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 standards. For example, different kinds of nuts, chocolate, gummy bears, or dried fruits are suitable.
<ol>
	<li>Insert a small Petri dish with different food rewards (pieces of 0.5 cm3) into the home-cage (Figure 1).</li>
	<li>After 10 min, check for leftovers.</li>
	<li>If all rewards are eaten after 10 min, shorten the time span until a preference can be estimated.</li>
	<li>On days 4 and 5 (e.g., Thursday and Friday), add small amounts of the preferred (&#34;winner&#34;) reward to the home-cage to establish an affectation of all mice in the cage to the reward.</li>
	<li>If the mice are not habituated to mouse tunnels, add a tunnel to the cage (at least 2 days before beginning the training, e.g., over the weekend).</li>
	<li>Repeat steps 1.2 - 1.4 on 3 consecutive days (e.g., Monday to Wednesday).</li>
</ol>
2. Clicker Training
<ol>
	<li>Preparations for each training session 
	NOTE: Every time a mouse needs to be lifted up in this protocol, lift it in a calm and gentle manner. Put a hand under the mouse to lift it up. During the first few times, the mice might be agitated, but the habituation process will be finished after several days. If this step creates difficulties, it is useful to refer to the protocol for &#34;cupping on the open hand&#34;28.
	<ol>
		<li>Prepare a cage with bedding material.</li>
		<li>Transfer the cage of the mouse being trained to a quiet place.</li>
		<li>Prepare the reward and have a timer and the clicker/target stick combination ready. 
		NOTE: If the mice are habituated to handling and already have a relationship of trust with the experimenter, the mice can be fed by hand. Otherwise, attach the reward to a stick or forceps in such a way that the mice can keep a distance from the experimenter.</li>
		<li>Remove all objects from the home-cage, which now serves as the training area (e.g., mouse houses, nest-building material, etc.).</li>
		<li>Transfer the cage companions to a prepared cage.</li>
	</ol>
	</li>
	<li>General rules that apply for all training sessions 
	NOTE: The following general rules apply for all sessions and will not be mentioned again throughout the protocol.
	<ol>
		<li>Train each mouse for 5 min. Alternate between 30-s training and 15-s break periods.</li>
		<li>Let the mouse gnaw on the reward no longer than a second. Then, take the stick (with the reward) out of the cage.</li>
		<li>Execute the following pattern of rewarding: 
		1 - 10 performances of behavior: reward every time. 
		11 - 21 performance of behavior: reward every second time. 
		From 22 performance of behavior on: reward every third time.</li>
		<li>After 4 min of training, the next performance of the desired behavior is reinforced by a jackpot reward. Allow the mouse to gnaw at the food reward three times longer than before.</li>
	</ol>
	</li>
	<li>Training session Day 1: &#34;Linking the secondary reinforcer with the food reward&#34;
	<ol>
		<li>Set the timer to 5 min, press start, and add the mouse tunnel to the home-cage. 
		NOTE: Take advantage of the innate thigmotaxis of mice and place the tunnel next to a wall (Figure 2). For the first few sessions, this will enhance the probability of the mouse entering the tunnel.</li>
		<li>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.</li>
		<li>Let the mouse feed on the reward while sitting in the tunnel.</li>
		<li>Click continuously for 15 s while the mouse is sitting in the tunnel.</li>
		<li>As soon as the mouse leaves the tunnel, go back to step 3.2.</li>
	</ol>
	</li>
	<li>Training session Days 2 - 5: &#34;Running through a tunnel&#34;
	<ol>
		<li>Set the timer to 5 min, press start, and add the mouse tunnel to the home-cage.</li>
		<li>As soon as the mouse enters the tunnel, click and present the reward at the end of the tunnel.</li>
		<li>As long as the mouse is in the tunnel, click and immediately present the reward in the same manner.</li>
		<li>Repeat this for the next 30 s, and then pause for 15 s to take the tunnel out of the cage.</li>
		<li>Add the tunnel again and click immediately when the mouse reenters the tunnel.</li>
		<li>Present the reward at the end of the tunnel.</li>
		<li>Allow the mouse to gnaw for up to 1 s.</li>
		<li>Take away the reward.</li>
		<li>As soon as the mouse starts re-entering the tunnel by itself, present the reward in front of the end of the tunnel (Figure 3).</li>
	</ol>
	</li>
	<li>Training session week 2: &#34;Following a target stick&#34;
	<ol>
		<li>Set the timer to 5 min, press start, and place the globe at the end of the target stick in the cage.</li>
		<li>As soon as the mouse shows interest in the globe, click and present the reward next to the globe (Figures 4 and 5).</li>
		<li>Reward the mouse only after it touches the globe with its nose.</li>
		<li>When the mouse has linked the performed action to the reward, change the position of the globe during the training session (Figure 6).</li>
		<li>Wait for the mouse to meet this last challenge.</li>
		<li>Place the globe in the cage.</li>
		<li>Shortly before the mouse touches the globe, slowly and carefully change the position of the globe by 1 cm.</li>
		<li>Click and reward if the mouse has crossed the distance and touches the globe.</li>
		<li>Repeat this procedure until the mouse follows reliably.</li>
		<li>Slowly extend the distance the mouse has to cross.</li>
