Name: introducing clicker training as a cognitive enrichment for laboratory mice
Uploaded: 2017-03-06T15:00:00
Description: Watch this Scientific Journal Video about Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice at JoVE.com

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.

Ethics Statement: The handling of the mice and the experimental procedures were conducted in accordance with European, national, and institutional guidelines for animal care.
NOTE: The protocol includes five days of interventions (Monday to Friday), with breaks on the weekends (Saturday and Sunday). The protocol can be easily adapted to meet specific needs.
1. Determining a Reward Suitable as a Second Reinforcer
NOTE: Use food rewards, such as vacuum-packed food or animal feed that meets food safety st...

<ol>
	<li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , 25-27 (1959).</li><li>European Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Official Journal of the European Union. , (2010).</li><li>Balcombe, J. P., Barnard, N. D., Sandusky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+routines+cause+animal+stress.">Laboratory routines cause animal stress.</a> Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 43 (6), 42-51 (2004).</li><li>Meijer, M. K., Sommer, R., Spruijt, B. M., van Zutphen, L. F. M., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+environmental+enrichment+and+handling+on+the+acute+stress+response+in+individually+housed+mice.">Influence of environmental enrichment and handling on the acute stress response in individually housed mice.</a> Laboratory animals. 41 (2), 161-173 (2007).</li><li>Gouveia, K., Hurst, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+mouse+anxiety+during+handling:+effect+of+experience+with+handling+tunnels.">Reducing mouse anxiety during handling: effect of experience with handling tunnels.</a> PloS one. 8 (6), e66401 (2013).</li><li>Meaney, M. J., Diorio, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+environmental+regulation+of+forebrain+glucocorticoid+receptor+gene+expression:+implications+for+adrenocortical+responses+to+stress.">Early environmental regulation of forebrain glucocorticoid receptor gene expression: implications for adrenocortical responses to stress.</a> Developmental neuroscience. 18 (1-2), 49-72 (1996).</li><li>G&#228;rtner, K., B&#252;ttner, D., D&#246;hler, K., Friedel, R., Lindena, J., Trautschold, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+response+of+rats+to+handling+and+experimental+procedures.">Stress response of rats to handling and experimental procedures.</a> Laboratory animals. 14 (3), 267-274 (1980).</li><li>Izumi, J., Hayashi-kuwabara, Y. U., Yoshinaga, K., Tanaka, Y., Ikeda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+Depressive-like+State+Induced+by+Repeated+Saline+Injections+in+Fischer+344+Rats.">Evidence for a Depressive-like State Induced by Repeated Saline Injections in Fischer 344 Rats.</a> Nature. 57 (4), 883-888 (1997).</li><li>Fridgeirsdottir, G. A., Hillered, L., Clausen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escalated+handling+of+young+C57BL/6+mice+results+in+altered+Morris+water+maze+performance.">Escalated handling of young C57BL/6 mice results in altered Morris water maze performance.</a> Upsala journal of medical sciences. 119 (1), 1-9 (2014).</li><li>Heredia, L., Torrente, M., Domingo, J. L., Colomina, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+housing+and+handling+procedures+modify+anxiety+levels+of+Tg2576+mice+assessed+in+the+zero+maze+test.">Individual housing and handling procedures modify anxiety levels of Tg2576 mice assessed in the zero maze test.</a> Physiology and Behavior. 107 (2), 187-191 (2012).</li><li>Maurer, B. M., D&#246;ring, D., Scheipl, F., K&#252;chenhoff, H., Erhard, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+gentling+programme+on+the+behaviour+of+laboratory+rats+towards+humans.">Effects of a gentling programme on the behaviour of laboratory rats towards humans.</a> Applied Animal Behaviour Science. 114 (3-4), 554-571 (2008).</li><li>Skinner, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+Teach+Animals.">How to Teach Animals.</a> Scientific American. 185, 26-29 (1951).</li><li>Bailey, R. E., Gillaspy, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operant+psychology+goes+to+the+fair:+Marian+and+Keller+Breland+in+the+popular+press,+1947-1966.">Operant psychology goes to the fair: Marian and Keller Breland in the popular press, 1947-1966.</a> The Behavior Analyst. 28 (2), 143-159 (2005).</li><li>McGreevy, P. D., Boakes, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Carrots and sticks: principles of animal training. , (2007).</li><li>Gillis, T. E., Janes, A. C., Kaufman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+reinforcement+training+in+squirrel+monkeys+using+clicker+training.">Positive reinforcement training in squirrel monkeys using clicker training.</a> American journal of primatology. 74 (8), 712-720 (2012).</li><li>Schapiro, S. J., Bloomsmith, M. A., Laule, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+reinforcement+training+as+a+technique+to+alter+nonhuman+primate+behavior:+quantitative+assessments+of+effectiveness.">Positive reinforcement training as a technique to alter nonhuman primate behavior: quantitative assessments of effectiveness.