体外刺激对龟脑神经的运动测量
Summary
本协议描述如何使用体外分离制剂来测量其眼球运动的运动学。大脑从颅骨切除后, 可以用电流刺激颅神经, 以量化眼睛的旋转和瞳孔大小的变化。
Abstract
在动物被安乐死后, 他们的组织开始死亡。海龟提供了一个优势, 因为他们的组织的生存时间较长, 特别是与热血脊椎动物相比。正因为如此, 在海龟体内进行的体外实验可以在较长时间内进行, 以调查神经信号和控制其目标动作。采用分离头制备方法, 测量了龟眼运动的运动学, 并对其进行了神经电信号调制。大脑从头骨中取出后, 将颅骨神经完整, 解剖头被放置在一个框架内以校准眼球运动。玻璃电极附着于颅神经 (动眼神经、滑车和展), 并以电流刺激以唤起眼球运动。我们用红外线视频跟踪系统和眼睛的量化旋转来监测眼球运动。电流脉冲的幅度, 频率, 和列车持续时间被用来观察对反应的影响。由于制剂与大脑分离, 进入肌肉靶的传出通路可以单独检查, 以便在没有中央处理的感官信息的情况下研究神经信号。
Introduction
在电生理实验中使用红耳滑块龟的基本原理:
红耳滑块龟 (Trachemys 龟线虫) 被认为是世界上最严重的入侵物种之一1 , 可以表明生态系统有问题。红耳滑块龟之所以如此成功的原因是很不清楚, 但它可能部分是由于他们的耐受生理和拥有的神经组织, 可以生存在缺氧条件下2,3,4.使用它们进行实验并不会威胁到他们的数量, 并以最小的努力, 电生理准备可以保持在延长的持续时间内可行, 只要18小时5,6。这种好处类似于使用无脊椎动物 (如小龙虾7) 的优势, 它也有能力抵御低氧水平的8。
眼球运动测量技术:
用非人类灵长类测量额眼动物眼球运动的方法得到了很好的发展9。眼睛在轨道旋转大约三个轴: 水平, 垂直和扭转。磁搜索线圈方法通常被认为是最可靠的测量旋转, 但侵入, 要求小线圈插入到动物的 scleras10,11。基于视频的系统也可以测量旋转, 具有非侵入性的优点。随着新的图像处理技术的发展, 增强了其功能, 使基于视频的系统成为考虑12、13、14的一种有吸引力的替代方案。
为测量 nonmammals 的眼球运动而开发的技术已经没有那么重要了。度量值要么是低分辨率, 要么仅描述部分旋转15、16、17、18。缺乏发展可以部分地归咎于训练 nonmammals 遵循视觉目标的困难。虽然眼球运动已在红耳滑块龟19,20,21,22,23,24,25 ,26,27,28,29,30, 由于训练动物跟踪目标的挑战, 他们的眼球运动的精确运动学是不好的理解。
红耳滑块龟一般被认为是侧眼脊椎动物, 但因为他们可以完全收回他们的头到他们的外壳31, 由甲壳的侧面视觉场显著闭塞发生32。结果是他们的视觉视线被逼向前方, 使它们的行为更像额眼哺乳动物。因此, 它们作为开发测量眼球运动方法的模型也提供了一个独特的进化视角。
本工作中描述的协议使用体外分离头准备, 以确定红耳滑块龟眼运动的运动学。大脑从头骨中解剖, 使脑神经完好无损。头部被放置在一个框架内, 以校准眼球运动, 并通过电刺激颅神经支配眼睛肌肉唤起反应。眼睛旋转的措施是由一个基于视频的系统, 使用软件算法, 跟踪黑暗的瞳孔和虹膜的标记。该制剂为测量眼外 (即、水平、垂直和扭转旋转) 的运动提供了一个机会:32和人工 (即, 瞳孔改变)33运动。
传出神经通路分析模型系统:
更普遍地说, 该方法为调查人员提供了一个机会, 研究传出神经信号如何产生眼球运动时, 肌肉从他们的放松状态开始, 并在缺乏综合感官信息处理的大脑32, 33。因此, 眼睛的运动学可以被检查在一个模型系统中, 他们是单独处理的传出神经通路离开大脑和 synapsing 到肌肉。
Protocol
注: 红耳滑块龟, 男性和女性, 从供应商购买。海龟被安置在一个温暖的动物套房里, 里面有两个60加仑的浴缸, 装有砖群岛, 在 250 W 红外线灯下晒黑。环境保持在一个14/10 小时的光/暗循环, 水温在22摄氏度。灯在凌晨6:00 打开, 在下午8:00 关闭。装有过滤系统的坦克每周都被清理干净, 海龟每隔一天就被喂食ad 随意。对红耳滑块龟的护理和以下所描述的所有实验过程32,<…
Representative Results
图 1显示从描述解剖的视频中拍摄的图像的剧照。图像提供的神经的典型位置之前, 从大脑切割。 图 1: 从解剖视频中捕捉到的图像的剧照, 以显示视神经 (nII)、动眼神经 (nIII)、滑车神经 (展) 的位置, 以及神经 (nVI).(A) …
Discussion
关键步骤:
本议定书内的关键步骤如下: 1) 解剖和护理, 以保持横断神经的生存能力;2) 将吸入电极大小与颅神经匹配, 提供一致的反应;3) 将头部放置在框架内, 为眼睛的旋转提供适当的校准。
故障 排除：
解剖可能具有挑战性, 但在完成了几次之后, 这些步骤就应该变得相对笔直。如果神经出现不反应, 最可能的原因是解剖失败。在大脑的切?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢波莱特和丽莎 Pezzino 在这项研究中为秘书提供支持, 菲尔奥尔巴赫先生为技术支持。作者还感谢 Drs 和迈克尔. 琼斯 (圣路易斯大学医学院) 介绍我们到体外孤立的头部准备。支持这一合作的资金是由生物学系 (罗伯特-s 大通基金)、学术研究委员会和拉斐特大学的神经科学项目提供的。最后, 这项工作献给2016年9月28日去世的菲尔奥尔巴赫先生;他退役了扫描电子显微镜, 并确认其5轴阶段的用处, 以便在本议定书中使用。他的友谊和足智多谋将被大大地怀念。
Materials
| Red-eared slider turtles | Kons Scientific | Trachemys scripta elegans | Large size (carapace length 15-20 cm) |
| Sodium chloride | Sigma-Aldrich Co. LLC. | S5886 | |
| Potassium chloride | Sigma-Aldrich Co. LLC. | P5405 | |
| Magnesium choride | Sigma-Aldrich Co. LLC. | M7304 | |
| Sodium bicarbonate | Sigma-Aldrich Co. LLC. | S5761 | |
| Dextrose | Sigma-Aldrich Co. LLC. | C5767 | |
| Concentrated hydrochloric acid | Sigma-Aldrich Co. LLC. | H7020 | |
| Calcium chloride | Sigma-Aldrich Co. LLC. | C7902 | |
| pH meter | Oakton | pH 6+ | |