	</ol>
	</li>
	<li>Training session week 3: &#34;Following the target stick to the experimenter&#39;s hand&#34;
	<ol>
		<li>Place one hand in the training cage while holding the clicker/target stick combination and the reward with the other hand.</li>
		<li>Set the timer to 5 min, press start, and place the globe at the end of the target stick next to the hand.</li>
		<li>As soon as the mouse shows interest in the globe, click and present the reward next to the globe.</li>
		<li>As soon as the mouse has met this challenge, move the globe several steps closer to the hand.</li>
		<li>Place the globe on the palm of the hand. Click and reward while the mouse is sitting on the palm of the hand (Figure 7).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 critical step within this protocol is the timing of the second reinforcer, which is very important to establishing a connection between the displayed behavior and the reinforcement. As mice are very agitated, it is slightly difficult to mark an exact performance while they are scampering around. The more experienced the trainer is, the faster training success can be achieved. We observed that the mice learned quite quickly, even with unexperienced trainers. Even little mistakes could be compensated for in the course of the clicker training protocol. Common sources of error included reinforcing a wrong behavior and a lack of interest in the food reward after clicking.
An uncontrollable factor is the individual character of each mouse. Although this study was performed with inbred mice, born and raised under the same conditions, they develop a variety of different characters. Due to previous research and our own empirical data, we know that this includes different tastes, different expressions of appetite, and different exploration behavior29,30. The first critical step was to find an appropriate food reward. The mice were offered different kinds of nuts, a sugar solution, marmalade, and different kinds of chocolate. For the mice in this study, white chocolate proved to be the most appropriate reward. The success of this protocol depends on the interest of the mice in the training. If anything else arouses their interest more, the training reaches its limits. The more equipment that was added to the cage to fulfill a training session, the less the mice participated in the training. The elimination of all attention-grabbing items in the surrounding is essential (e.g., leaving even a small amount of bedding material in the cage to prevent the mice from burrowing). Sometimes the mice gain more interest in the training after a habituating to the new environment for several minutes.
Gentle handling protocols have already been determined to benefit the wellbeing of mice when treated amicably9,10,11. The results of the present study indicate that a closer human-animal relationship contributes to mouse tolerance to treatments. Under manipulation, trained animals displayed fewer anxiety-related behaviors (i.e., urination, defecation, and vocalization)31. In the Morris Water Maze Test, the untrained but gently handled mice expressed a significantly higher prevalence of floating behavior. Trained mice displayed less of this depression-related behavior32. It could be argued that the positive results were due to increased well-being in the trained mice.
A limitation of this study is that only one strain of inbred mice was studied. Due to high behavioral differences between different strains of mice, additional inbred strains, as well as outbred strains, should be investigated29. In further studies, supplementary information about mouse wellbeing, such as a whole behavioral battery, physiological data (e.g., corticosterone levels), or the impact of training on brain development, should be gathered.
Further applications of clicker training could assist in improving health and hygiene conditions in animal facilities. By applying the clicker training protocol, we succeeded in teaching mice to follow a target stick and to cross a bridge from one cage to another (Figure 13). If this technique is perfected, it makes manual cage-changing unnecessary. In addition, mice could possibly be trained to voluntarily walk onto measurement equipment (e.g., scale or behavioral areas). This could provide reliable protection against anthropozoonotic disease transmission, as direct contact with the animals will no longer be necessary.
Positive reinforcement training is highly recommended for captive animals, as it contributes to their mental health33. If mice experience a high level of wellbeing, they display few individually different coping strategies. Clicker training, therefore, makes a noteworthy contribution to the 3Rs, as previous work shows that a reduction in the variability of mice phenotypes leads to a reduced number of animals necessary to meet statistical requirements27. This study has demonstrated that clicker training in mice strongly correlates with a reduced fear of human-mice interactions and matches results observed in earlier studies with different species34. Clicker training has further potential, as it could be considered cognitive enrichment. The broad implementation of positive reinforcement training in laboratory animal facilities could make a valuable contribution to the 3R principle, as it refines the keeping and biomedical research of mice.