</a> Journal of applied animal welfare science: JAAWS. 6 (3), 175-187 (2003).</li><li>Martin, L., Iceberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Social+Motivation+in+Mice+Using+Operant+Conditioning.">Quantifying Social Motivation in Mice Using Operant Conditioning.</a> Journal of visualized experiments : JoVE. (102), e53009 (2015).</li><li>Sclafani, A., Ackroff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operant+licking+for+intragastric+sugar+infusions:+Differential+reinforcing+actions+of+glucose,+sucrose+and+fructose+in+mice.">Operant licking for intragastric sugar infusions: Differential reinforcing actions of glucose, sucrose and fructose in mice.</a> Physiology and Behavior. , 115-124 (2016).</li><li>Bathellier, B., Tee, S. P., Hrovat, C., Rumpel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplicative+reinforcement+learning+model+capturing+learning+dynamics+and+interindividual+variability+in+mice.">A multiplicative reinforcement learning model capturing learning dynamics and interindividual variability in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (49), 19950-19955 (2013).</li><li>Clark, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Can cognitive challenge enhance the psychological well-being of large-brained mammals in zoos? [unpublished doctoral thesis]. , (1999).</li><li>Shettleworth, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cognition, evolution, and behavior. , (2010).</li><li>Hagen, K., Broom, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotional+reactions+to+learning+in+cattle.">Emotional reactions to learning in cattle.</a> Applied Animal Behaviour Science. 85 (3-4), 203-213 (2004).</li><li>Ernst, K., Puppe, B., Sch&#246;n, P., Manteuffel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+automatic+feeding+system+for+pigs+aimed+to+induce+successful+behavioural+coping+by+cognitive+adaptation.">A complex automatic feeding system for pigs aimed to induce successful behavioural coping by cognitive adaptation.</a> Applied Animal Behaviour Science. 91, 205-281 (2005).</li><li>Puppe, B., Ernst, K., Schn, P. C., Manteuffel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+enrichment+affects+behavioural+reactivity+in+domestic+pigs.">Cognitive enrichment affects behavioural reactivity in domestic pigs.</a> Applied Animal Behaviour Science. 105 (1-3), 75-86 (2007).</li><li>Zebunke, M., Puppe, B., Langbein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cognitive+enrichment+on+behavioural+and+physiological+reactions+of+pigs.">Effects of cognitive enrichment on behavioural and physiological reactions of pigs.</a> Physiology and Behavior. 118, 70-79 (2013).</li><li>Manteuffel, G., Langbein, J., Puppe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+farm+animal+welfare+by+positively+motivated+instrumental+behaviour.">Increasing farm animal welfare by positively motivated instrumental behaviour.</a> Applied Animal Behaviour Science. 118 (3-4), 191-198 (2009).</li><li>Bayne, K., W&#252;rbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+environmental+enrichment+on+the+outcome+variability+and+scientific+validity+of+laboratory+animal+studies.">The impact of environmental enrichment on the outcome variability and scientific validity of laboratory animal studies.</a> Science Direct. 33 (1), 273-280 (2014).</li><li>Hurst, J. L., West, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+anxiety+in+laboratory+mice.">Taming anxiety in laboratory mice.</a> Nature Methods. 7 (10), 825-826 (2010).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's Wrong With My Mouse?. , (2007).</li><li>Loos, M., Koopmans, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-strain+variation+in+behavior+differs+consistently+between+common+inbred+strains+of+mice.">Within-strain variation in behavior differs consistently between common inbred strains of mice.</a> Mammalian genome : official journal of the International Mammalian Genome Society. 26 (7-8), 348-354 (2015).</li><li>Seibenhener, M. L., Wooten, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Open+Field+Maze+to+Measure+Locomotor+and+Anxiety-like+Behavior+in+Mice.">Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice.</a> Journal of visualized experiments : JoVE. (96), e1-e6 (2015).</li><li>Can, A., Dao, D. T., Arad, M., Terrillion, C. E., Piantadosi, S. C., Gould, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mouse+Forced+Swim+Test.">The Mouse Forced Swim Test.</a> Journal of Visualized Experiments. , 4-8 (2011).</li><li>Clayton, L. A., Tynes, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+the+Exotic+Pet+Mentally+Healthy.">Keeping the Exotic Pet Mentally Healthy.</a> Veterinary Clinics of North America: Exotic Animal Practice. 18 (2), 187-195 (2015).</li><li>Ward, S. J., Melfi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implications+of+husbandry+training+on+zoo+animal+response+rates.">The implications of husbandry training on zoo animal response rates.</a> Applied Animal Behaviour Science. 147 (1-2), 179-185 (2013).</li></ol>

