皮洛卡平诱导癫痫小鼠记忆功能评估
Summary
本文介绍了评估皮洛卡平诱导癫痫小鼠记忆损伤的实验程序。该协议可用于研究癫痫相关认知衰退的病理生理学机制，这是癫痫最常见的合并症之一。
Abstract
认知障碍是叶癫痫最常见的合并症之一。为了在癫痫动物模型中重述癫痫相关认知衰退，我们产生了由皮洛卡平治疗的慢性癫痫小鼠。我们使用这些癫痫小鼠为三种不同的行为测试提供了一个协议：新对象位置 （NL）、新对象识别 （NO） 和模式分离 （PS） 测试，分别用于评估地点、对象和上下文的学习和记忆。我们解释了如何在运动场测试后，为NL、NO和PS测试设置行为装置并提供实验程序，该测试测量动物的基底运动活动。我们还描述了NL、NO和PS测试在评估癫痫小鼠记忆功能的其他行为测试方面的技术优势。最后，我们讨论了癫痫小鼠在熟悉会话期间未能与物体进行良好接触的可能原因和解决方案，这是成功进行记忆测试的关键步骤。因此，该协议提供了有关如何使用小鼠评估癫痫相关记忆损伤的详细信息。NL、NO 和 PS 测试简单、高效的检测，适用于评估癫痫小鼠中不同类型的记忆。
Introduction
癫痫是一种慢性疾病，其特征是自发性复发性发作11、2、3。2,3由于重复性癫痫发作可引起大脑11、2、32,3的结构和功能异常，异常发作活动可引起认知功能障碍，这是最常见的癫痫相关合并症4,4、5、6。,6与慢性癫痫发作事件相反，这些发作是短暂的和短暂的，认知障碍会持续到癫痫患者的生活中，从而恶化他们的生活质量。因此，了解癫痫相关认知衰退的病理生理学机制非常重要。
各种癫痫实验动物模型被用来证明与慢性癫痫相关的学习和记忆缺陷787，8，9，10，11，12。,9,10,11,12,例如，莫里斯水迷宫、上下文恐惧调理、孔板、新型物体位置（NL）和新颖的物体识别（NO）测试经常被用来评估叶癫痫（TLE）的记忆功能障碍。由于海马是TLE显示病理学的主要区域之一，因此通常优先选择能够评估海马依赖记忆功能的行为测试。然而，鉴于癫痫发作可以诱发异常海马神经发生，并促成癫痫相关认知衰退10，测试变性新生儿神经元功能的行为范式（即空间模式分离，PS）8，13也可以提供有关癫痫记忆损伤细胞机制8,13的宝贵信息。
在本文中，我们演示了针对癫痫小鼠的一组内存测试 NL、NO 和 PS。测试简单且易于访问，不需要复杂的系统。
Protocol
所有实验程序均经韩国天主教大学伦理委员会批准，并根据《国家卫生研究院实验室动物护理和使用指南》（NIH出版物第80-23号）进行。 1. 新颖对象位置测试 （NL） 在皮洛卡平注射4-6周后准备癫痫C57BL/6或转基因小鼠。注：急性癫痫发作是由腹内（IP）皮洛卡平注射引起的，遵循我们上次报告14中详述的协议。 在行为测试开始前一天，将癫?…
Representative Results
图1显示了评估认知功能的一般实验时间表和设置。在引入皮洛卡平引起的急性癫痫发作六周后，小鼠接受NL、NO和PS测试，按该顺序在测试之间间隔3天休息期（图1A）。对于 NL 测试，在熟悉会话 （F1） 期间，两个相同的对象放置在开放字段中，第二天，一个对象移动到新位置。在 NO 测试中，一个对象在测试会话期间被替换为新对象。对于 PS 测试，两?…
Discussion
本研究描述了评估慢性癫痫小鼠认知功能的实验程序。许多不同的行为测试范式用于评估小鼠18中的学习和记忆功能。莫里斯水迷宫、径向臂迷宫、Y迷宫、上下文恐惧调节和基于对象的测试是最常用的行为测试，并提供可靠的结果。其中，NL、NO和PS测试是评价癫痫小鼠,8、10学习和记忆的有效、简单的方法。8由于癫痫小鼠在行为过程中…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢李杰敏博士的技术支持。这项工作得到了韩国政府资助的韩国国家研究基金会（NRF-2019R1A2C1003958，NRF-2019K2A9A2A2A08000167）的支持。
Materials
| 1 ml syringe | Sung-shim | Use with the 26 or 30 gauge needle | |
| 70% Ethanol | Duksan | UN1170 | Spray to clean the box and objects |
| black curtain | For avoiding unnecessary visual cues | ||
| Cresyl violet | Sigma | C5042 | For Cresyl violet staining |
| cryotome | Leica | E21040041 | For tissue sectioning |
| double-sided sticky tape | For the firm placement of the objects | ||
| DPX mounting medium | Sigma | 06522 | |
| ethanol series | Duksan | UN1170 | Make 100%, 95%, 90%, 80%, 70% ethanol solutions |
| floor plate with narrow grid patterns | Leehyo-bio | Behavioral experiment equipment, plate size: 42.5 x 42.5 x 0.5 cm, grid size: 2.75 x 2.75 cm | |
| floor plate with wide grid patterns | Leehyo-bio | Behavioral experiment equipment, plate size: 42.5 x 42.5 x 0.5 cm, grid size: 5.5 x 5.5 cm | |