| Suction stimulation electrode | A-M Systems | 573000 | Bipolar suction electrode. Note that 573000 has been replaced with 573050. |
| Capillary glass | A-M systems | 626000 | Single-barrel borosilicate capillary glass without microfilament, length 10 cm, outside diameter 1.0 mm, inner diameter 0.50 mm |
| Alternative suction stimulation electrode | A-M Systems | 573050 | Bipolar suction electrode. Requires larger diameter capillary glass: 627000, outside diameter 1.2 mm, inner diameter 0.68 mm |
| Stereoscope | Lieca | GZ7 | Magnification range, 10x – 70x |
| Fiber optic light source | Amscope | HL250-A | 150W Fiber optical microscope illuminator light box |
| Rongeurs | Carolina Biological Supply Company | 625654 | stainless steel, straight spring, 5.25" |
| Blunt dissection probe | Carolina Biological Supply Company | 627405 | Huber mall probe, double-ended probe and seeker, 6" |
| Microscissors | Carolina Biological Supply Company | 623555 | Iris microdissecting scissors, stainless steel, 0.5" blades, 4.75" long |
| Fine forceps | Sigma-Aldrich Co. LLC. | F6521 | Jewelers forceps, dumont No. 5, inox alloy, 4.25" |
| Curved forceps | Sigma-Aldrich Co. LLC. | Z168696 | Medium tip, curved forceps, stainless steel, 4" |
| Scalpel handle | Sigma-Aldrich Co. LLC. | S2896 | Scalpel handles, No. 3, stainless steel |
| Scalpel blade | Sigma-Aldrich Co. LLC. | S2771 | Scalpel blades, No. 11, steel |
| Guillotine | Harvard Apparatus | 73-1918 | Kleine guillotine type 7575 |
| Spatula | Sigma | Z648299 | Micro spoon and spatula weighing set. Use small spatula: 5.9” long x 0.07” diameter handle with square end: 0.17” x 1.3” long, other end round: 0.17” x 1.27” long |
| Hook | Autozone | 98069 | SureBilt hook and pick set. Use grinder to dull sharp points of hook to prevent injury to animals mouth. |
| 95/5% O2/CO2 | Airgas, Inc. | X02OX95C2003102 | 5% Carbon dioxide balance oxygen certified standard gas mixture, size 200 Cylinder, CGA-296 |
| Regulator | Airgas, Inc. | Y11244D296-AG | Single stage brass 0-100 psi analytical cylinder regulator CGA-296 with needle outlet. Use brass adjustable airline pipe valve to go from 3/8", inner diameter, vinyl airline tubing connected to regulator to a 3/16", inner diameter, airline connection going to airstone or glass pasteur pipette. |
| Adjustable airline pipe valve | Doctors Foster and Smith | CD-12061 | Brass valve |
| Rigid table | Unknown | Unknown | Auto-clave door laid on top of a sturdy table. Nine 5" diameter tennis balls isolate vibrations from the top surface of the table. |
| 5" tennis ball | Petco Animal Supplies, Inc. | 712868 | Petco Jumbo Pet Tennis Ball: balls are unsliced and held within an integrated frame on the underside part of the autoclave door. |
| Alternative vibration isolation table | Newport Corporation | INT1-36-6-N | Rigid vibration control system, integrity 1: Surface dimensions, 3' x 6' |
| Gimbal | ISI, International Scientific Instruments, Inc. | Stage from SUPER III-A Scanning EM | 5-axis eucentric stage: X, Y, and Z linear movements, ±20 mm, 0.1 mm precision; Rotations, vertical, ±10°, and horizontal, ±12.5°, with 1.25° precision. Note: from decommission instrument. |
