Documentation

Introduction

The Environmental Enrichment (EE) cage is used to house subjects and contains a variety of enrichment objects. Environmental enrichment helps provide rodents with enhanced sensory, motor, and cognitive stimulation as compared to standard housing conditions (Bailoo et al., 2018). Studies have used EE cages for different experimental protocols involving age-related cognition, anxiety, stress, brain plasticity, as well as for operant conditioning (Morano, Hoskins O, Smith, & Herman, 2019; He, Tsipis, LaManna, & Xu, 2017). The EE cage provides adequate environmental enrichment and stimulation and consists of three floors, two balconies, and four flights. The task usually involves dividing the subjects into control and environmental enrichment groups and subjecting them to different behavioral tasks to observe if the EE cage produces any behavioral differences. Furthermore, environmental enrichment helps increase novelty-seeking behaviors and object exploration, therefore, the enrichment objects in the cage can easily be replaced or moved around so that the subjects do not lose interest in them.

Environmental enrichment has also shown to reduce anxiety behaviors and abnormal repetitive behaviors in laboratory mice. Therefore, the EE cage can also be used to improve animal welfare in standard laboratory conditions. Since the EE cage is spacious enough to occupy 8 to 10 subjects easily, it can also be used to provide rodents with social stimulation. 

Other apparatus used to study rodent behaviors include the Visual Burrow System, the Dig Task, the Linear Maze, and the Social Reward Chamber. 

Apparatus and Equipment

The EE cage measures 79 x 52 x 140 cm and consists of three floors, two balconies, and four flights. The cage is filled with several safe and non-toxic enrichment objects such as plastic tunnels, wooden sticks, toys, balls, paper nesting material, and a running wheel. Feeding boxes and water bottles are also present. 

Training Protocol

Clean the apparatus regularly to remove any waste droppings. Change or move enrichment objects twice per week. Clean the apparatuses used for behavioral tests after every trial to avoid the influence of olfactory cues from previous trials. A tracking and recording system such as the Noldus Ethovision XT can be used to assist with observations.

The following is a sample protocol for the assessment of cognitive performances in aging animals using the EE cage:

Habituation and Pre-training 

Allow the subject to explore and habituate to the test room for 5 minutes a day for five consecutive days. 

Open Field Test

Release a subject into a corner of the open field arena. Allow the subject to explore the arena for 10 minutes. 

Step-through Passive Avoidance Test 

Release the subject into the Passive Avoidance Test apparatus. Allow the subject to explore the apparatus for 10 minutes freely. On the next day, restrict the subject to the dark compartment of the apparatus for 3 minutes. On the third day, release the subject into the illuminated compartment of the apparatus. Open the sliding door after 10 seconds. After the subject enters the dark compartment, deliver an electric foot shock. On the fourth day, release the subjects into the illuminated compartment. Open the sliding door after 10 seconds. Allow the subjects to enter the dark compartment within 300 seconds. 

EE Cage Test 

After the subjects perform the open field test and the step-through passive avoidance screening tests divide the subjects to either EE cage or standard housing groups. Place 8 to 10 subjects in the EE cage from the EE cage group. Allow the subjects to continue to live in standard housing with minimal enrichment for the other group. In addition, divide naïve subjects into standard housing or EE cage groups without performing the screening tests. After 12 weeks, allow the subjects to repeat testing of the behavioral tasks or perform testing for the first time (naïve group). 

Data Analysis

The following parameters can be observed using the EE cage:

• Change of exploratory behaviors after living in an EE cage versus standard housing 
• Memory retention abilities after living in an EE cage versus standard housing 

