引进响片训练为实验室老鼠认知富集
Summary
The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.
Abstract
Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the “click” sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.
Introduction
新的细化策略实验室老鼠的发展是一个具有挑战性的任务，有助于3R原则的实验动物科学1的履行（替代，减少和细化）。在细化方面的改进可以进一步促进数以百万计的动物被用于实验目的福祉。因此，需要在该领域的深入研究。这也是一个定义的欧盟指令六十三分之二千零十的目的。指令63分之2010/ EU指出，实验动物的生存经验已得到加强，而且“机构应当建立适合于动物的习惯和培训计划，程序和项目的长度”（第3.7条）2。
而他们被关押和饲养实验，实验动物可以经历许多紧张的情况下。一般，劳动力之间的相互作用atory小鼠和负责人是相当有限的。因此，互相信任的关系就不能发展。这可引起在反应处理增加的焦虑和压力，这是不利的动物3，4，5，6的行为和生理的，因此，福祉。此外，常规进行的实验室程序像一般处理，克制，血液或组织取样可引起应激反应，这可以通过测量不同的参数，如压力荷尔蒙或行为7,8进行检查。它已经显示，处理的程序可以有效地降低朝向研究者焦虑在实验室啮齿动物9，10，11。处理程序，因此可以提高动物条件和可能大大有助于动物福利5。
这项研究的目的是引入小鼠作为特定处理程序积极强化训练。正强化训练是操作性条件反射的一种形式，让研究者塑造动物行为的一种手段。当动物执行所期望的行为，它后面是一个正刺激（这里，食物奖励）。我们的目的是，动物链接奖励到相应的行为。响片训练是使用空调的二级强化物正强化培训的形式，“咔嚓”一唱首歌声音，并已被证明为加强特定的行为12。
更具体而言，点击声用作行为和即将到来的奖励13之间的“时间桥”。培训师点击正是当动物执行所需的行为，没有任何时间延迟14，然后介绍了食物奖励。这增强了奖励行为，这将具有更高的频率来执行。响片训练是广泛与伴侣动物使用，并作出了自己的方式进入实验动物科学，它已与非人灵长类动物13，15，16已成功实施。由于老鼠当操作性条件反射范式的挑战很快就学会相反，引入第二增强剂不应过度紧张他们的认知能力5，17，18，19。
通过引入响片训练小鼠的饲养，我们只能够体验到认知富集。认知富集的设计必须使老鼠用他们的认知技能SOLVE麻烦并得到控制他们的环境20,21。不同种类的几项研究证明认知富集对圈养动物22，23，24福利的积极影响。加强动物与环境挑战成功应对的能力，有助于他们的福利25,26。
此外，如果动物在其一生中会遇到的应力水平较低，它们不易于开发当与发生在生物医学研究中的压力源面临不利的应对策略。因此，始终如一地贯彻认知富集可以向受试者的表型的同质化。这将有助于减少的3R原则，因为它可以减少所需的对象的数目以满足统计要求27。
通过开发可靠的协议，可以轻松集成到饲养实验室老鼠的作息，我们可以显着提高福利的一生经历。
Protocol
Representative Results
Discussion
有效实施与男女各12只BALB / c近交系小鼠的队列唱首歌培训协议被证明是简单的。以前的研究已经证实唱首歌培训的效果与几个品种，我们扩展了这个小鼠。由于老鼠是实验动物中最低的开发哺乳动物，他们的能力常常被低估。因此，数据的最令人吃惊的一方面是，训练成功可以与几乎所有小鼠来实现。
此协议中的一个关键步骤是第二加强件，这是为了建立显示行为和加强之?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Translational Animal Research Center of the University Medical Center of the Johannes Gutenberg-University Mainz. The authors are most grateful to Thomas Wacker for his technical support.
Materials
| Lid for open housing | Tecniplast | GM500LID117 | |
| SealSAfe Plus top for open housing | Tecniplast | GM500400SU | |
| Type II long, filter top cages | Tecniplast | GM500PFSPC | |
| Aspen bedding material | Lab & Vet Service GmbH | H0234-300 | Environmental enrichment |
| Red polycarbonate Mouse House | Tecniplast | ACRE011 | Environmental enrichment |
| Tissue papers | Tork, SCA Hygiene Products GmbH | 290179 | Environmental enrichment |
| Food – ssniff M-H Extrudat | ssniff | V1126-000 | ad libitum |
| Target Stick with Clicker | Trixie | 2282 | |
| PVC Tube (Tunnels) | Thyssen Krupp | RTPVCU04003005 | |
| White Chocolate/ white chocolate cream | Company doesn't matter, preferable organic quality | ||
| Forceps | FineScienceTools | e.g. 11150-10 | Or any other tool to fixate chocolate |
| Prism Version 6.0 for Windows | GraphPad Software |
References
- Russell, W. M. S., Burch, R. L. . The Principles of Humane Experimental Technique. , 25-27 (1959).
- European Commission. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union. , (2010).
- Balcombe, J. P., Barnard, N. D., Sandusky, C. Laboratory routines cause animal stress. Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 43 (6), 42-51 (2004).
- Meijer, M. K., Sommer, R., Spruijt, B. M., van Zutphen, L. F. M., Baumans, V. Influence of environmental enrichment and handling on the acute stress response in individually housed mice. Laboratory animals. 41 (2), 161-173 (2007).
- Gouveia, K., Hurst, J. L. Reducing mouse anxiety during handling: effect of experience with handling tunnels. PloS one. 8 (6), e66401 (2013).
