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><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&amp;nbsp;</td><td>Lab &amp;amp; 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)&amp;nbsp;</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&#039;t matter, preferable organic quality</td></tr><tr><td>Forceps</td><td>FineScienceTools</td><td>&lt;em&gt;e.g.&lt;/em&gt; 11150-10</td><td>Or any other tool to fixate chocolate</td></tr><tr><td>Prism Version 6.0 for Windows&amp;nbsp;</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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

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 ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Neuroscience

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 ...

Evaluating Skilled Prehension in Mice Using an Auto-Trainer

We describe a method to introduce na&#239;ve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that permits the mouse to reach out of its cage and retrieve food pellets. Minimal food restriction is employed to encourage the mice to perform the food retrieval from the slot. As the mice begin to associate coming to the slot for food, the pellets are manually pulled away to stimulate extension and pronation of their paw to grasp and retrieve the pellet through the frontal slot. When the mice begin to reach for the pellets as they arrive at the slot, the behavioral assay can be performed by measuring the rate at which they successfully grasp and retrieve the desired pellet. They are then introduced to an auto-trainer that automates both the process of providing food pellets for the mouse to grasp, and the recording of successful and failed reaching and grasping attempts. This allows for the collection of reaching data for multiple mice with minimal effort, to be used in experimental analysis as appropriate.

Method to assess the impact of training on motor skills is a useful tool. Unfortunately, most behavioral assessments can be labor intensive and/or expensive.We describe here a robotic method of assessing prehension (reach-to-grasp) skill in mice.

We describe a method to introduce na&#239;ve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that ...

Positive Reinforcement-Based Training for Routine Rabbit Husbandry Procedures

Training Laboratory Rabbits to Refine Routine Husbandry Procedures

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving both animal welfare and scientific quality. Clicker training was utilized to develop training protocols for rabbits to refine stressful routine husbandry procedures usually associated with lifting (i.e., being picked up from the floor)/restraining (i.e., being held in the arms of a human) them. Thirteen female New Zealand White rabbits were trained over three weeks. All rabbits learned the predefined goal behaviors: they followed the target stick, jumped onto the weighing scale, entered a transport box, and reared while placing their front paws onto the trainer&#39;s hand. In addition, ten animals jumped from the floor onto the sitting trainer&#39;s lap and allowed the trainer to lift their paws off the surface while sitting on the trainer&#39;s lap. For some individuals, the protocols had to be adapted by additional interim steps. At the end of the training, the rabbits reliably showed the expected goal behaviors, even after short and long training breaks. With few exceptions, a familiar person other than the trainer could elicit the goal behaviors from the rabbits (generalization), though further sessions were required for generalization. In the voluntary approach test, the rabbits preferred interacting with the trainer in the 1st trial but spent as much time with an unfamiliar person as with the trainer in the 2nd trial. The behavioral observations suggested that picking the rabbits up with the transport box, as described in the protocol, instead of restraining them with the scruff of their neck and lifting them on the arm, was less aversive. All in all, the training protocols were feasible and can serve as a refinement strategy in laboratory animal facilities. In the interest of animal welfare, the training protocols should be applied wherever possible.

Author Spotlight: Training of Laboratory Animals for Gentle and Stress-Free Handling

Training protocols involving positive reinforcement for routine husbandry procedures in rabbits were developed and proved to be successful, as the rabbits reliably showed the trained behaviors at the weekly general examination. Picking the rabbits up with the transport box was less aversive than the conventional handling technique.

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving ...

Behavioral Training Procedures for Head-fixed Virtual Reality in Mice

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays to be performed in head-fixed mice. This enables precise behavioral recordings while incorporating various neurophysiological techniques that require head-fixation to minimize movement-related signal noise during neural recordings. However, despite the growing use of VR, there is little published data on the detailed methodology of how to implement it. In this study, a training protocol is developed whereby male and female C57B16/J mice are trained to run down a virtual linear corridor, the length of which is increased from 1-3 m over multiple training sessions. Building upon this foundation, this study investigated the feasibility of mice performing complex behaviors within VR using a Y-maze paradigm. The task required navigating to the arm with black walls from the choice point in the Y-maze. After reaching a criterion of two consecutive days equal to or greater than 70% correct, the mice progressed to increasingly difficult sensory discrimination. The findings provide important details on the methodologies useful for the successful training of mice in VR and demonstrate that mice exhibit learning capabilities in navigating the Y-maze. The methodology presented not only offers insights into training duration in VR-based assays but also underscores the potential for probing intricate behaviors in mice, opening avenues for more comprehensive neuroscience investigations.

The article describes the experimental procedures for the commonly used linear track virtual reality (VR) paradigm in mice as well as determining the feasibility of running complex VR tasks by testing a Y-shaped signal discrimination task.

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays ...

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