Literature Review

Investigation of the effect of environmental enrichment given to late adult rats 

Balietti, Pugliese, Fabbietti, Di Rosa, and Conti (2019) investigated the effect of the EE cage on the behavior of late adult rats. Male Sprague Dawley rats (17 months) were used in the study. The subjects were divided into control and experimental groups. The subjects from the experimental group were pre-screened on the Open Field Test and the Step-through Passive Avoidance Test to obtain baseline results. On the open field test, the subjects were required to explore the open field arena for 10 minutes to observe their locomotor activity and anxiety behaviors. The step-through passive avoidance test involved testing the subject on the passive avoidance test apparatus to classify their aversive memory as impaired or non-impaired. The subjects were then divided into two groups and were either placed in the EE cage (EE group) or continued to standard housing (SH group) for 12 weeks. Eight to ten subjects were placed together in the EE cage, while two subjects were placed in standard housing that had minimal enrichment. After 12 weeks, the subjects were retested on the two behavioral tasks to observe their post-housing behavior.  Naïve subjects who had not performed pre-screening tests were used as the control group. The subjects were divided into standard housing and EE cage groups and were required to perform the behavioral tasks after 12 weeks. Results from the open field test in the experimental group indicated a significant decrease in the total distance moved between baseline and post-housing conditions. The experimental groups were further subdivided into low performing and high performing subjects. Results indicated that the low performers from the EE group had a significant increase in exploration change at post housing conditions. In contrast, the high performers from the EE group had a significant decrease in exploration change in post housing conditions. Subjects from the SH group had a significant decrease in post housing conditions in both low performing and high performing groups. Observations from the naïve group included a significantly higher total distance moved in the EE group as compared to the SH group. Results from the step-through passive avoidance test indicated a significant decrease in impaired animals in the SH experimental group in post-housing conditions as compared to baseline conditions. In contrast, the number of impaired animals did not change in the EE experimental group. Impaired subjects at baseline from both experimental groups converted to not impaired at post housing conditions. Non-impaired subjects from the SH group remained unchanged at post housing. However, non-impaired subjects from the EE group significantly converted to impaired subjects post housing. Results from the naïve group indicated a significantly higher percentage of impaired EE subjects as compared to SH subjects. Overall results indicate that environmental enrichment had a significant effect on aging rats with different levels of cognitive performance depending on the type of stimulus. 

Investigation of the effect of environmental enrichment removal on the behaviors of female rats

Morano, Hoskins, Smith, and Herman (2019) tested the effect of environmental enrichment removal on the behaviors of eighty female Sprague-Dawley rats. The subjects were divided into two cohorts. Cohort 1 had five days of habituation to their housing condition, while cohort 2 had seven days of habituation. Each cohort was further equally divided into four treatment groups depending on their housing manipulations: single-housed animals, paired-house control animals, continuously enriched animals (EE), and enrichment removal animals (ER). Housing manipulations began at the end of the five days and seven days habituation for cohort 1 and 2. The rats were subjected to housing manipulations during their 12-hour dark (active) cycle and placed in standard housing during their 12-hour inactive cycle. Housing manipulations continued until the end of the experiment. However, subjects from the enrichment removed groups were removed after four weeks and were placed in single-housing conditions before behavioral tests began. Acute restraint tests were performed on the subjects seven days post-removal. The subjects were restrained in plexiglass tubes, and their behaviors were recorded. Their blood was then collected to determine their hypothalamic-pituitary-adrenocortical (HPA) axis. Results indicated an HPA axis dysregulation. Behavioral tests included sucrose preference test (SPT) and forced swim test (FST). In the SPT, a water bottle with 1% sucrose and a standard water bottle was placed in the home cage for three days after habituation. Results indicated that the ER animals had a decrease in sucrose preference as compared to single-housing and paired-housing animals. The EE animals had a decrease in total sucrose intake as well as a decrease in water intake as compared to the ER and paired-housing animals. The FST was conducted 14 days post-removal to test the subjects’ passive coping behavior. During the test, the subjects were placed in a cylindrical plexiglass tank that contained water. The subjects’ swimming, diving, immobility, and climbing behaviors were monitored and assessed. Results indicated that the ER animals spent more time immobile as compared to the single-housing and paired-housing groups. Overall results indicate that enrichment removal increases passive coping behavior and anhedonia like behavior.  

