用于监测小脑依赖性感觉联想学习的灵活平台
Summary
我们开发了一个单一的平台来跟踪动物在两个攀爬纤维依赖性联想学习任务中的行为。低成本设计允许与光遗传学或成像实验集成，旨在攀登纤维相关的小脑活动。
Abstract
攀爬浦肯野细胞的纤维输入为小脑依赖性联想学习提供了至关重要的指导性信号。在头部固定小鼠中研究这些信号有助于使用成像，电生理学和光遗传学方法。在这里，开发了一个低成本的行为平台（约1000美元），可以跟踪头部固定的小鼠的联想学习，这些小鼠在跑步轮上自由运动。该平台结合了两种常见的联想学习范式：眨眼调节和延迟触觉惊吓调节。使用摄像头跟踪行为，并由检测器跟踪车轮运动。我们描述了组件和设置，并为训练和数据分析提供了详细的协议。该平台允许结合光遗传学刺激和荧光成像。该设计允许一台主机控制多个平台，以同时训练多只动物。
Introduction
巴甫洛夫条件反射刺激之间的亚秒级关联以引发条件反应长期以来一直用于探究小脑依赖性学习。例如，在经典的延迟眨眼调节（DEC）中，动物学会在响应中性条件刺激（CS;例如，闪光或听觉音调）时，当它与无条件刺激（美国;例如，施加在角膜上的空气）反复配对时，做出适时的保护性眨眼，这总是引起反射性眨眼， 并且出现在 CS 的末尾或接近末尾。习得的反应被称为条件反应（CR），而反射反应被称为无条件反应（UR）。在兔子中，小脑特异性病变破坏了这种形式的学习1，2，3，4。此外，浦肯野细胞复合物尖峰，由其爬升光纤输入5驱动，提供必要的6，7 和足够的8，9 信号，用于采集适当定时的CR。
最近，已经为头部固定的小鼠开发了攀爬纤维依赖性联想学习范式。DEC是第一个适应这种配置的关联学习范式10，11。头固定小鼠中的DEC已被用于识别小脑区域11，12，13，14，15，16，17 和电路元件11，12，13，14，15，18，19 任务获取和消亡所必需的。这种方法也被用来证明任务参数的细胞水平生理表示如何随着学习13，15，16而进化。
除了眨眼之外，延迟惊吓触觉调节（DTSC）范式最近被开发为一种用于头部固定小鼠的新型联想学习任务20。在概念上与DEC类似，DTSC涉及用US呈现中性CS，轻敲面部的强度足以使惊吓反射21，22 作为UR。在DTSC范式中，UR和CR都被读出为车轮上的向后运动。DTSC现在已被用于揭示联想学习如何改变小脑活动和基因表达模式20。
在这项工作中，开发了一种在单个平台中灵活应用DEC或DTSC的方法。激励和平台属性在 图 1 中进行了架构化。该设计结合了使用摄像头跟踪动物行为的能力，以及用于跟踪鼠标在轮子上的运动的旋转编码器。数据记录和试验结构的所有方面都由配对的微控制器（Arduino）和单板计算机（SBC;树莓派）。可以通过提供的图形用户界面访问这些设备。在这里，我们介绍了用于设置、实验准备和执行的工作流程，以及用于数据可视化的自定义分析管道。
Protocol
这里描述的动物协议已获得普林斯顿大学动物护理和使用委员会的批准。 1. 设置 SBC 将相机串行接口 （CSI） 电缆连接到 Raspberry NoIR V2 相机和 SBC 上的相机端口。 将 SBC 的操作系统下载到主机上。将操作系统映像写入微型安全数字 （microSD） 卡。注意：有关树莓派SBC的这些程序的详细说明，可以在别处找到23。该系统已使用以下…
Representative Results
DEC实验和分析的工作流程正确的实验参数选择对于成功进行延迟眨眼调节 （DEC） 训练非常重要。对于此处提供的数据，GUI 用于选择 350 ms 的 CS 持续时间和 50 ms 的美国持续时间。这种配对导致300毫秒的刺激间间隔：足够长以防止低振幅CR产生10 ，足够短以避免进入不良学习或痕量条件调节的制度，这一过程涉及额外的大脑区域11。试验之间的时?…
Discussion
此处概述的具有相关协议的平台可用于可靠地跟踪两个感官联想学习任务中的动物行为。每个任务都依赖于通过攀爬纤维路径的完整通信。在这里描述的设计中，我们加入了促进小脑反应的学习和记录/扰动的元素。这些包括一个允许自由运动的轮子11，18 以及头部固定。滚轮允许小鼠受试者自由运动，这已被观察到对于DEC采集18至关重…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国家心理健康研究所NRSA F32 MH120887-03（G.J.B.）和R01 NS045193和R01 MH115750（S.S-H.W.）的资助。我们感谢Bas Koekkoek博士和Henk-Jan Boele博士为优化DEC设置而进行的有益讨论，并感谢Yue Wang博士和Xiaoying Chen博士为优化DTSC设置而进行的有益讨论。
Materials
| "B" Quick Base For C&B METABOND – 10 mL bottle | Parkell | S398 | Dental cement solvent |
| "C" Universal TBB Catalyst – 0.7 mL | Parkell | S371 | Catalyst |
| #8 Washers | Thorlabs | W8S038 | Washers |
| 0.250" (1/4") x 8.00" Stainless Steel Precision Shafting | Servocity | 634172 | 1/4" shaft |
| 0.250” (0.770") Clamping Hub | Servocity | 545588 | Clamping hub |
| 1/4" to 6 mm Set Screw Shaft Coupler- 5 pack | Actobotics | 625106 | Shaft-coupling sleeve |
| 1/4"-20 Cap Screws, 3/4" Long | Thorlabs | SH25S075 | 1/4" bolt |
| 100 pcs 5 mm 395–400 nm UV Ultraviolet LED Light Emitting Diode Clear Round Lens 29 mm Long Lead (DC 3V) LEDs Lights +100 pcs Resistors | EDGELEC | ED_YT05_U_100Pcs | CS LEDs |
| 2 m Micro HDMI to DVI-D Cable – M/M – 2 m Micro HDMI to DVI Cable – 19 pin HDMI (D) Male to DVI-D Male – 1920 x 1200 Video | Star-tech | HDDDVIMM2M | Raspberry Pi4B to monitor cable |
