体内 熟练运动行为的无线光遗传学控制
概要
本实验方案描述了如何在单个颗粒到达抓取任务中使用无线光遗传学与高速摄像相结合，以表征参与自由移动小鼠中熟练运动行为表现的神经回路。
Abstract
精细运动技能在日常生活中是必不可少的，在几种神经系统疾病中可能会受到损害。这些任务的获取和执行需要感觉 – 运动整合，并涉及对双侧脑回路的精确控制。在动物模型中实施单手动行为范式将提高对大脑结构（如纹状体）对复杂运动行为的贡献的理解，因为它允许在任务执行期间在控制条件和疾病中操纵和记录特定细胞核的神经活动。
自创建以来，光遗传学一直是通过对神经元群体进行选择性和靶向激活或抑制来询问大脑的主要工具。光遗传学与行为测定的结合揭示了特定大脑功能的潜在机制。带有小型发光二极管（LED）的无线头戴式系统允许在完全自由移动的动物中进行远程光遗传学控制。这避免了有线系统对动物行为限制较少的限制，而不会影响发光效率。目前的协议将无线光遗传学方法与高速摄像相结合，用于单手动灵巧性任务，以剖析特定神经元群体对精细运动行为的贡献。
Introduction
在我们执行的大多数运动中都存在运动技能行为，并且已知在几种脑部疾病中受到影响1，2，3，4，5，6。实施允许研究熟练运动的发展，学习和表现的任务对于理解运动功能的神经生物学基础至关重要，特别是在脑损伤，神经退行性和神经发育障碍的模型中2，7，8，9，10，11，12，13.在日常生活行动中，伸手和取回物体是常规的，这是在早期发育过程中获得的首批运动技能之一，然后在5，6年中得到完善。它包括一种复杂的行为，需要感觉运动过程，例如对物体特征的感知，运动计划，动作选择，运动执行，身体协调和速度调制7，14，15，16。因此，单手动高灵活性任务需要两个半球的许多大脑结构的参与16，17，18，19，20，21，22。在小鼠中，单个颗粒伸手抓取任务的特征是可以单独控制和分析的几个阶段7，13，23。该特征允许研究特定神经元亚群在不同阶段的习得和行为表现的贡献，并为运动系统的详细研究13，23，24提供平台。运动发生在几秒钟内;因此，高速摄像应用于熟练运动轨迹7，25的不同阶段的运动学分析。可以从视频中提取几个参数，包括身体姿势，轨迹，速度和错误类型25。运动学分析可用于检测无线光遗传学操作过程中的细微变化7，23。
使用小型化发光二极管（LED）通过无线头戴式系统传递光，可以在动物执行任务时进行远程光遗传学控制。无线光遗传学控制器接受来自刺激器的单脉冲或连续触发命令，并将红外（IR）信号发送到连接到微型LED23，26的接收器。目前的方案将这种无线光遗传学方法与灵巧性任务的高速摄像相结合，以剖析特定神经元群体在精细运动行为执行期间的作用23。由于这是一项单项任务，因此可以评估两个半球结构的参与情况。传统上，大脑以高度不对称的方式控制身体运动;然而，高灵巧性任务需要许多大脑结构的仔细协调和控制，包括同侧核和细胞核10，20，21，22，23内神经元亚群的差异贡献。该协议表明，来自两个半球的皮质下结构控制前肢23的轨迹。这种范式可以适用于研究脑部疾病的其他大脑区域和模型。
Protocol
涉及动物使用的程序按照当地和国家指南进行，并得到相应的机构动物护理和使用委员会（细胞生理学研究所IACUC协议VLH151-19）的批准。Drd1-Cre转基因雄性小鼠27，产后35-40天，C57BL / 6背景在当前方案中使用。小鼠在以下条件下保存：温度22±1°C;湿度 55%;光照计划12/12小时，晚上7点熄灯，并在产后第21天断奶。断奶的幼崽被安置在2-5的同性组中。动物被安置在带有微屏障顶部的静态?…
Representative Results
伸手可及的任务是一种范式，广泛用于研究不同实验操作下精细技能运动的塑造，学习，表现和运动学。小鼠在几天内学会执行任务，并在训练5天后达到平台，准确率超过55%（图2A，B）。与之前报道的类似，一定比例的动物没有适当地执行任务（29.62%），这些动物应从进一步分析中排除30。这些包括非学习小鼠的子集（6/54只小鼠，11.1%），从?…
Discussion
在明确定义的行为范式中使用神经元群体的光遗传学操作正在推进我们对运动控制机制的知识7，23。无线方法特别适用于需要对多种动物进行测试或自由移动的任务34，35。然而，随着技术和设备的改进，它应该是任何行为任务与光遗传学相结合的首选34，36。
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The authors have nothing to disclose.