| illuminometer | TES Electrical Electronic Corp. | 1334A | For the measurement of the room lighting (60 Lux) |
| Intensive care unit | Thermocare | #W-1 | |
| ketamine hydrochloride | Yuhan | 7003 | Use to anesthetize the mouse for transcardial perfusion |
| LED lamp | Lungo | P13A-0422-WW-04 | Lighting for the behavioral test room |
| objects | Rubber doll, 50 ml plastic tube, glass Coplin jar, plastic T-flask, glass bottle | ||
| open field box | Leehyo-bio | Behavioral experiment equipment, size: 44 x 44 x 31 cm | |
| paper towel | Yuhan-Kimberly | 47201 | Use to dry open field box and objects |
| paraformaldehyde | Merck Millipore | 104005 | Make 4% solution |
| pilocarpine hydrochloride | Sigma | P6503 | |
| ruler | Use to locate the objects in the open field box | ||
| scopolamine methyl nitrate | Sigma | S2250 | Make 10X stock |
| Smart system 3.0 | Panlab | Video tracking system | |
| stopwatch | Junso | JS-307 | For the measurement of explorative activities of mice |
| sucrose | Sigma | S9378 | For cryoprotection of tissue sections |
| terbutaline hemisulfate salt | Sigma | T2528 | Make 10X stock |
| video camera (CCD camera) | Vision | VCE56HQ-12 | Place the camera directly overhead of the open field box |
| xylazine (Rompun) | Bayer korea | KR10381 | Use to anesthetize the mouse for transcardial perfusion |
| xylene | Duksan | UN1307 | For Cresyl violet staining |
Riferimenti
- Chang, B. S., Lowenstein, D. H. Mechanisms of disease – Epilepsy. New England Journal of Medicine. 349 (13), 1257-1266 (2003).
- Scharfman, H. E. The neurobiology of epilepsy. Current Neurology and Neuroscience Report. 7 (4), 348-354 (2007).
- Rakhade, S. N., Jensen, F. E. Epileptogenesis in the immature brain: emerging mechanisms. Nature Reviews in Neurology. 5 (7), 380-391 (2009).
- Breuer, L. E., et al. Cognitive deterioration in adult epilepsy: Does accelerated cognitive ageing exist. Neuroscience and Biobehavior Reviews. 64, 1-11 (2016).
- Leeman-Markowski, B. A., Schachter, S. C. Treatment of Cognitive Deficits in Epilepsy. Neurology Clinics. 34 (1), 183-204 (2016).
- Helmstaedter, C., Elger, C. E. Chronic temporal lobe epilepsy: a neurodevelopmental or progressively dementing disease. Brain. 132, 2822-2830 (2009).
- Groticke, I., Hoffmann, K., Loscher, W. Behavioral alterations in the pilocarpine model of temporal lobe epilepsy in mice. Experimental Neurology. 207 (2), 329-349 (2007).