| 256 GB Ultra Fit USB 3.1 Flash Drive | SanDisk | SDCZ430-256G-G46 | USB thumb drive |
| 3.3 V–5 V 4 Channels Logic Level Converter Bi-Directional Shifter Module | Amazon | B00ZC6B8VM | Logic level shifter |
| 32 GB 95 MB/s (U1) microSDHC EVO Select Memory Card | Samsung | MB-ME32GA/AM | microSD card |
| 4.50" Aluminum Channel | Servocity | 585444 | 4.5" aluminum channel |
| 48-LED CCTV Ir Infrared Night Vision Illuminator | Towallmark | SODIAL | Infrared light array |
| 4PCS Breadboards Kit Include 2PCS 830 Point 2PCS 400 Point Solderless Breadboards for Proto Shield Distribution Connecting Blocks | REXQualis | B07DL13RZH | Breadboard |
| 5 Port Gigabit Unmanaged Ethernet Network Switch | TP-Link | TL-SG105 | Ethernet switch |
| 5 V 2.5 A Raspberry Pi 3 B+ Power Supply/Adapter | Canakit | DCAR-RSP-2A5 | Power supply for Raspberry Pi 3B+ |
| 5-0 ETHILON BLACK 1 x 18" C-3 | Ethicon | 668G | Sutures |
| 6 mm Shaft Encoder 2000 PPR Pushpull Line Driver Universal Output Line Driver Output 5-26 V dc Supply | Calt | B01EWER68I | Rotary encoder |
| Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 1", 5 Pack | Thorlabs | TR1-P5 | Optical posts |
| Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 2", 5 Pack | Thorlabs | TR2-P5 | Optical posts |
| Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 4", 5 Pack | Thorlabs | TR4-P5 | Optical posts |
| Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 6", 5 Pack | Thorlabs | TR6-P5 | Optical posts |
| Ø1/2" Post Holder, Spring-Loaded Hex-Locking Thumbscrew, L = 2" | Thorlabs | PH2 | Optical post holder |
| Adapter-062-M X LUER LOCK-F | The Lee Co. | TMRA3201950Z | Solenoid valve luer adapter |
| Aeromat Foam Roller Size: 36" Length | Aeromat | B002H3CMUE | Foam roller |
| Aluminum Breadboard 10" x 12" x 1/2", 1/4"-20 Taps | Thorlabs | MB1012 | Aluminum breadboard |
| Amazon Basics HDMI to DVI Adapter Cable, Black, 6 Feet, 1-Pack | Amazon | HL-007347 | Raspberry Pi3B+ to monitor cable |
| Arduino Uno R3 | Arduino | A000066 | Arduino Uno (microcontroller board) |
| Arduino Due | Arduino | A000062 | Arduino Due (microcontroller board) |
| Bench Power Supply, Single, Adjustable, 3 Output, 0 V, 24 V, 0 A, 2 A | Tenma | 72-8335A | Power supply |
| Clear Scratch- and UV-Resistant Cast Acrylic Sheet, 12" x 24" x 1/8" | McMaster Carr | 8560K257 | Acrylic sheet |
| CNC Stepper Motor Driver 1.0–4.2 A 20–50 V DC 1/128 Micro-Step Resolutions for Nema 17 and 23 Stepper Motor | Stepper Online | B06Y5VPSFN | Stepper motor driver |
| Compact Compressed Air Regulator, Inline Relieving, Brass Housing, 1/4 NPT | McMaster Carr | 6763K13 | Air source regulator |
| Cotton Swab | Puritan | 806-WC | Cotton swab |
| Dell 1908FP 19" Flat Panel Monitor – 1908FPC | Dell | 1908FPC | Computer monitor |