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 training was implemented with a cohort of 12 BALB/c inbred mice of each sex. All mice were highly interested in the training. For the evaluation of training success, we checked for the proper performance of the desired behavior: &#34;following the target stick&#34; (Figure 4). The vast majority of the trained mice-all female mice and 83% of the male mice-followed the target stick (Figure 8). Female mice displayed a higher motivation for training in general and performed the respective behavior with a higher frequency throughout the training sessions. After completing 4 days of training, female mice followed the target stick with a mean of 64 times per 5 min, whereas male mice displayed this behavior only 50 times per 5 min. In 5 min, the task &#34;following to the palm of the hand&#34; was performed on average 55 times by female mice and 35 times by male mice (Figure 9).
To check whether the training had a positive effect on the well-being of the trained mice, we evaluated the tolerance to manipulations after completing the training. Therefore, we analyzed anxiety-related behaviors while the mice were singlehandedly restrained (i.e., grasping the scruff of the neck and the base of the tail with one hand) for 15 s. Spontaneous urination, defecation and vocalization were recorded. An untrained but gently handled cohort of 12 BALB/c inbred mice of each sex served as a control group. Trained mice displayed a significantly lower frequency of anxiety related behaviors than untrained mice. (Figure 10). Similar results were obtained when recording the vocalization linked to handling while performing the Morris Water Maze Test (Figures 11 and 12). The trained mice squeaked significantly less than the untrained mice (Figure 11) and the total number of squeaks per squeaking mouse was significantly reduced. To further evaluate this issue, we analyzed the floating behavior during the Morris Water Maze Test. Floating behavior during the Morris Water Maze Test is described as periods of time when the mice are not swimming, but are merely floating on the surface. This depression-related floating behavior turned out to be significantly reduced in the trained group (Figure 12).
<img alt="Figure 1" src="/files/ftp_upload/55415/55415fig1.jpg" /> 
Figure 1. Presentation of Possible Rewards. Six positions for the presentation of rewards are marked in a Petri dish.
<img alt="Figure 3" src="/files/ftp_upload/55415/55415fig2.jpg" /> 
Figure 2. Position of Mouse Tunnel. To enhance the probability of the mouse entering the tunnel, the tunnel is placed next to a cage wall. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55415/55415fig3.jpg" /> 
Figure 3. Presenting the Reward. The appropriate position for presenting the reward outside the end of the tunnel is shown. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55415/55415fig4.jpg" /> 
Figure 4. Training Success. A trained mouse follows the target stick while being trained. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55415/55415fig5.jpg" /> 
Figure 5. Rewarding. A mouse is rewarded next to the target stick during the training session. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55415/55415fig6.jpg" /> 
Figure 6. Alternating Target Stick Positions in the Second Week of Training. The positions of the training stick are indicated in this schematic drawing. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/55415/55415fig7.jpg" /> 
Figure 7. Training Success. A mouse has followed the target stick onto the hand of the trainer. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55415/55415fig8.jpg" /> 
Figure 8. Success of Training Protocol. A cohort of 12 BALB/c inbred strain mice of each sex was trained. By the end of training session week 2: &#34;Following a target stick,&#34; all female mice and 83% of the male mice successfully overcame the challenge. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55415/55415fig9.jpg" /> 
Figure 9. Repetitions of Respective Behavior after One Week of Training. A cohort of 12 BALB/cin bred mice of each sex was trained. On the last day of training session week 2: &#34;Following a target stick&#34; and 3: &#34;Following the target stick to the hand&#34;, the repetitions of the respective behavior were counted during a 5-min training session. In both weeks, female mice displayed the strengthened behavior with a significantly higher frequency than male mice (&#34;Target stick&#34;:63.92 &#177; 3.72, p = 0.0300; &#34;On hand&#34;: 54.92 &#177; 4.01, p = 0.0069). The frequency of the strengthened behavior did not significantly vary in female mice from week two to week three. However, male mice showed a significant decrease in repetitions from week two to week three (&#34;Target stick&#34;: 50.42 &#177; 4.48; &#34;On hand&#34;: 35.92 &#177; 5; p = 0.0408). A Mann-Whitney U-test was performed. The results of the data were expressed as the mean &#177; S.E.M. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55415/55415fig10.jpg" /> 
Figure 10. Displayed Behavior Triggered by Scruff Holding. A cohort of 12 BALB/c inbred mice of each sex was trained following the clicker training protocol. An untrained but gently handled cohort of 12 BALB/c inbred mice of each sex served as a control group. The mice were singlehandedly restrained (i.e., grasping the scruff of the neck and the base of the tail with one hand) for 15 s. Spontaneous urination, defecation, and vocalization were recorded. There was a profound difference between the displayed behavior of the trained and the control group. Trained mice displayed all behaviors with a significantly lower frequency than untrained mice. (&#34;Urination&#34;: p &#60; 0.001; &#34;Defecation&#34;: p &#60; 0.001; &#34;Vocalization&#34;: p &#60; 0.001). A Mann-Whitney U-test was performed. The columns are expressed as the percent of all tested mice. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55415/55415fig11.jpg" /> 
Figure 11. Displayed Behaviors During the Morris Water Maze Test. A cohort of 12 BALB/c inbred mice of each sex was trained following the clicker training protocol. An untrained but gently handled cohort of 12 BALB/c inbred mice of each sex served as a control group. A Morris Water Maze Test was performed with all mice after the third week of training. Floating behavior and vocalization linked to handling were recorded. Floating behavior occurred significantly less in trained mice (p &#60; 0.001). Further, there was a profound difference in the vocalization of the trained and the control group. Trained mice squeaked significantly less when handled than mice of the untrained control group (p &#60; 0.001). A Mann-Whitney U-test was performed. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55415/55415fig12.jpg" /> 
Figure 12. Prevalence of Squeaks per Mouse. A cohort of 12 BALB/c inbred mice of each sex was trained following the clicker training protocol. An untrained but gently handled cohort of 12 BALB/c inbred mice of each sex served as control group. A Morris Water Maze Test was performed with all mice after the third week of training. The vocalization linked to the handling of the mice was recorded. Untrained mice squeaked significantly more often than trained mice. Untrained mice displayed vocalization 1.167 &#177; 0.7 times and untrained mice 4.071 &#177; 0.83 times. An unpaired t-test with Welch&#39;s correction was performed. The results of the data were expressed as the mean &#177; S.E.M. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" src="/files/ftp_upload/55415/55415fig13.jpg" /> 
Figure 13. Further Applications. A mouse is following a target stick to cross a bridge from one cage to another. <a href="http://cloudflare.jove.com/files/ftp_upload/55415/55415fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Behavior

