在头固定小鼠胡须信号眨眼经典条件反射
Summary
The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.
Abstract
Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse’s head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.
Introduction
眨眼调理巴甫洛夫条件反射的一种形式，调查联想学习和记忆的神经机制的模型系统。它已在各种物种，包括人类，兔，猫，大鼠和小鼠中进行了研究。该模式包括两个配对的刺激的呈现：中性条件刺激（CS; 例如 ，一个音，一道闪光，或晶须刺激），以及一个突出的非条件刺激（US; 例如 ，吹气的眼睛，或眶周震）。美国引发的无条件的，反身眨眼反应（ 即 ，UR）。最终，经过配对的CS-美国的一些演讲，主题学习到CS与美国相关联。这个学习表现为一个条件反应（CR），仅由在CS在先于美国的演示引起一个眨眼的形式。
在跟踪的形式眨眼调理包括一些H的无刺激间隔undred毫秒分隔CS和美国（ 图1）。跟踪空调是声明学习的一种形式，因为它需要刺激偶然性1意识。颞间隙要求动物保持CS的神经'跟踪'前脑区域如海马，以便美国和CS成为相关1-6。随着前脑区域，痕迹条件也依赖于小脑7。
眨眼调理，因此，对于存储器的多个方面，包括采集，合并，和检索的调查一个有用的范例。期间眨眼空调，动物的对照组呈现不成对的刺激以随机顺序来测试pseudoconditioning或致敏反应到CS可以由美国介绍单独而不是学习的CS-US关联而引起的。
常用的appara土族的眨眼调理的啮齿类动物的调查是其中啮齿动物被允许在训练过程中8-10自由行动室。与这种类型的装置中，系绳通常附连到被固定在啮齿动物的头骨头饰。系绳允许与美国（有时在CS）的递送和用于发送动物的对这些刺激（ 即眨眼反应）10的响应。系绳本身可以基于刺激的类型来修改递送和眨眼响应如何被记录。
之所以使用“自由移动”拴小鼠眨眼条件是，对小鼠克制斗争。虽然其他物种可能是更适合的约束，在眨眼调理实验使用的小鼠的主要优点是，大多数可用的遗传修饰的突变体菌株的是小鼠品系。除了挣扎，完整的资源小鼠traint导致急性窘迫。最大限度地减少应力的头固定鼠标准备将增加可眨眼调理过程中获得的生理信息。例如，该系统将允许用2-光子显微镜11的皮层神经元的成像。
头固定制剂已在以前的实验用于通过可拆卸颅植入的皮质光学成像被使用， 在啮齿类动物的脑与四极管阵列，双光子钙成像的体内电生理记录，也可作为在小鼠中11为眨眼调理的平台-16。
在头部固定系统，可靠的刺激和录音，确保没有鼠标（ 图2）完全克制。像中的自由移动系统中使用的一项所述的头盔固定到小鼠的颅骨。在训练期间，头状被固定到被连接到棒上的连接器为了一圆柱形的跑步机，以稳定的啮齿动物的头部（ 图2A）。圆柱形跑步机允许鼠标舒适休息，但如果鼠标愿意，也允许其运行或行走。与使用该系统，小鼠可以用晶须振动作为CS和温和的眶周触电，因为美国的（ 图1）进行培训。美国正在通过导线手术放置在皮肤侧至眼睛下方输送。在CS经由附加到一个2层为矩形的弯曲致动器（ 图2B）的梳递送。梳子和弯曲致动器，然后附着到正在训练期间移动到适当的位置，并调整为每个单独的动物的最佳递送的磁性底座。梳子被定位成跨在所选晶须。期间交付的CS，一个信号被发送到弯曲致动器位移的梳子和导致晶须17的振动。
<p class="“jove_content”">其他刺激如音调或光的闪光灯已被用作在小鼠中有效条件刺激过去16,18,19。究其原因晶须刺激选择了CS这个实验范式是鼠类动物的勘探过程中他们的触须对躯体感觉信息输入的依赖。晶须刺激已被证明是可靠的和有效的CS 20。此外，鉴于触须系统（ 即 ，桶皮质），晶须刺激的成熟和有组织的皮质衬底作为CS提供映射与学习眨眼空调20,21相关的皮层变化和可塑性优雅的工具。头部固定系统允许对选定的晶须的精确刺激比较刺激神经元和非刺激晶须接收输入神经元之间的反应。最后，小鼠多株表现出与年龄相关的听力损失是相对年轻的成年人<su在空调闪烁期间P> 22，且眼睑闭合改变一个可视化的CS（虽然视觉CS确实改善问题与惊跳反应16）。晶须刺激不受任一这些并发症。这里介绍的是在对方头部固定筹备眨眼空调，包括CS和美国交付方式独特而重要的修改，并收购了眨眼反应。此装置和在眨眼调理训练模式的可靠性通过学习从空调小鼠和从pseudoconditioned对照动物（ 图7A）的相对平坦的学习曲线的曲线证明。
Protocol
Representative Results
Discussion
经典眨眼调理联想学习有助于理解基础的学习和记忆的神经基板的有用工具的一种形式。如小鼠在啮齿类眨眼调理采用以前的方法涉及，允许用于动物至约自由移动的腔室。用于在小鼠中眨眼调理的磁头固定制剂，使用在西格尔等人在光诱发痕量眨眼调理由Chettih 等人和Heiney 等人 ，最近利用所描述的装置提供了几个优点，如允许某些眨眼调理该没有可能或在过去已被限制（ <e…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国防部（W81XWH-13-01-0243）和美国国立卫生研究院（R37 AG008796）的资助。我们感谢艾伦·贝克在美国西北大学的机械加工车间建设头固定筒设备。我们感谢Shoai服部博士，他在MATLAB和Solidworks指导。我们感谢约翰功率博士对于控制实验中的LabVIEW软件。
Materials
| Exervo TeraNova Foam Roller 36" x 6" | Amazon | B002ONUM0E | For cylinder |
| Plexiglas | Custom-made; 1 cm thick | ||
| Metal Rods (12.7mm diameter) | Custom-made | ||
| 4-40 machine screw (.25 in long) | Amazon Supply | B00F33Q8QO | For cylinder |
| Classic Design Hair Comb | Conair | 93505WG-320 | For whisker stimulation |
| 2-Layer Rectangular Bending Actuator | Piezo Systems | T220-A4-303X | For whisker stimulation |
| Solder and Flux Kit | Piezo Systems | MSF-003-NI | For whisker stimulation |
| Magnetic Base | Thor Labs | MB175 | For whisker stimulation |
| Threaded rod for magnetic base | Custom-made | ||
| Strips based on 221 series nylon strip connectors from Electronic Connector Corp. | Custom-made, based on Weiss and Disterhoft, 2008 | ||
| TO-220 Style Transistor | Amazon Supply | B0002ZPZYO | For connector; for the wings |
| Relia-Tac Sockets | Electronic Connector Corp. | 220-S02 | For connector |
| Relia-Tac Pins | Electronic Connector Corp. | 220-P02 | For headpiece |