| Flex Cable for Raspberry Pi Camera | Adafruit | 2144 | camera serial interface cable |
| High Torque Nema 17 Bipolar Stepper Motor 92 oz·in/65 N·cm 2.1 A Extruder Motor | Stepper Online | 17HS24-2104S | Stepper motor |
| Isoflurane | Henry Schein | 66794001725 | Isoflurane |
| Krazy Maximum Bond Permanent Glue, 0.18 oz. | Krazy Glue | KG483 | Cyanoacrylate glue |
| Lidocaine HCl | VetOne | 510212 | Lidocaine |
| Low-Strength Steel Hex Nut, Grade 2, Zinc-Plated, 1/4"-20 Thread Size | McMaster Carr | 90473A029 | Nuts |
| M3 x 50 mm Partially Threaded Hex Key Socket Cap Head Screws 10 pcs | Uxcell | A16040100ux1380 | M3 bolt |
| NEMA 17 Stepper Motor Mount | ACTOBOTICS | 555152 | Stepper motor mount |
| Official Raspberry Pi Power Supply 5.1 V 3 A with USB C – 1.5 m long | Adafruit | 4298 | Power supply for Raspberry Pi 4B |
| Optixcare Dog & Cat Eye Lube Lubricating Gel, 0.70-oz tube | Optixcare | 142422 | Opthalimic ointment |
| Precision Stainless Steel Ball Bearing, Shielded, Trade No. R188-2Z, 13000 rpm Maximum Speed | McMaster-Carr | 3759T57 | Bearing |
| Premium Female/Female Jumper Wires – 40 x 6" | Adafruit | 266 | Wires |
| Premium Female/Male 'Extension' Jumper Wires – 40 x 6" (150 mm) | Adafruit | 826 | Wires |
| Premium Male/Male Jumper Wires – 40 x 6" | Adafruit | 758 | Wires |
| Radiopaque L-Powder for C&B METABOND – 5 g | Parkell | S396 | Dental cement powder |
| Raspberry Pi (3B+ or 4B) | Adafruit | 3775 or 4295 | Raspberry Pi |
| Raspberry Pi NoIR Camera Module V2 – 8MP 1080P30 | Raspberry Pi Foundation | RPI3-NOIR-V2 | Raspberry NoIR V2 camera |
| Right-Angle Bracket, 1/4" (M6) Counterbored Slot, 8-32 Taps | Thorlabs | AB90E | Right-angle bracket |
| Right-Angle Clamp for Ø1/2" Posts, 3/16" Hex | Thorlabs | RA90 | Right-angle optical post clamp |
| Right-Angle End Clamp for Ø1/2" Posts, 1/4"-20 Stud and 3/16" Hex | Thorlabs | RA180 | Right-angle end clamp |
| RJ45 Cat-6 Ethernet Patch Internet Cable | Amazon | CAT6-7FT-5P-BLUE | Ethernet cable |
| Rotating Clamp for Ø1/2" Posts, 360° Continuously Adjustable, 3/16" Hex | Thorlabs | SWC | Rotating optical post clamps |
| Spike & Hold Driver-0.1 TO 5 MS | The Lee Co. | IECX0501350A | Solenoid valve driver |
| Swivel Base Adapter | Thorlabs | UPHA | Post holder adapter |
| USB 2.0 A-Male to Micro B Cable, 6 feet | Amazon | 7T9MV4 | USB2 type A to USB2 micro cable |
| USB 2.0 Printer Cable – A-Male to B-Male, 6 Feet (1.8 m) | Amazon | B072L34SZS | USB2 type B to USB2 type A cable |
| VHS-M/SP-12 V | The Lee Co. | INKX0514900A | Solenoid valve |
| Zinc-Plated Steel 1/4" washer, OD 1.000" | McMaster Carr | 91090A108 | Washers |
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Citar este artículo
Broussard, G. J., Kislin, M., Jung, C., Wang, S. S. -. A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning. J. Vis. Exp. (179), e63205, doi:10.3791/63205 (2022).