小鼠视动反射视觉特征选择性的量化
Summary
在这里，我们描述了一种用于量化视动反射的标准方案。它结合了虚拟鼓刺激和视频眼部成像，因此可以精确评估行为的特征选择性及其自适应可塑性。
Abstract
视动反射 （OKR） 是一种重要的先天性眼球运动，由视觉环境的整体运动触发，用于稳定视网膜图像。由于其重要性和稳健性，OKR 已被用于研究视觉运动学习并评估具有不同遗传背景、年龄和药物治疗的小鼠的视觉功能。在这里，我们介绍了一种高精度评估头部固定小鼠OKR响应的程序。头部固定可以排除前庭刺激对眼球运动的影响，从而可以测量仅由视觉运动触发的眼球运动。OKR 由虚拟鼓系统引发，其中三个计算机显示器上呈现的垂直光栅以振荡方式水平漂移或以恒定速度单向漂移。通过这个虚拟现实系统，我们可以系统地改变视觉参数，如空间频率、时间/振荡频率、对比度、亮度和光栅方向，并量化视觉特征选择性的调谐曲线。高速红外视频眼球造影可确保准确测量眼球运动的轨迹。对个体小鼠的眼睛进行校准，以提供比较不同年龄、性别和遗传背景的动物之间的OKR的机会。该技术的定量能力使其能够检测 OKR 的变化，当这种行为由于衰老、感官体验或运动学习而塑性地适应时;因此，它使该技术成为用于研究眼睛行为可塑性的工具库的宝贵补充。
Introduction
为了响应环境中的视觉刺激，我们的眼睛会移动以转移我们的视线、稳定视网膜图像、跟踪移动目标或将两只眼睛的中央凹与距离观察者不同距离的目标对齐，这对正常视力至关重要 1,2。动眼神经行为已被广泛用作有吸引力的感觉运动整合模型，以了解健康和疾病中的神经回路，至少部分原因是动眼神经系统的简单性3。在三对眼外肌的控制下，眼睛在眼眶中主要围绕三个相应的轴旋转：沿横轴的抬高和凹陷，沿垂直轴的内收和外展，以及沿前后轴的内收和外展1,2。这种简单的系统使研究人员能够在实验室环境中轻松准确地评估小鼠的动眼神经行为。
一种主要的动眼神经行为是视动反射 （OKR）。这种不自主的眼球运动是由视网膜上图像的缓慢漂移或滑动触发的，当动物的头部或其周围环境移动时，用于稳定视网膜图像2,4。OKR 作为一种行为范式，出于多种原因对研究人员来说很有趣。首先，它可以被可靠地刺激并准确量化5,6。其次，量化这种行为的程序相对简单和标准化，可用于评估大型动物群体的视觉功能7。第三，这种先天行为具有高度的可塑性5,8,9。当长时间重复性视网膜滑脱时，其振幅可以增强 5,8,9，或者当其工作伙伴前庭眼反射 （VOR）（由前庭输入2 触发的另一种稳定视网膜图像的机制）受损时5。这些OKR增强的实验范式使研究人员能够揭示动眼神经学习的电路基础。
在以前的研究中，主要使用两种非侵入性方法评估 OKR：（1） 视频眼部造影结合物理鼓 7,10,11,12,13 或 （2） 任意确定头部转动结合虚拟鼓6,14,15,16.尽管它们的应用在理解动眼神经可塑性的分子和电路机制方面取得了丰硕的发现，但这两种方法都存在一些缺点，限制了它们在定量检查OKR特性方面的能力。首先，带有黑白条纹或圆点印刷图案的物理鼓不允许轻松快速地改变视觉图案，这在很大程度上限制了 OKR 对某些视觉特征的依赖性的测量，例如移动光栅的空间频率、方向和对比度 8,17。相反，OKR 对这些视觉特征的选择性测试可以从计算机化的视觉刺激中受益，其中视觉特征可以方便地从试验到试验进行修改。通过这种方式，研究人员可以系统地研究多维视觉参数空间中的OKR行为。此外，OKR 测定的第二种方法仅报告触发可识别 OKR 的视觉参数的阈值，而不报告眼睛或头部运动的幅度6、14、15、16。因此，缺乏定量能力无法分析调谐曲线的形状和首选的视觉特征，或检测正常和病理条件下个体小鼠之间的细微差异。为了克服上述局限性，在最近的研究中，视频眼部造影和计算机虚拟视觉刺激相结合来检测 OKR 行为 5,17,18,19,20。然而，这些先前发表的研究没有提供足够的技术细节或分步说明，因此研究人员为自己的研究建立这样的OKR测试仍然具有挑战性。
在这里，我们提出了一种协议，通过视频眼部造影和计算机化虚拟视觉刺激的组合，精确量化明视或暗视条件下OKR行为的视觉特征选择性。将小鼠头部固定以避免前庭刺激引起的眼球运动。高速摄像机用于记录观察具有变化视觉参数的移动光栅的小鼠的眼球运动。校准单个小鼠眼球的物理大小，以确保推导眼球运动角度的准确性21。这种定量方法允许比较不同年龄或遗传背景的动物之间的OKR行为，或监测其由药物治疗或视觉运动学习引起的变化。
Protocol
Representative Results
Discussion
此处介绍的 OKR 行为测定方法具有几个优点。首先，计算机生成的视觉刺激解决了物理鼓的内在问题。针对物理鼓不支持空间频率、方向或对比度调谐的系统检查8的问题，虚拟鼓允许在逐个试验的基础上改变这些视觉参数，从而有助于对OKR行为的特征选择性进行系统和定量分析（图4A）;当物理鼓受到外部光源23的不均匀照明时，虚拟鼓可?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢何颖天分享方向调整数据。这项工作得到了加拿大创新基金会和安大略省研究基金（CFI/ORF 项目编号 37597）、NSERC （RGPIN-2019-06479）、CIHR（项目资助 437007）和康诺特新研究员奖的资助。
Materials
| 2D translational stage | Thorlabs | XYT1 | |
| Acrylic resin | Lang Dental | B1356 | For fixing headplate on skull and protecting skull |
| Bupivacaine | STERIMAX | ST-BX223 | Bupivacaine Injection BP 0.5%. Local anesthesia |
| Carprofen | RIMADYL | 8507-14-1 | Analgesia |
| Compressed air | Dust-Off | ||
| Eye ointment | Alcon | Systane | For maintaining moisture of eyes |
| Graphic card | NVIDIA | Geforce GTX 1650 or Quadro P620. | For generating single screen among three monitors |
| Heating pad | Kent Scientific | HTP-1500 | For maintaining body temperature |
| High-speed infrared (IR) camera | Teledyne Dalsa | G3-GM12-M0640 | For recording eye rotation |
