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>
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(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. 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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.

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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 ...

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 ...

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 ...

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

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 ...

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

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 ...

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 ...