Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Summary

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Abstract

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

Introduction

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 "establishments shall set up habituation and training programs suitable for the animals, the procedures and length of the project" (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 animals^3,4,5,6. 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 behavior^7,8. It has been shown that handling programs can efficiently decrease the anxiety toward the investigator in laboratory rodents^9,10,11. Handling programs can therefore improve the animals' 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 "click" sound of a clicker, and has been proven to strengthen a specific behavior¹².

More specifically, the click sound serves as a "time bridge" 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 primates^13,15,16. As mice learn rather quickly when challenged with operant conditioning paradigms, the introduction of a second reinforcer should not overstrain their cognitive abilities^5,17,18,19.

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 environment^20,21. Several studies with different species prove the positive impact of cognitive enrichment on the welfare of captive animals^22,23,24. Enhancing the ability of the animals to successfully cope with environmental challenges contributes to their well-being^25,26.

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' 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.

Protocol

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.

1. Insert a small Petri dish with different food rewards (pieces of 0.5 cm³) into the home-cage (Figure 1).
2. After 10 min, check for leftovers.
3. If all rewards are eaten after 10 min, shorten the time span until a preference can be estimated.
4. On days 4 and 5 (e.g., Thursday and Friday), add small amounts of the preferred ("winner") reward to the home-cage to establish an affectation of all mice in the cage to the reward.
5. 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).
6. Repeat steps 1.2 - 1.4 on 3 consecutive days (e.g., Monday to Wednesday).

2. Clicker Training

1. 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 "cupping on the open hand"²⁸.
   1. Prepare a cage with bedding material.
   2. Transfer the cage of the mouse being trained to a quiet place.
   3. 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.
   4. Remove all objects from the home-cage, which now serves as the training area (e.g., mouse houses, nest-building material, etc.).
   5. Transfer the cage companions to a prepared cage.
2. 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.
   1. Train each mouse for 5 min. Alternate between 30-s training and 15-s break periods.
   2. Let the mouse gnaw on the reward no longer than a second. Then, take the stick (with the reward) out of the cage.
   3. 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.
   4. 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.
3. Training session Day 1: "Linking the secondary reinforcer with the food reward"
   1. 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.
   2. 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.
   3. Let the mouse feed on the reward while sitting in the tunnel.
   4. Click continuously for 15 s while the mouse is sitting in the tunnel.
   5. As soon as the mouse leaves the tunnel, go back to step 3.2.
4. Training session Days 2 - 5: "Running through a tunnel"
   1. Set the timer to 5 min, press start, and add the mouse tunnel to the home-cage.
   2. As soon as the mouse enters the tunnel, click and present the reward at the end of the tunnel.
   3. As long as the mouse is in the tunnel, click and immediately present the reward in the same manner.
   4. Repeat this for the next 30 s, and then pause for 15 s to take the tunnel out of the cage.
   5. Add the tunnel again and click immediately when the mouse reenters the tunnel.
   6. Present the reward at the end of the tunnel.
   7. Allow the mouse to gnaw for up to 1 s.
   8. Take away the reward.
   9. 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).
5. Training session week 2: "Following a target stick"
   1. Set the timer to 5 min, press start, and place the globe at the end of the target stick in the cage.
   2. As soon as the mouse shows interest in the globe, click and present the reward next to the globe (Figures 4 and 5).
   3. Reward the mouse only after it touches the globe with its nose.
   4. When the mouse has linked the performed action to the reward, change the position of the globe during the training session (Figure 6).
   5. Wait for the mouse to meet this last challenge.
   6. Place the globe in the cage.
   7. Shortly before the mouse touches the globe, slowly and carefully change the position of the globe by 1 cm.
   8. Click and reward if the mouse has crossed the distance and touches the globe.
   9. Repeat this procedure until the mouse follows reliably.
   10. Slowly extend the distance the mouse has to cross.
6. Training session week 3: "Following the target stick to the experimenter's hand"
   1. Place one hand in the training cage while holding the clicker/target stick combination and the reward with the other hand.
   2. Set the timer to 5 min, press start, and place the globe at the end of the target stick next to the hand.
   3. As soon as the mouse shows interest in the globe, click and present the reward next to the globe.
   4. As soon as the mouse has met this challenge, move the globe several steps closer to the hand.
   5. 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).

Results

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

The actual trainin...

Discussion

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

One critica...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lid for open housing	Tecniplast	GM500LID117
SealSAfe Plus top for open housing	Tecniplast	GM500400SU
Type II long, filter top cages	Tecniplast	GM500PFSPC
Aspen bedding material	Lab & Vet Service GmbH	H0234-300	Environmental enrichment
Red polycarbonate Mouse House	Tecniplast	ACRE011	Environmental enrichment
Tissue papers	Tork, SCA Hygiene Products GmbH	290179	Environmental enrichment
Food - ssniff M-H Extrudat	ssniff	V1126-000	ad libitum
Target Stick with Clicker	Trixie	2282
PVC Tube (Tunnels)	Thyssen Krupp	RTPVCU04003005
White Chocolate/ white chocolate cream			Company doesn't matter, preferable organic quality
Forceps	FineScienceTools	e.g. 11150-10	Or any other tool to fixate chocolate
Prism Version 6.0 for Windows	GraphPad Software