| IR LED | Digikey | PDI-E803-ND | For CR reference and the illumination of the eye |
| IR mirror | Edmund optics | 64-471 | For reflecting image of eye |
| Isoflurane | FRESENIUS KABI | CP0406V2 | |
| Labview | National instruments | version 2014 | eye tracking |
| Lactated ringer | BAXTER | JB2324 | Water and energy supply |
| Lidocaine and epinephrine mix | Dentsply Sirona | 82215-1 | XYLOCAINE. Local anesthesia |
| Luminance Meter | Konica Minolta | LS-150 | for calibration of monitors |
| Matlab | MathWorks | version xxx | analysis of eye movements |
| Meyhoefer Curette | World Precision Instruments | 501773 | For scraping skull and removing fascia |
| Microscope calibration slide | Amscope | MR095 | to measure the magnification of video-oculography |
| Monitors | Acer | B247W | Visual stimulation |
| Neutral density filter | Lee filters | 299 | to generate scotopic visual stimulation |
| Nigh vision goggle | Alpha optics | AO-3277 | for scotopic OKR |
| Photodiode | Digikey | TSL254-R-LF-ND | to synchronize visual stimulation and video-oculography |
| Pilocarpine hydrochloride | Sigma-Aldrich | P6503 | |
| Post | Thorlabs | TR1.5 | |
| Post holder | Thorlabs | PH1 | |
| PsychoPy | open source software | version xxx | visual stimulation toolkit |
| Scissor | RWD | S12003-09 | For skin removal |
| Superglue | Krazy Glue | Type: All purpose. For adhering headplate on the skull |
References
- Gerhard, D. Neuroscience. 5th Edition. Yale Journal of Biology and Medicine. , (2013).
- Distler, C., Hoffmann, K. P. . The Oxford Handbook of Eye Movement. , 65-83 (2011).
- Sereno, A. B., Bolding, M. S. . Executive Functions: Eye Movements and Human Neurological Disorders. , (2017).
- Giolli, R. A., Blanks, R. H. I., Lui, F. The accessory optic system: basic organization with an update on connectivity, neurochemistry, and function. Progress in Brain Research. 151, 407-440 (2006).
- Liu, B. H., Huberman, A. D., Scanziani, M. Cortico-fugal output from visual cortex promotes plasticity of innate motor behaviour. Nature. 538 (7625), 383-387 (2016).
- Prusky, G. T., Alam, N. M., Beekman, S., Douglas, R. M. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Investigative Ophthalmology & Visual Science. 45 (12), 4611-4616 (2004).
- Stahl, J. S., van Alphen, A. M., De Zeeuw, C. I. A comparison of video and magnetic search coil recordings of mouse eye movements. Journal of Neuroscience Methods. 99 (1-2), 101-110 (2000).
- Faulstich, B. M., Onori, K. A., du Lac, S. Comparison of plasticity and development of mouse optokinetic and vestibulo-ocular reflexes suggests differential gain control mechanisms. Vision Research. 44 (28), 3419-3427 (2004).
- Katoh, A., Kitazawa, H., Itohara, S., Nagao, S. Dynamic characteristics and adaptability of mouse vestibulo-ocular and optokinetic response eye movements and the role of the flocculo-olivary system revealed by chemical lesions. Proceedings of the National Academy of Sciences. 95 (13), 7705-7710 (1998).