The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. Previous studies have confirmed the effectiveness of clicker training with several species, and we extended this to mice. As mice are the lowest developed mammals among laboratory animals, their abilities are often underestimated. Therefore, the most surprising aspect of the data is that training success could be achieved with almost all mice.
One critica...

The development of new refinement strategies for laboratory mice is a challenging task that contributes to the fulfillment of the 3Rs (replacement, reduction, and refinement) of laboratory animal science1. Improvements in the field of refinement can further contribute to the well-being of the millions of animals that are used for experimental purposes. Therefore, intensive research is needed in the field. This is also a defined aim of Directive 2010/63 of the European Union. Directive 2010/63/EU points out that the lifetime experience of laboratory animals has to be enhanced and that &#34;establishments shall set up habituation and training programs suitable for the animals, the procedures and length of the project&#34; (Article 3.7)2.
Laboratory animals can experience many stressful situations while they are being kept and bred for experiments. Usually, the interaction between the laboratory mice and the responsible persons is rather limited. Therefore, a trusting relationship cannot develop. This can elicit increased anxiety and stress in reaction to handling, which is detrimental to the behavior and physiology, and therefore, the well-being, of the animals3,4,5,6. Furthermore, routinely performed laboratory procedures like general handling, restraint, and blood or tissue sampling can cause stress responses, which can be examined by measuring different parameters, such as stress hormones or behavior7,8. It has been shown that handling programs can efficiently decrease the anxiety toward the investigator in laboratory rodents9,10,11. Handling programs can therefore improve the animals&#39; conditions and could contribute considerably to animal welfare5.
The goal of this study is to introduce positive reinforcement training for mice as a specific handling program. Positive reinforcement training is a form of operant conditioning that gives the investigator a means to shape animal behavior. When the animal performs a desired behavior, it is followed by a positive stimulus (here, a food reward). The intention is that the animal links the reward to the respective behavior. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the &#34;click&#34; sound of a clicker, and has been proven to strengthen a specific behavior12.
More specifically, the click sound serves as a &#34;time bridge&#34; between the behavior and the upcoming reward13. The trainer clicks precisely when the animal performs the desired behavior, without any time delay14, and then presents the food reward. This strengthens the rewarded behavior, which will be performed with a higher frequency. Clicker training is widely used with companion animals and has made its way into laboratory animal science, where it has been successfully implemented with nonhuman primates13,15,16. As mice learn rather quickly when challenged with operant conditioning paradigms, the introduction of a second reinforcer should not overstrain their cognitive abilities5,17,18,19.