Acknowledgements
这项工作得到了UNAM-PAPIIT项目 IA203520的支持。我们感谢IFC动物设施在小鼠菌落维护方面的帮助和IT支持的计算单元，特别是Francisco Perez-Eugenio。
Materials
| Anaesthesia machine | RWD | R583S | Isoflurane vaporizer |
| Anesket | PiSA | Ketamine | |
| Breadboard | Thorlabs | MB3090/M | Solid aluminum optical breadboard |
| Camera lense | Canon | 50mmf/ 1.4 manual focus lenses (c-mount) | |
| Camera system | BrainVision | MiCAM02 | Camera controller and synchronizer |
| Cotton swabs | |||
| CS solution | PiSA | Sodium chloride solution 9% | |
| Customized training chamber | In house | ||
| Drill bit #105 | Dremel | 2 615 010 5AE | Engraving cutter |
| Dustless precission chocolate pellets | Bio-Serv | F05301 | |
| Ethyl Alcohol | J.T. Baker | 9000-02 | Ethanol |
| Eyespears | Ultracell | 40400-8 | Eyespears of absorbent PVA material |
| Fluriso | VetOne | V1 502017-250 | Isoflurane |
| Glass capillaries | Drumond Scientific | 3-000-203-G/X | Pipettes for NanoJect II |
| Hidrogen peroxide | Farmacom | Antiseptic | |
| High-speed camera | BrainVision | MiCAM02-CMOS | Monochrome high-speed cameras |
| Infrared emmiter | Teleopto | ||
| Insulin syringe | |||
| LED cannula | Teleopto | TelC-c-l-d | LED cannula 250um 487nm light |
| Micropipette 10 uL | Eppendorf | Z740436 | |
| Micro-pipette puller | Sutter | P-87 | Horizontal puller |
| Microscope LSM780 | Zeiss | Confocal microscope | |
| Microtome | |||
| Mock receiver | Teleopto | ||
| NanoJect II | Drumond Scientific | 3-000-204 | Micro injector |
| Oxygen tank | Infra | na | |
| pAAV-EF1a-double.floxed-hChR2(H134R)-mCherry-WPRE- HGHpA | Addgene | 20297 | Viral vector for ChR-2 expression |
| Parafilm | |||
| Paraformaldehyde | Sigma | P-6148 | |
| Phosphate saline buffer | Sigma | P-4417 | Phosphate saline buffer tablets |
| Pipette tips 10 uL | ThermoFisher | AM12635 | 0.5-10 uL volume |
| Pisabental | PiSA | Sodium pentobarbital | |
| Plexiglass | commercial | Acrylic sheet | |
| Povidone iodine | Farmacom | Antiseptic | |
| Procin | PiSA | Xylacine | |
| Puralube | Perrigo pharma | 1228112 | Eye lubricant 15% mineral oil/85% petrolatum |
| Rotary tool | Kmoon | Mini grinder | Standard |
| Scalpel | |||
| Scalpel blade | |||
| Stereotaxic apparatus | Stoelting | 51730D | Digital apparatus |
| Super-Bond C&B | Sun Medical | Dental cement | |
| Surgical dispossable cap | |||
| Teleopto remote controller | Teleopto | ||
| Tg Drd1-Cre mouse line | Gensat | 036916-UCD | Transgene insertion FK150Gsat |
| Tissue adhesive | 3M Vetbond | 1469SB | |
| TPI Vibratome 1000 plus | Peico | Microtome | |
| Vectashield mounting media with DAPI | Vector laboratories | H-1200 | Mounting media |
| Wireless receiver | Teleopto | TELER-1-P |
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