利用视觉刺激技术评价小鼠视力
Summary
为了检查鼠标的视觉, 我们进行了一个即将到来的测试。老鼠被安置在一个大的广场竞技场上, 天花板上有一个显示器。隐约约的视觉刺激一直会在小鼠体内引起冷冻或飞行反应。
Abstract
中枢神经系统中的视觉系统处理不同的视觉信号。虽然整体结构的特点是从视网膜通过外侧膝状核到视觉皮层, 系统是复杂的。已经进行了细胞和分子研究, 以阐明支持视觉处理的机制, 进而阐明疾病机制。这些研究可能有助于人工视觉系统的发展。为了验证这些研究的结果, 行为视觉测试是必要的。在这里, 我们表明, 隐约刺激实验是一个可靠的鼠标视觉测试, 需要一个相对简单的设置。即将进行的实验是在一个大的外壳中进行的, 一个角落有一个掩体, 天花板上有一个电脑显示器。位于计算机显示器旁边的 CCD 摄像机用于观察鼠标行为。一只老鼠被放置在圈内 10分钟, 并允许它适应和探索周围的环境。然后, 监视器将程序产生的隐约刺激投射了10倍。老鼠对刺激的反应不是冷冻, 就是逃到藏身之处。记录了鼠标在隐约刺激前后的行为, 并使用运动跟踪软件对视频进行了分析。在隐约出现的刺激后, 老鼠的运动速度发生了显著变化。相比之下, 在盲鼠中没有观察到任何反应。我们的结果表明, 简单的隐约实验是对老鼠视觉的可靠测试。
Introduction
视觉系统从视网膜开始, 视觉信号被光感受器捕捉, 引导到双极细胞 (2 阶神经元), 最后传递到神经节细胞 (第3 阶神经元)。视网膜2阶和第3级神经元被认为形成多个神经通路, 传达视觉信号的特定方面, 如颜色、运动或形状。这些不同的视觉特征被传递到外侧膝状核和视觉皮层。相反, 导致眼球运动的视觉信号被发送到上大肠杆菌。经典地, 两个视网膜皮质通路已被确定: 磁细胞和副细胞通路。这些路径分别编码移动和静止物体, 它们的存在体现了并行处理1,2,3,4,5, 6。最近, 超过15种双极细胞7,8,9, 10,11和神经节细胞 12,13,14在包括灵长类视网膜在内的许多物种的视网膜中, 有15,16例。这些细胞的区别不仅在于形态方面, 而且还表现出不同的标记和基因8,10,17, 18,这表明, 各种特征视觉信号是平行处理的, 比最初预期的要复杂得多。
细胞和分子技术有助于我们了解视觉处理和异常视觉处理可能产生的潜在疾病机制。这样的理解可能有助于人工眼睛的发育。尽管细胞检查和分析在细胞层面提供了深入的知识, 但行为实验和细胞实验的结合将显著增强我们目前对微小视觉过程的理解。例如, Yoshida 等人 19发现, 星爆 amacrine 细胞是小鼠视网膜运动检测的关键神经元。在进行细胞实验后, 他们进行了视动性眼球震颤 (OKN) 行为实验, 以表明星爆 amacrine 细胞功能失调的突变小鼠对运动物体没有反应, 从而证实了它们的细胞调查。此外, Pearson 等人20 在小鼠视网膜进行光感受器移植, 以恢复患病小鼠的视力。他们不仅进行了细胞实验, 而且还通过使用光爆反应记录和水迷宫任务测量了老鼠的行为, 从而使皮尔逊等人能够验证移植的光感受器是否恢复了以前失明的视力小 鼠。总之, 行为实验是评估老鼠视力的有力工具。
有多种方法可用于测量鼠标视觉。这些方法有其优点和局限性。在体内 ERG 提供有关小鼠视网膜, 特别是光感受器和双极细胞, 是否对光刺激做出适当反应的信息。Erg 可以在苏格兰或光子条件下进行测试 21,22。然而, ERG 需要麻醉, 这可能会影响输出测量23。视感反射 (OKR) 或光耦合器响应 (OMR) 是一种用于评估对比度灵敏度和空间分辨率的可靠方法, 这两个功能成分都是小鼠视觉的组成部分。然而, OKR 需要手术给老鼠头骨24 上安装固定装置。OMR 既不需要手术, 也不需要小鼠训练;然而, 它需要训练, 让实验者主观地检测到微妙的鼠标头部运动, 以响应一个移动的光栅在一个光学鼓 25,26。小学生光反射测量瞳孔收缩对光刺激的反应, 这不需要麻醉, 并表现出客观和稳健的反应19。虽然瞳孔反射在体内模拟视网膜光的反应, 但反射主要是由本质上光敏视网膜神经节细胞 (弹性角膜) 27介导的。由于 ipRGCs 只占 Rgc 的一小部分, 并不作为传统的形成图像的神经节细胞, 因此这种测量不能提供与大多数神经节细胞有关的信息。
这种隐约出现的光实验此前并不被认为是测量老鼠视力的主要测试。然而, 它也是一个强大和可靠的视觉测试, 对各种物种, 如老鼠28,29, 斑马鱼30, 蝗虫31,32, 和人类33,34,35. 重要的是, 即将到来的实验是测试图像形成途径的仅有的方法之一—-它不是反射路径—-因为中枢神经系统中的视觉和边缘系统参与了这一电路 36, 37,38。我们已经建立了一个隐约的视觉刺激系统, 并已证明它的能力, 以诱导运动检测在鼠标, 我们使用它作为一个代理来评估鼠标视觉系统的不变性。