- Long, Q., et al. Intranasal MSC-derived A1-exosomes ease inflammation, and prevent abnormal neurogenesis and memory dysfunction after status epilepticus. Proceedings of the National Academy of Science U. S. A. 114 (17), 3536-3545 (2017).
- Lima, I. V. A., et al. Postictal alterations induced by intrahippocampal injection of pilocarpine in C57BL/6 mice. Epilepsy & Behavior. 64, 83-89 (2016).
- Cho, K. O., et al. Aberrant hippocampal neurogenesis contributes to epilepsy and associated cognitive decline. Nature Communication. 6, 6606 (2015).
- Zhou, Q., et al. Adenosine A1 Receptors Play an Important Protective Role Against Cognitive Impairment and Long-Term Potentiation Inhibition in a Pentylenetetrazol Mouse Model of Epilepsy. Molecular Neurobiology. 55 (4), 3316-3327 (2018).
- Jiang, Y., et al. Ketogenic diet attenuates spatial and item memory impairment in pentylenetetrazol-kindled rats. Brain Research. 1646, 451-458 (2016).
- Zhuo, J. M., et al. Young adult born neurons enhance hippocampal dependent performance via influences on bilateral networks. Elife. 5, 22429 (2016).
- Kim, J. E., Cho, K. O. The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice. Journal of Visualized Experiments. (132), e56831 (2018).
- Gage, G. J., Kipke, D. R., Shain, W. Whole animal perfusion fixation for rodents. Journal of Visualized Experiments. (65), e3564 (2012).
- Muller, C. J., Groticke, I., Bankstahl, M., Loscher, W. Behavioral and cognitive alterations, spontaneous seizures, and neuropathology developing after a pilocarpine-induced status epilepticus in C57BL/6 mice. Experimental Neurology. 219 (1), 284-297 (2009).
- Brandt, C., Gastens, A. M., Sun, M., Hausknecht, M., Loscher, W. Treatment with valproate after status epilepticus: effect on neuronal damage, epileptogenesis, and behavioral alterations in rats. Neuropharmacology. 51 (4), 789-804 (2006).
- Wolf, A., Bauer, B., Abner, E. L., Ashkenazy-Frolinger, T., Hartz, A. M. A Comprehensive Behavioral Test Battery to Assess Learning and Memory in 129S6/Tg2576 Mice. PLoS One. 11 (1), 0147733 (2016).
- Lueptow, L. M. Novel Object Recognition Test for the Investigation of Learning and Memory in Mice. Journal of Visualized Experiments. (126), e55718 (2017).
- Antunes, M., Biala, G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cognitive Processing. 13 (2), 93-110 (2012).
- van Goethem, N. P., van Hagen, B. T. J., Prickaerts, J. Assessing spatial pattern separation in rodents using the object pattern separation task. Nature Protocols. 13 (8), 1763-1792 (2018).
- Leger, M., et al. Object recognition test in mice. Nature Protocols. 8 (12), 2531-2537 (2013).
- Moscovitch, M., Cabeza, R., Winocur, G., Nadel, L. Episodic Memory and Beyond: The Hippocampus and Neocortex in Transformation. Annual Reviews in Psychology. 67, 105-134 (2016).
- Eichenbaum, H. A cortical-hippocampal system for declarative memory. Nature Reviews Neuroscience. 1 (1), 41-50 (2000).
- Brown, M. W., Aggleton, J. P. Recognition memory: What are the roles of the perirhinal cortex and hippocampus. Nature Reviews Neuroscience. 2 (1), 51-61 (2001).
- Winters, B. D., Forwood, S. E., Cowell, R. A., Saksida, L. M., Bussey, T. J. Double dissociation between the effects of peri-postrhinal cortex and hippocampal lesions on tests of object recognition and spatial memory: Heterogeneity of function within the temporal lobe. Journal of Neuroscience. 24 (26), 5901-5908 (2004).
- Winters, B. D., Bussey, T. J. Transient inactivation of perirhinal cortex disrupts encoding, retrieval, and consolidation of object recognition memory. Journal of Neuroscience. 25 (1), 52-61 (2005).
- Bermudez-Rattoni, F., Okuda, S., Roozendaal, B., McGaugh, J. L. Insular cortex is involved in consolidation of object recognition memory. Learning & Memory. 12 (5), 447-449 (2005).