- Meaney, M. J., Diorio, J., et al. Early environmental regulation of forebrain glucocorticoid receptor gene expression: implications for adrenocortical responses to stress. Developmental neuroscience. 18 (1-2), 49-72 (1996).
- Gärtner, K., Büttner, D., Döhler, K., Friedel, R., Lindena, J., Trautschold, I. Stress response of rats to handling and experimental procedures. Laboratory animals. 14 (3), 267-274 (1980).
- Izumi, J., Hayashi-kuwabara, Y. U., Yoshinaga, K., Tanaka, Y., Ikeda, Y. Evidence for a Depressive-like State Induced by Repeated Saline Injections in Fischer 344 Rats. Nature. 57 (4), 883-888 (1997).
- Fridgeirsdottir, G. A., Hillered, L., Clausen, F. Escalated handling of young C57BL/6 mice results in altered Morris water maze performance. Upsala journal of medical sciences. 119 (1), 1-9 (2014).
- Heredia, L., Torrente, M., Domingo, J. L., Colomina, M. T. Individual housing and handling procedures modify anxiety levels of Tg2576 mice assessed in the zero maze test. Physiology and Behavior. 107 (2), 187-191 (2012).
- Maurer, B. M., Döring, D., Scheipl, F., Küchenhoff, H., Erhard, M. H. Effects of a gentling programme on the behaviour of laboratory rats towards humans. Applied Animal Behaviour Science. 114 (3-4), 554-571 (2008).
- Skinner, B. F. How to Teach Animals. Scientific American. 185, 26-29 (1951).
- Bailey, R. E., Gillaspy, J. A. Operant psychology goes to the fair: Marian and Keller Breland in the popular press, 1947-1966. The Behavior Analyst. 28 (2), 143-159 (2005).
- McGreevy, P. D., Boakes, R. A. . Carrots and sticks: principles of animal training. , (2007).
- Gillis, T. E., Janes, A. C., Kaufman, M. J. Positive reinforcement training in squirrel monkeys using clicker training. American journal of primatology. 74 (8), 712-720 (2012).
- Schapiro, S. J., Bloomsmith, M. A., Laule, G. E. Positive reinforcement training as a technique to alter nonhuman primate behavior: quantitative assessments of effectiveness. Journal of applied animal welfare science: JAAWS. 6 (3), 175-187 (2003).
- Martin, L., Iceberg, E. Quantifying Social Motivation in Mice Using Operant Conditioning. Journal of visualized experiments : JoVE. (102), e53009 (2015).
- Sclafani, A., Ackroff, K. Operant licking for intragastric sugar infusions: Differential reinforcing actions of glucose, sucrose and fructose in mice. Physiology and Behavior. , 115-124 (2016).
- Bathellier, B., Tee, S. P., Hrovat, C., Rumpel, S. A multiplicative reinforcement learning model capturing learning dynamics and interindividual variability in mice. Proceedings of the National Academy of Sciences of the United States of America. 110 (49), 19950-19955 (2013).
- Clark, F. E. . Can cognitive challenge enhance the psychological well-being of large-brained mammals in zoos? [unpublished doctoral thesis]. , (1999).
- Shettleworth, S. J., et al. . Cognition, evolution, and behavior. , (2010).
- Hagen, K., Broom, D. M. Emotional reactions to learning in cattle. Applied Animal Behaviour Science. 85 (3-4), 203-213 (2004).
- Ernst, K., Puppe, B., Schön, P., Manteuffel, G. A complex automatic feeding system for pigs aimed to induce successful behavioural coping by cognitive adaptation. Applied Animal Behaviour Science. 91, 205-281 (2005).
- Puppe, B., Ernst, K., Schn, P. C., Manteuffel, G. Cognitive enrichment affects behavioural reactivity in domestic pigs. Applied Animal Behaviour Science. 105 (1-3), 75-86 (2007).
- Zebunke, M., Puppe, B., Langbein, J. Effects of cognitive enrichment on behavioural and physiological reactions of pigs. Physiology and Behavior. 118, 70-79 (2013).
- Manteuffel, G., Langbein, J., Puppe, B. Increasing farm animal welfare by positively motivated instrumental behaviour. Applied Animal Behaviour Science. 118 (3-4), 191-198 (2009).
- Bayne, K., Würbel, H. The impact of environmental enrichment on the outcome variability and scientific validity of laboratory animal studies. Science Direct. 33 (1), 273-280 (2014).
- Hurst, J. L., West, R. S. Taming anxiety in laboratory mice. Nature Methods. 7 (10), 825-826 (2010).
- Crawley, J. N. . What’s Wrong With My Mouse?. , (2007).
- Loos, M., Koopmans, B., et al. Within-strain variation in behavior differs consistently between common inbred strains of mice. Mammalian genome : official journal of the International Mammalian Genome Society. 26 (7-8), 348-354 (2015).
- Seibenhener, M. L., Wooten, M. C. Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice. Journal of visualized experiments : JoVE. (96), e1-e6 (2015).
- Can, A., Dao, D. T., Arad, M., Terrillion, C. E., Piantadosi, S. C., Gould, T. D. The Mouse Forced Swim Test. Journal of Visualized Experiments. , 4-8 (2011).
- Clayton, L. A., Tynes, V. V. Keeping the Exotic Pet Mentally Healthy. Veterinary Clinics of North America: Exotic Animal Practice. 18 (2), 187-195 (2015).
- Ward, S. J., Melfi, V. The implications of husbandry training on zoo animal response rates. Applied Animal Behaviour Science. 147 (1-2), 179-185 (2013).