Protocol
Representative Results
Discussion
随着视觉刺激系统的临近, 大多数 (97%)健康的眼鼠表现出飞行反应。29只老鼠中的一只没有表现出明显的飞行反应。然而, 老鼠向圆顶走去, 一直呆在圆顶附近, 直到隐约消失, 这表明当即将到来的刺激发生时, 老鼠至少是谨慎的。因此, 即将到来的刺激不断引起健康眼鼠天生的恐惧反应。另一方面, 三只盲鼠并没有对即将到来的 (初步结果) 做出任何反应。总之, 我们证明了隐约出现的实验是对小鼠有?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 NIH R01 Y028915 (TI) 和 RPB 赠款的支持。
Materials
| 10.1" monitor (2° display) | Elecrow | Elecrow 10.1 Inch Raspberry Pi 1920x1080p Resolution Display | |
| 14" Business Class Laptop 5490 | Dell | 84 / rcrc961481-4860836 | |
| 20" x 50" Absorbant Liners | Fisher Scientific | AL2050 | works well to protect floor of arena, could use any type of liner |
| 21.5" monitor (1° display) | Acer | Acer R221Q bid 21.5-inch IPS Full HD Display | |
| CCD Camera | Lumenera Corporation | Infiniyy3S-1UR | excellent for behavioral studies due to high fps rate (60 fps) |
| Enclosure (alminum frames and PVC panels) | 80/20 Inc. | 4x cat.#9010, 4x cat.#9005, 1x cat.#9000, 5x cat.#65-2616 | excellent, used quick build tab to find PVC, joints, and frame |
| Ethanol | Fisher Scientific | 22-032-601 | |
| Excel Spreadsheet Software | Microsoft Office | user friendly and widespread knowledge of Microsoft Office software | |
| Freearm | Amazon | used to mount camera to the table, could use any mountable extendable arm | |
| ImagePro Premiere 3D | Media Cybernetics | version 9.3 | good program, could use some updating with the automated tracking feature |
| Matlab software (Psychotoolbox 3) | MathWorks | Matlab R2018b 64-bit (9.5.0.944444) | excellent software to generate pattern stimuli of any conditions |
| SteamPix sorftware | Norpix | StreamPix 7 64-bit Single Camera | works well, a few problems with frame dropping but good customer service |
| WD My Book External Hard Drive | Western Digital | WDBBGB0080HBK hard drive 8 TB USB 3.0 | necessary if using .avi files with no compression codec due to large size of files |
| Wide angle lens | Navitar | NMV-5M23 | excellent and necessary to capture entire arena |
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