- Cahill, H., Nathans, J. The optokinetic reflex as a tool for quantitative analyses of nervous system function in mice: application to genetic and drug-induced variation. PLoS One. 3 (4), 2055 (2008).
- Cameron, D. J., et al. The optokinetic response as a quantitative measure of visual acuity in zebrafish. Journal of Visualized Experiments. (80), 50832 (2013).
- de Jeu, M., De Zeeuw, C. I. Video-oculography in mice. Journal of Visualized Experiments. (65), e3971 (2012).
- Kodama, T., du Lac, S. Adaptive acceleration of visually evoked smooth eye movements in mice. The Journal of Neuroscience. 36 (25), 6836-6849 (2016).
- Doering, C. J., et al. Modified Ca(v)1.4 expression in the Cacna1f(nob2) mouse due to alternative splicing of an ETn inserted in exon 2. PLoS One. 3 (7), e2538 (2008).
- Shi, C., et al. Optimization of optomotor response-based visual function assessment in mice. Scientific Reports. 8 (1), 9708 (2018).
- Waldner, D. M., et al. Transgenic expression of Cacna1f rescues vision and retinal morphology in a mouse model of congenital stationary night blindness 2A (CSNB2A). Translational Vision Science & Technology. 9 (11), 19 (2020).
- Tabata, H., Shimizu, N., Wada, Y., Miura, K., Kawano, K. Initiation of the optokinetic response (OKR) in mice. Journal of Vision. 10 (1), 1-17 (2010).
- Al-Khindi, T., et al. The transcription factor Tbx5 regulates direction-selective retinal ganglion cell development and image stabilization. Current Biology. 32 (19), 4286-4298 (2022).
- Harris, S. C., Dunn, F. A. Asymmetric retinal direction tuning predicts optokinetic eye movements across stimulus conditions. eLife. 12, e81780 (2023).
- van Alphen, B., Winkelman, B. H., Frens, M. A. Three-dimensional optokinetic eye movements in the C57BL/6J mouse. Investigative Ophthalmology & Visual Science. 51 (1), 623-630 (2010).
- Stahl, J. S. Calcium channelopathy mutants and their role in ocular motor research. Annals of the New York Academy of Sciences. 956, 64-74 (2002).
- Endo, S., et al. Dual involvement of G-substrate in motor learning revealed by gene deletion. Proceedings of the National Academy of Sciences. 106 (9), 3525-3530 (2009).
- Thomas, B. B., Seiler, M. J., Sadda, S. R., Coffey, P. J., Aramant, R. B. Optokinetic test to evaluate visual acuity of each eye independently. Journal of Neuroscience Methods. 138 (1-2), 7-13 (2004).
- Burroughs, S. L., Kaja, S., Koulen, P. Quantification of deficits in spatial visual function of mouse models for glaucoma. Investigative Ophthalmology & Visual Science. 52 (6), 3654-3659 (2011).
- Wakita, R., et al. Differential regulations of vestibulo-ocular reflex and optokinetic response by β- and α2-adrenergic receptors in the cerebellar flocculus. Scientific Reports. 7 (1), 3944 (2017).
- Dehmelt, F. A., et al. Spherical arena reveals optokinetic response tuning to stimulus location, size, and frequency across entire visual field of larval zebrafish. eLife. 10, e63355 (2021).
- Magnusson, M., Pyykko, I., Jantti, V. Effect of alertness and visual attention on optokinetic nystagmus in humans. American Journal of Otolaryngology. 6 (6), 419-425 (1985).
- Collins, W. E., Schroeder, D. J., Elam, G. W. Effects of D-amphetamine and of secobarbital on optokinetic and rotation-induced nystagmus. Aviation, Space, and Environmental Medicine. 46 (4), 357-364 (1975).
- Reimer, J., et al. Pupil fluctuations track fast switching of cortical states during quiet wakefulness. Neuron. 84 (2), 355-362 (2014).
- Sakatani, T., Isa, T. PC-based high-speed video-oculography for measuring rapid eye movements in mice. Neuroscience Research. 49 (1), 123-131 (2004).
- Sakatani, T., Isa, T. Quantitative analysis of spontaneous saccade-like rapid eye movements in C57BL/6 mice. Neuroscience Research. 58 (3), 324-331 (2007).
- Vinck, M., Batista-Brito, R., Knoblich, U., Cardin, J. A. Arousal and locomotion make distinct contributions to cortical activity patterns and visual encoding. Neuron. 86 (3), 740-754 (2015).
- Bradley, M. M., Miccoli, L., Escrig, M. A., Lang, P. J. The pupil as a measure of emotional arousal and autonomic activation. Psychophysiology. 45 (4), 602-607 (2008).
- Hess, E. H., Polt, J. M. Pupil size as related to interest value of visual stimuli. Science. 132 (3423), 349-350 (1960).
- Di Stasi, L. L., Catena, A., Canas, J. J., Macknik, S. L., Martinez-Conde, S. Saccadic velocity as an arousal index in naturalistic tasks. Neuroscience and Biobehavioral Reviews. 37 (5), 968-975 (2013).