| 0-80 stainless steel machine screw (1 in. long) | Amazon Supply | B000FN68EE | Locking Screw |
| 0-80 stainless steel machine screw hex nut (5/32 in. thick) | Amazon Supply | B000N2TK7Y | Locking Screw Head |
| Loctite Super Glue-Liquid | Loctite | 1365896 | Cyanoacrylic glue; for the locking screw |
| Quick Setting Epoxy | Ace Hardware | 18613 | For connector and whisker stimulation system |
| Ethernet Cable Wires | Ethernet cable can be taken apart to use the individual wires for the connector | ||
| Polyimide coated stainless steel wires (2 in. long, .005 in. diameter) | PlasticsOne | 005sw/2.0 37365 S-S | For headpiece, EMG and shock wires |
| Stainless steel uncoated wire (.005 in. diameter) | AM Systems | 792800 | For headpiece, ground wires |
| Tenma Variable Autotransformer | Tenma | 72-110 | For the whisker stimulation; rheostat to adjust current to the bending actuator |
| Amplifier | A-M Systems | 1700 | Amplifier for filtering and amplifying EMG signals |
| WPI A385R stimulus isolator | World Precision Instruments | 31405 | For the electrical shock |
| Isothesia (Isoflurane) | Henry Schein: Animal Health | 50031 | For surgery; anesthesia |
| Buprenex Injectable CIII | Reckett Benckiser Pharmaceuticals Inc | NDC 12496-0757-1 | For surgery; analgesic |
| Akwa Tears: Lubricant Ophthalmic Ointment | Akorn | NDC 17478-062-35 | Artificial tear ointment to prevent dry eyes while under anesthesia |
| Povidine-Iodine Prep Pads | PDI | NDC 10819-3883-1 | For surgery; antiseptic |
| Alcohol Prep Pads | May be purchased from any standard pharmacy | ||
| Stainless steel surgical scalpel handles (no.3) | Integra Miltex | 4-7. | For surgery |
| Stainless steel surgical scalpel blades | Integra Miltex | 4-310 or 4-315 | For surgery; number 10 or 15 scalpel blade |
| 3% Hydrogen Peroxide | May be purchased from any standard pharmacy | ||
| Micro Clip | Roboz | RS-5459 | For surgery, to hold back skin |
| 00-90 stainless steel machine screw (0.0625 in. long) | Amazon Supply | B002SG89X4 | For surgery, to wrap ground wire around |
| Professional Rotary Tool | Walnut Hollow | 29637 | Hand drill for surgery, to drill holes in skull |
| Inverted Cone Burr | Roboz | RS-6282C-34 | Inverted cone burr size 34; for surgery, to drill holes in skull |
| Engraving Cutter Drill Bit | Dremel | 106 | Engraving cutter; 1.6 mm bit; for surgery, to drill holes in skull |
| C&B Metabond-Quick! Cement System "B" Quick Base | Parkell | S398 | For surgery; adhesive luting cement system; important to prevent headpiece avulsion |
| C&B Metabond-Quick! Cement System Clear L-Powder | Parkell | S399 | For surgery; adhesive luting cement system; important to prevent headpiece avulsion |
| C&B Metabond-Quick! Cement System "C" Universal TBB Catalyst 0.7 ml | Parkell | S371 | For surgery; adhesive luting cement system; important to prevent headpiece avulsion |
| C&B Metabond-Quick! Cement System Ceramic Mixing Dish with temperature strip | Parkell | S387 | For surgery; adhesive luting cement system; important to prevent headpiece avulsion |
| Swiss Tweezers, style #5 | World Precision Instruments | 504506 | For surgery |
| Puritan Cotton-Tipped Applicators | VWR International | 10806-005 | For surgery |
| Dental Caulk Grip Cement Kit | Dentsply | 675570 | For surgery; dental cement |
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