By introducing clicker training to the keeping of mice, we enable mice to experience cognitive enrichment. The design of cognitive enrichment must enable the mice to use their cognitive skills to solve problems and to gain control over their environment20,21. Several studies with different species prove the positive impact of cognitive enrichment on the welfare of captive animals22,23,24. Enhancing the ability of the animals to successfully cope with environmental challenges contributes to their well-being25,26.
In addition, if the animals experience low levels of stress during their lifetime, they are less prone to develop detrimental coping strategies when confronted with stressors that occur in biomedical research. Thus, the consistent implementation of cognitive enrichment can contribute to a homogenization of the subjects&#39; phenotypes. This will contribute to the 3R principle of reduction, as it can reduce the number of subjects required to meet statistical requirements27.
By developing a reliable protocol that can be easily integrated into the daily routine of keeping laboratory mice, we can substantially improve their lifetime experience of welfare.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lid for open housing</td>
			<td>Tecniplast</td>
			<td>GM500LID117</td>
			<td></td>
		</tr>
		<tr>
			<td>SealSAfe Plus top for open housing</td>
			<td>Tecniplast</td>
			<td>GM500400SU</td>
			<td></td>
		</tr>
		<tr>
			<td>Type II long, filter top cages</td>
			<td>Tecniplast</td>
			<td>GM500PFSPC</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspen bedding material&#160;</td>
			<td>Lab &#38; Vet Service GmbH</td>
			<td>H0234-300</td>
			<td>Environmental enrichment</td>
		</tr>
		<tr>
			<td>Red polycarbonate Mouse House</td>
			<td>Tecniplast</td>
			<td>ACRE011</td>
			<td>Environmental enrichment</td>
		</tr>
		<tr>
			<td>Tissue papers</td>
			<td>Tork, SCA Hygiene Products GmbH</td>
			<td>290179</td>
			<td>Environmental enrichment</td>
		</tr>
		<tr>
			<td>Food - ssniff M-H Extrudat</td>
			<td>ssniff</td>
			<td>V1126-000</td>
			<td>ad libitum</td>
		</tr>
		<tr>
			<td>Target Stick with Clicker</td>
			<td>Trixie</td>
			<td>2282</td>
			<td></td>
		</tr>
		<tr>
			<td>PVC Tube (Tunnels)&#160;</td>
			<td>Thyssen Krupp</td>
			<td>RTPVCU04003005</td>
			<td></td>
		</tr>
		<tr>
			<td>White Chocolate/ white chocolate cream</td>
			<td></td>
			<td></td>
			<td>Company doesn&#39;t matter, preferable organic quality</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FineScienceTools</td>
			<td>e.g. 11150-10</td>
			<td>Or any other tool to fixate chocolate</td>
		</tr>
		<tr>
			<td>Prism Version 6.0 for Windows&#160;</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

introducing clicker training as a cognitive enrichment for laboratory mice

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.

The first and also one of the most important steps was the determination of an appropriate food reward. Therefore, the mice were offered different kinds of nuts, a sugar solution, marmalade, and different kinds of chocolate in a Petri dish (Figure 1). In our experience, the mice showed an obvious preference for white chocolate. Hence, we used white chocolate for all further training processes.
The actual trainin...