- Akirav, I., Maroun, M. Ventromedial prefrontal cortex is obligatory for consolidation and reconsolidation of object recognition memory. Cerebral Cortex. 16 (12), 1759-1765 (2006).
- Cohen, S. J., Stackman, R. W. Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behavior Brain Research. 285, 105-117 (2015).
- Cohen, S. J., et al. The Rodent Hippocampus Is Essential for Nonspatial Object Memory. Current Biology. 23 (17), 1685-1690 (2013).
- Broadbent, N. J., Gaskin, S., Squire, L. R., Clark, R. E. Object recognition memory and the rodent hippocampus. Learning and Memory. 17 (1), 5-11 (2010).
- Tuscher, J. J., Taxier, L. R., Fortress, A. M., Frick, K. M. Chemogenetic inactivation of the dorsal hippocampus and medial prefrontal cortex, individually and concurrently, impairs object recognition and spatial memory consolidation in female mice. Neurobiology of Learning and Memory. 156, 103-116 (2018).
- de Lima, M. N., Luft, T., Roesler, R., Schroder, N. Temporary inactivation reveals an essential role of the dorsal hippocampus in consolidation of object recognition memory. Neuroscience Letters. 405 (1-2), 142-146 (2006).
- Hammond, R. S., Tull, L. E., Stackman, R. W. On the delay-dependent involvement of the hippocampus in object recognition memory. Neurobiology of Learning and Memory. 82 (1), 26-34 (2004).
- Clark, R. E., Zola, S. M., Squire, L. R. Impaired recognition memory in rats after damage to the hippocampus. Journal of Neuroscience. 20 (23), 8853-8860 (2000).
- Stackman, R. W., Cohen, S. J., Lora, J. C., Rios, L. M. Temporary inactivation reveals that the CA1 region of the mouse dorsal hippocampus plays an equivalent role in the retrieval of long-term object memory and spatial memory. Neurobiology of Learning and Memory. 133, 118-128 (2016).
- Mumby, D. G., Gaskin, S., Glenn, M. J., Schramek, T. E., Lehmann, H. Hippocampal damage and exploratory preferences in rats: memory for objects, places, and contexts. Learning & Memory. 9 (2), 49-57 (2002).
- Jeong, K. H., Lee, K. E., Kim, S. Y., Cho, K. O. Upregulation of Kruppel-Like Factor 6 in the Mouse Hippocampus after Pilocarpine-Induced Status Epilepticus. Neuroscienze. 186, 170-178 (2011).
- Kim, J. E., Cho, K. O. The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice. Journal of Visualized Experiments. (132), e56831 (2018).
- Jiang, Y., et al. Abnormal hippocampal functional network and related memory impairment in pilocarpine-treated rats. Epilepsia. 59 (9), 1785-1795 (2018).
- Wang, L., Liu, Y. H., Huang, Y. G., Chen, L. W. Time-course of neuronal death in the mouse pilocarpine model of chronic epilepsy using Fluoro-Jade C staining. Brain Research. 1241, 157-167 (2008).
- Detour, J., Schroeder, H., Desor, D., Nehlig, A. A 5-month period of epilepsy impairs spatial memory, decreases anxiety, but spares object recognition in the lithium-pilocarpine model in adult rats. Epilepsia. 46 (4), 499-508 (2005).
- Benini, R., Longo, D., Biagini, G., Avoli, M. Perirhinal Cortex Hyperexcitability in Pilocarpine-Treated Epileptic Rats. Hippocampus. 21 (7), 702-713 (2011).
- Yassa, M. A., Stark, C. E. Pattern separation in the hippocampus. Trends in Neurosciences. 34 (10), 515-525 (2011).
- Goncalves, J. T., Schafer, S. T., Gage, F. H. Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior. Cell. 167 (4), 897-914 (2016).
- Sahay, A., et al. Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature. 472 (7344), 466-539 (2011).
Citazione di questo articolo
Park, K., Kim, J., Choi, I., Cho, K. Assessment of Memory Function in Pilocarpine-induced Epileptic Mice. J. Vis. Exp. (160), e60751, doi:10.3791/60751 (2020).