| Chuck for gimbal | Unknown | Unknown | Chuck from an old microtome of unknown manufacture was machined to fit the shaft of the specimen holder of the Scanning EM stage |
| Alternative gimbal | ThorLabs, Inc. | GN2/M with MBT602/M | Dual-axis goniometer (GN2/M) mounted on 3-axis microblock stage with thumbscrew adjusters (MBT602/M): design a chuck to hold turtle head with eye at 12.7 mm above top surface of goniometer (distance to point of rotation) |
| Video-based eye tracking system | Arrington Research, Inc. | ViewPoint EyeTracker, PC-60 | Tracking method: Infrared video by dark pupil; Black and white camera (Item BC02): 30 Hz, 640 x 480; System requirements: Windows 2000, XP, 7, 8, 8.1, 10; Visual range: Horizontal +/- 44°; vertical +/- 20°; Accuracy ~0.5°; Spatial resolution ~0.15°; Pupil size resolution ~0.03 mm; Eye data: X, Y position of gaze, pupil height and width, torsion, delta time, total time, and regions of interest (ROI); Real-time communication (Item 0022): 4-Channel AnalogOut with eight TTL input channels to mark codes into the data file |
| Multi-position magnetic base | Harbor Freight Tools | Pittsburg, item #5645 | Magnetic holder reaches up to 12" and produces 45 lbs. of magnetic pull. Use to position camera. Machine thread holes onto the end of the rod to mount cameras. |
| Micromanipulator | Kopf | 900 | 5 axis manipulation for mount of suction electrode: X, Y, Z linear travel, 2 axis of rotation |
| Dissection scope on boom | Lieca | GZ6 | Magnification range, 6.7x – 40x |
| Nerve/muscle stimulator | Astro-Med Grass Telefactor | Grass S88 | Dual pulse voltage stimulator: two output channels that can be operated independently or synchronized to generate non-isolated constant voltage pulses (10 mv to 150 V). Pulses can be single (10 μsec to 10 sec), repetitive (0.01 Hz to 1 KHz), and trains (1 ms to 10 s) and synchronized with TTL inputs and output. Send TTL outputs via the output channels of a DB25 connector to the TTL input channels of the ViewPoint EyeTracker. Note: Astro-Med Grass Telefactor is no longer in business. |
| Current isolation device | Astro-Med Grass Telefactor | PSIU6 | Current stimulus isolation unit: enables safe delivery of constant currents by the S88 to the preparation. The PSIU6 connects by a BNC cable to one of the output channels of the S88. Multiplier switches on the PSIU6 allow the S88 to generate a wide array of current amplitudes ranging from 0.1 µA to 15 mA. |
| Alternative nerve/muscle stimulator with isolation | A-M Systems | 2100 | Isolated Pulse Stimulator: Unit has built-in isolator to produce constant currents. |
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Cite This Article
Cano Garcia, M., Nesbit, S. C., Le, C. C., Dearworth Jr., J. R. Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle. J. Vis. Exp. (136), e56864, doi:10.3791/56864 (2018).