Watch this Scientific Journal Video about Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice at JoVE.com

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

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.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

The overall goal of this study is to establish a positive reinforcement training for mice as a specific handling program. This method can answer key questions in the emerging field of refinement strategies for laboratory mice such as how to reduce stress levels in keeping and performing experiments. The main advantage of this technique is that is can be modified to various purposes and that it can help to improve the well being of laboratory mice.We first had the idea for this method when we thought about ergonomic alternatives to transfer mice between cages without touching them. Visual demonstration of this method is critical as the technique is challenging to learn because the timing and overall handling of the mice are essential. Before beginning the experiment, transfer the home cage to a quiet environment.Then remove all objects form the home cage to use it as a training area. Then transfer the cage companions out of the home cage leaving only one training mouse. To start the training, set the timer to five minutes and press start.Add the mouse tunnel to the home cage near a wall. Then wait until the mouse inspects the tunnel. As soon as the mouse enters the tunnel, click and present the reward at the end of the tunnel.While the mouse sits and feeds on the reward in the tunnel tenuously click for 15 seconds. As soon as the mouse leaves the tunnel, repeat the session again. On days two through five, set the timer to five minutes.Press start and add the mouse tunnel to the home cage. As soon as the mouse enters the tunnel, click and present the reward at the end of the tunnel. Repeat this for the next 30 seconds and then pause for 15 seconds to take the tunnel out of the cage.Then add the tunnel again and click immediately when the mouse reenters the tunnel. Present the reward at the end of the tunnel and allow the mouse to gnaw on it for up to one second. Then take away the reward.Finally, as soon as the mouse starts reentering the tunnel by itself, present the reward in front of the end of the tunnel. During week two, begin by setting the timer to five minutes. Press start and place the globe at the end of the target stick in the cage.As soon as the mouse touches the globe with its nose, click and present the reward next to the globe. After the mouse has linked the performed action to the reward, change the position of the globe during the training session. Reward the mouse after it touches the globe with its nose in the new position.Next place the globe in the cage and shortly before the mouse touches the globe, slowly and carefully change the position of the globe by one centimeter. Click and reward if the mouse has crossed the distance and touches the globe. Slowly extend the distance the mouse has to cross and repeat this procedure until the mouse follows reliably.During week three begin by placing one hand in the training cage while holding the clicker target stick combination and the reward with the other hand. Set the timer to five minutes. Press start and place the globe at the end of the target stick next to the hand.As soon as the mouse shows interest in the globe, click and present the reward next to the globe. Finally, place the globe on the palm of the hand. Then click and reward while the mouse is sitting on the palm of the hand.After clicker training, the vast majority of trained mice all female mice and 83 percent of male mice followed the target stick. Female mice displayed higher motivation for training in general form the respective behavior with the higher frequency throughout the training sessions. Trained mice displayed a significantly lower frequency of anxiety related behaviors than untrained mice.Furthermore, depression related floating behavior during the Morris water maze was significantly reduced in the trained group. Once mastered, this technique can be completed in three to five minutes per mouse, per day if it is performed properly. While attempting this procedure, it's important to remember to handle mice in a gentle, calm, and confident manner.Following this procedure, other methods like teaching mice to run over a bridge to transfer on another cage independently can be performed to improve health and hygiene conditions in animal facilities. After its development, clicker training contributes to refinement as it can be considered as a form of cognitive enrichment. After watching this video, you should have a good understanding of how to implement positive reinforcement training for mice in laboratory animal facilities.

introducing-clicker-training-as-cognitive-enrichment-for-laboratory

Research

JoVE Journal

Behavior

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many neuropsychiatric and neurodevelopmental disorders, including depression, post-traumatic stress disorder, schizophrenia and autism spectrum disorder. Deficits in cognitive function also represent the most difficult symptom domain&#160;to successfully treat, as serotonin reuptake inhibitors and tricyclic antidepressants have only modest effects. Functional neuroimaging studies and postmortem analysis of human brain tissue implicate the PFC as being a primary region of dysregulation in patients with these disorders. However, preclinical behavioral assays used to assess these deficits in mouse models which can be readily manipulated genetically and could provide the basis for studies of new treatment avenues have been underutilized. Here we describe the adaptation of a behavioral assay, the attentional set shifting task (AST), to be performed in mice to assess prefrontal cortex mediated cognitive deficits. The neural circuits underlying behavior during the AST are highly conserved across humans, nonhuman primates and rodents, providing excellent face, construct and predictive validity.