12.8K Views.  Johannes Gutenberg-Universität Mainz. 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. 

Video: Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

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 ...

A Method for Investigating Age-related Differences in the Functional Connectivity of Cognitive Control Networks Associated with Dimensional Change Card Sort Performance

The ability to adjust behavior to sudden changes in the environment develops gradually in childhood and adolescence. For example, in the Dimensional Change Card Sort task, participants switch from sorting cards one way, such as shape, to sorting them a different way, such as color. Adjusting behavior in this way exacts a small performance cost, or switch cost, such that responses are typically slower and more error-prone on switch trials in which the sorting rule changes as compared to repeat trials in which the sorting rule remains the same. The ability to flexibly adjust behavior is often said to develop gradually, in part because behavioral costs such as switch costs typically decrease with increasing age. Why aspects of higher-order cognition, such as behavioral flexibility, develop so gradually remains an open question. One hypothesis is that these changes occur in association with functional changes in broad-scale cognitive control networks. On this view, complex mental operations, such as switching, involve rapid interactions between several distributed brain regions, including those that update and maintain task rules, re-orient attention, and select behaviors. With development, functional connections between these regions strengthen, leading to faster and more efficient switching operations. The current video describes a method of testing this hypothesis through the collection and multivariate analysis of fMRI data from participants of different ages.

This video presents a method of examining age-related changes in functional connectivity of cognitive control networks engaged by targeted tasks/processes. The technique is based on multi-variate analysis of fMRI data.

The ability to adjust behavior to sudden changes in the environment develops gradually in childhood and adolescence. For example, in the Dimensional ...

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 ...

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.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer&#8217;s disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM&#8211;8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely ...