#34;, matte two sides</td>
		</tr>
		<tr>
			<td>cabinet and custom frame materials</td>
			<td>various (Home Depot, etc.)</td>
			<td></td>
			<td>3/4&#34; fiber board (see protocol for dimensions of each structure)</td>
		</tr>
		<tr>
			<td>acoustic foam</td>
			<td>Acoustic First</td>
			<td></td>
			<td>FireFlex Wedge Acoustical Foam (2&#34; Thick)</td>
		</tr>
		<tr>
			<td>ventilation fans</td>
			<td>Cooler Master (Amazon)</td>
			<td>B002R9RBO0</td>
			<td>Rifle Bearing 80mm Silent Cooling Fan for Computer Cases and CPU Coolers</td>
		</tr>
		<tr>
			<td>cabinet door hinges</td>
			<td>Everbilt (Home Depot</td>
			<td>#14609</td>
			<td>continuous steel hinge (1.4&#34; x 48&#34;)</td>
		</tr>
		<tr>
			<td>cabinet wheels</td>
			<td>Everbilt (Home Depot</td>
			<td>#49509</td>
			<td>Soft rubber swivel plate caster with 90 lb. load rating and side brake</td>
		</tr>
		<tr>
			<td>cabinet door handle</td>
			<td>Everbilt (Home Depot</td>
			<td>#15094</td>
			<td>White light duty door pull (4.5&#34;)</td>
		</tr>
		<tr>
			<td>computer</td>
			<td>Hewlett Packard</td>
			<td>Z620</td>
			<td>HP Z620 Desktop Workstation</td>
		</tr>
		<tr>
			<td>Camera Link Frame Grabber</td>
			<td>National Instruments</td>
			<td>#781585-01</td>
			<td>PCIe-1473 Virtex-5 LX50 Camera Link - Full</td>
		</tr>
		<tr>
			<td>Multifunction RIO Board</td>
			<td>National Instruments</td>
			<td>#781100-01</td>
			<td>PCIe-17841R</td>
		</tr>
		<tr>
			<td>Analog RIO Board Cable</td>
			<td>National Instruments</td>
			<td>SCH68M-68F-RMIO</td>
			<td>Multifunction Cable</td>
		</tr>
		<tr>
			<td>Digital RIO Board Cable</td>
			<td>National Instruments</td>
			<td>#191667-01</td>
			<td>SHC68-68-RDIO Digital Cable for R Series</td>
		</tr>
		<tr>
			<td>Analog Terminal Block</td>
			<td>National Instruments</td>
			<td>#782536-01</td>
			<td>SCB-68A Noise Rejecting, Shielded I/O Connector Block</td>
		</tr>
		<tr>
			<td>Digital Terminal Block</td>
			<td>National Instruments</td>
			<td>#782536-01</td>
			<td>SCB-68A Noise Rejecting, Shielded I/O Connector Block</td>
		</tr>
		<tr>
			<td>24 position relay rack</td>
			<td>Measurement Computing Corp.</td>
			<td>SSR-RACK24</td>
			<td>Solid state relay backplane (Gordos/OPTO-22 type relays), 24-channel</td>
		</tr>
		<tr>
			<td>DC switch</td>
			<td>Measurement Computing Corp.</td>
			<td>SSR-ODC-05</td>
			<td>Solid state relay module, single, DC switch, 3 to 60 VDC @ 3.5 A</td>
		</tr>
		<tr>
			<td>DC Sense</td>
			<td>Measurement Computing Corp.</td>
			<td>SSR-IDC-05</td>
			<td>solid state relay module, single, DC sense, 3 to 32 VDC</td>
		</tr>
		<tr>
			<td>DC Power Supply</td>
			<td>BK Precision</td>
			<td>1671A</td>
			<td>Triple-Output 30V, 5A Digital Display DC Power Supply</td>
		</tr>
		<tr>
			<td>sugar pellets</td>
			<td>Bio Serv</td>
			<td>F0023</td>
			<td>Dustless Precision Pellets, 45 mg, Sucrose (Unflavored)</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National Instruments</td>
			<td>LabVIEW 2014 SP1, 64 and 32-bit versions</td>
			<td>64-bit LabVIEW is required to access enough memory to stream videos, but FPGA coding must be performed in 32-bit LabVIEW</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>Matlab R2019a</td>
			<td>box calibration and trajectory reconstruction software is written in Matlab and requires the Computer Vision toolbox</td>
		</tr>
	</tbody>
</table>

automated rat single-pellet reaching with 3-dimensional reconstruction of paw and digit trajectories

啮齿动物技能达到通常用于研究灵巧的技能,但需要大量的时间和精力来实现任务和分析行为。最近开发了几个自动的熟练接触版本。在这里,我们描述了一个版本,它会自动向大鼠显示颗粒,同时以高帧速率(300 fps)从多个角度录制高清视频。爪子和单个数字使用 DeepLabCut 进行跟踪,DeepLabCut 是一种用于无标记姿势估计的机器学习算法。该系统也可以与生理记录同步,或用于触发生理干预(例如,电刺激或光学刺激)。

一旦适应了仪器,大鼠就会迅速获得熟练的到达任务,在1⁄2周内达到达到和精度方面,其性能趋于稳定(图5)。图 6显示了指示 DeepLabCut 标识的结构的示例视频帧,图 7显示了单个会话中叠加的单个到达轨迹。最后,在图8中,我们说明了如果爪子检测触发器(步骤4.3.4~4.3.6)未准确设置会发生什么情况。爪子突破到达槽?...

Watch this Scientific Journal Video about Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories at JoVE.com

Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories

自动大鼠单丸到达与3维重建的爪子和数字轨迹

啮齿动物技能达到通常用于研究灵巧的技能,但需要大量的时间和精力来实现任务和分析行为。我们描述了一个自动化版本的熟练到达与运动跟踪和三维重建的到达轨迹。

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the ...

此方法基于已建立的大鼠熟练到达协议，以自动化培训和测试，为相对快速获取大型数据集提供了有效手段。它最大限度地减少了实验者的努力，同时允许前肢运动学的三维重建。运动学可用于评估精确定时干预的效果，或与生理记录相关。通过一些调整，该技术也可以应用于小鼠。首先打开灯，把老鼠放在熟练的接触室。使用钳子将颗粒通过盒子前面的到达槽。允许大鼠从钳子中吃掉三粒。下次大鼠尝试从钳子中吃掉颗粒时，将颗粒拉回来，然后重复，直到大鼠用一只爪子到达颗粒，其中 11 只最达到指示爪子偏好。接下来，将颗粒输送杆与接触槽的侧面与大鼠首选的爪子相对对齐。在输送棒上放置一个颗粒。然后用钳子用小丸子引诱大鼠，但将大鼠指向输送杆，以便其爪子击中杆上的颗粒。在大鼠试图达到10到达传递杆而不被诱饵后，将大鼠推进到下一阶段。接下来，根据大鼠的爪子偏好定位颗粒输送杆，并设置其位置为二。使用执行器遥控器设置颗粒输送杆的高度位置。一旦输送杆达到正确的高度，按住所需的数字，直到指示灯呈红色闪烁以设置。然后把老鼠放在房间里，用小丸子把老鼠引诱到后面。当大鼠向腔室的背面移动足够远，如果自动版本正在运行，它会断开红外光束，则将颗粒输送杆移动到位置三。最后，等待大鼠伸手寻找颗粒，然后将颗粒输送杆移回位置二。如果脱落，在输送杆上放置新颗粒。重复这些步骤，逐渐诱饵大鼠越来越少，直到大鼠开始一个，移动到后面要求一个颗粒，而不被诱饵，和两个，立即移动到前面后，要求一个颗粒在后面。大鼠完成此操作 10 次后，开始执行自动化任务的培训。要设置自动系统，请打开腔室中的灯并重新填充颗粒储液罐。根据大鼠的爪子偏好放置颗粒输送杆。检查执行器位置是否设置正确。接下来，打开计算机并打开熟练的伸手程序。在主题下输入大鼠 ID 号，然后从手下拉菜单中选择爪子首选项。指定视频的保存路径。然后设置会话时间和最大视频数。也设置颗粒提升持续时间，并启用早期到达惩罚。接下来，要拍摄校准图像，将伸出援助之手放在伸手室内，然后将鳄鱼夹戳穿到达槽。用鳄鱼夹将立方体放在到达槽的前面。然后定位立方体，使红色一面出现在顶部镜像中，绿色面出现在左镜像中，蓝色面显示在右镜像中。在行为程序中，确保 ROI 阈值设置为非常大的值。单击运行按钮，一旦相机初始化按钮变为绿色，按开始开始视频采集。单击"卡路里模式"，然后单击"拍摄卡尔图像"以拍摄图像。稍微移动立方体，然后拍摄另一个图像。对总共三个图像重复重复。最后，通过单击"停止"停止，然后单击停止标志按钮来停止程序。从包装盒中取下援助之手和立方体。当天拍摄校准图像后，注意不要撞到行为室中的任何东西。首先，将大鼠放在熟练的接触室中。单击白色箭头以运行程序。然后通过调整 x 偏移、y 偏移、ROI 宽度和 ROI 高度来设置爪子检测 ROI 的位置。将 ROI 放在侧镜中，该侧镜直接在到达槽前面显示爪子的 动头，然后单击"开始"以开始程序。当大鼠未到达时，调整低 ROI 阈值，直到实时 ROI 触发值在 0 和 1 之间振荡。然后将 ROI 阈值设置为显著大于鼻子戳期间的实时 ROI 触发值，并在大鼠到达时低于实时 ROI 触发值。调整，直到视频在老鼠到达时持续触发，但当它通过插槽戳鼻子时，则不触发。监视前几个试验，以确保一切工作正常。当大鼠在请求颗粒之前到达时，观察早期达到数量的增加。当大鼠在请求颗粒后到达时，观察视频数量增加，同时将视频副本保存为垃圾箱文件。最后，在到达会话时间或最大视频数后，按停止标志按钮。结果表明，20天后，大鼠获得熟练的到达任务。此处显示单个会话的到达轨迹。这仅代表初始爪子提升，便于演示。此外，如果爪子检测触发器未准确设置，则爪子破坏接触槽的框架存在显著变化，这可能导致在接触运动过程中触发干预时发生变异。除了小心处理动物，本协议中不会采取任何预防措施。最重要的是正确设置 ROI 阈值，以便在适当的时候触发视频或其他干预。此方法可以与多种其他技术（如光遗传学、钙成像和遗传模型）配对，这些技术可以解决从基本运动控制到运动障碍病理生理学等各种问题。这项技术允许详细的接触和掌握运动学与生理记录或干预的相关性，使研究人员不仅回答是否，但运动如何改变与神经电路的变化。

Research

JoVE Journal

Behavior

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

小鼠自动化，定量认知/行为筛选：对于遗传学，药理学，动物认知与本科教学

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

科研

行为学

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

为感觉运动整合与认知控制实验完全自动化的啮齿动物调理协议

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

Study Motor Skill Learning by Single-pellet Reaching Tasks in Mice

学习运动技能通过单颗粒到达任务小鼠学习

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

使用自动图像处理方法鼠标社交性的实验评估

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

PAW-拖动：一本小说，鼠标缸试验敏感性分析

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device (ALDM) Test Systems

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device ALDM Test Systems

社会认知的非人类灵长类动物评估使用电脑自动学习设备（ALDM）测试系统的网络

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys ...

Acquisition of a High-precision Skilled Forelimb Reaching Task in Rats

收购高精度熟练的前肢到达任务大鼠

Movements are the main measurable output of central nervous system function. Developing behavioral paradigms that allow detailed analysis of motor ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

墨西哥河鱼睡眠和睡眠活动的自动测量

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep ...

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

对比度增强记录 (PrAnCER) 的爪打印分析:用于评估电机缺陷的低成本、开放访问自动步态分析系统

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...