The goal of this protocol is to perform a behavioral assay such as the attentional set shifting task (AST) to assess prefrontal cortex-mediated cognitive flexibility in mice.

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many ...

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington&#8217;s disease 1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box 5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent&#8217;s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter&#8217;s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple &#8220;Puzzle Box&#8221; method for assessing an animal&#8217;s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Environmental enrichment can dramatically influence the development and function of neural circuits.  Further, enrichment has been shown to successfully ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

A Pressure Injection System for Investigating the Neuropharmacology of Information Processing in Awake Behaving Macaque Monkey Cortex

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation of sensory information processing by attention. However, little is known about which neurotransmitter systems are involved in such modulations. A practical way to address this question is to combine single-cell recording with local and temporary neuropharmacological manipulation in a suitable animal model. Here we demonstrate a technique combining acute single-cell recordings with the injection of neuropharmacological agents in the direct vicinity of the recording electrode. The video shows the preparation of the pressure injection/recording system, including preparation of the substance to be injected. We show a rhesus monkey performing a visual attention task and the procedure of single-unit recording with block-wise pharmacological manipulations.

Here, we show the pressure injection of neuropharmacological substances during single-cell recording in an awake, behaving macaque monkey. This procedure allows pharmacological manipulation in the direct vicinity of a cortical recording site.

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation ...

The Deese-Roediger-McDermott (DRM) Task: A Simple Cognitive Paradigm to Investigate False Memories in the Laboratory

The Deese,&#160;Roediger and McDermott&#160;(DRM) task is a false memory paradigm in which subjects are presented with lists of semantically related words (e.g., nurse, hospital, etc.) at encoding. After a delay, subjects are asked to recall or recognize these words. In the recognition memory version of the task, subjects are asked whether they remember previously presented words, as well as related (but never presented) critical lure words (&#39;doctor&#39;). Typically, the critical word is recognized with high probability and confidence. This false memory effect has been robustly demonstrated across short (e.g., immediate, 20 min) and long (e.g., 1, 7, 60 d) delays between encoding and memory testing. A strength of using this task to study false memory is its simplicity and short duration. If encoding and retrieval components of the task occur in the same session, the entire task can take as little as 2 - 30 min. However, although the DRM task is widely considered a &#39;false memory&#39; paradigm, some researchers consider DRM illusions to be based on the activation of semantic memory networks in the brain, and argue that such semantic gist-based false memory errors may actually be useful in some scenarios (e.g., remembering the forest for the trees; remembering that a word list was about &#34;doctors&#34;, even though the actual word &#34;doctor&#34; was never presented for study). Remembering the gist of experience (instead of or along with individual details) is arguably an adaptive process and this task has provided a great deal of knowledge about the constructive, adaptive nature of memory. Therefore, researchers should use caution when discussing the overall reach and implications of their experiments when using this task to study &#39;false memory&#39;, as DRM memory errors may not adequately reflect false memories in the real world, such as false memory in eyewitness testimony, or false memories of sexual abuse.

The Deese-Roediger-McDermott DRM Task: A Simple Cognitive Paradigm to Investigate False Memories in the Laboratory

Here we present the Deese,&#160;Roediger and McDermott&#160;(DRM) task, a tool to study false memories in the laboratory. Subjects study lists of semantically related words (e.g.,&#160;nurse, sick, etc.), and later falsely remember an unstudied word (doctor) that represents the gist, or theme, of the word list.

The Deese,&#160;Roediger and McDermott&#160;(DRM) task is a false memory paradigm in which subjects are presented with lists of semantically related words ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.