Investigation of the effect of environmental enrichment on brain plasticity and cognitive function in mice

He, Tsipis, LaManna, and Xu (2017) investigated the effect of environmental enrichment on brain plasticity and cognitive function in 3-month-old and 18-month-old mice. The mice were randomly placed in either environmental enriched or non-enriched cages for three weeks. The environmental enriched group was housed in groups of 3 in enriched cages while the non-enriched group was housed in standard cages. After the three weeks, the capillary density was determined in the 3-month-old mice from both enriched and non-enriched groups. Results indicated that the subjects from the enriched group exhibited a 29% increase in capillary density as compared to the non-enriched group. In the 18-month-old mice, their cognitive function was assessed before and after four weeks of enrichment using a Y-maze test and novel object recognition test. A Y-maze was used to test the subject’s innate preferences for spontaneous alterations. The novel object recognition test was used to test the subject’s tendency to approach a novel object instead of a familiar one. Results indicated that the enriched group had a significantly higher alteration rate after four weeks of enrichment as compared to the non-enriched group. However, in the novel object recognition test, no significant differences were seen in the novel object exploration rate in both groups before and after the four weeks of enrichment. 

Investigation of the use of environmental enrichment to induce abstinence in heroin-seeking rats 

Peck, Galaj, Eshak, and Ranaldi (2015) investigated the use of environmental enrichment as a treatment strategy in heroin-seeking rats using the abstinence conflict model. The abstinence conflict model uses aversive stimuli for subjects seeking drugs to induce abstinence. Male Long Evan rats were used in the study. The subjects were tested on an operant conditioning chamber that was equipped with two levers (one active and one inactive). Two-thirds of the floor of the chamber served as the electric barrier that administered constant-current as aversive stimuli, while one-third of the floor served as the no shock zone where no current was administered.  The experiment was divided into drug self-administration and electric barrier application phases. During the drug administration phase, the subjects were placed in the operant conditioning chambers for 3-hour sessions daily. When the subjects pressed on the active lever, it activated the pump that delivered an injection of 0.05 mg/kg of heroin in a 0.125 ml volume of saline over 4.5 seconds. A press on the inactive lever issued no drug administration.  It was observed that the subjects pressed the active lever more than the inactive one. After the subjects achieved the learning criteria, they were randomly assigned to either environmental enrichment (EE) or no environmental enrichment (NEE) groups for 48 hours. The subjects then performed the electric barrier application phase. During the first session, the current was set to 0.25 mA and was increased by an increment of 0.04 mA after each session if the subject pressed on the active lever. However, the current did not exceed 1.00 mA. Results indicated that the EE subjects pressed the active lever less than the NEE subjects in the first session. The final shock intensities, as well as the total number of sessions that lead to abstinence, was also greater in the NEE group. 

Strengths and Limitations

Strengths 

The EE cage can be used in various experimental protocols that test the effect of environmental enrichment on learning, anxiety, or age-related cognitive behaviors in rodents. It contains numerous forms of enrichment such as plastic tunnels, nesting materials, toys, and a running wheel and also has multiple floors, balconies, and flights. The enrichment objects can easily be replaced with new objects or moved around. The EE cage is spacious and can easily occupy 8 to 10 subjects to provide social stimulation. Apart from being used in various experimental protocols, it can also be used to house rodents to improve their welfare in standard laboratory conditions. 

Limitations 

Enrichment objects have to be routinely changed for the subjects not to get bored with them. The EE cage may provide different stimulation depending on factors such as age, gender, and strain of the subjects.

Summary

• The EE cage is used to house subjects in an enriched environment.
• It is a large cage that contains three floors, two balconies, and four flights and can occupy 8 to 10 subjects. 
• It contains numerous forms of enrichment, such as plastic tunnels, wooden sticks, toys, balls, paper nesting material, and a running wheel.
• The EE cage can be applied to different experimental protocols that test the effect of environmental enrichment on learning, anxiety, age-related behaviors, cognition, operant conditioning, and brain plasticity. 
• The EE cage can also be used to provide rodents with welfare in standard laboratory conditions. 

References

1. Balietti, M., Pugliese, A., Fabbietti, P., Di Rosa, M., & Conti, F. (2019). Aged rats with different performances at environmental enrichment onset display different modulation of habituation and aversive memory. Neurobiology of Learning and Memory, 161, 83–91. doi:10.1016/j.nlm.2019.04.001 
2. Morano, R., Hoskins, O., Smith, B. L., & Herman, J. P. (2019). Loss of environmental enrichment elicits behavioral and physiological dysregulation in female rats. Frontiers in behavioral neuroscience, 12, 287. doi:10.3389/fnbeh.2018.00287
3. Bailoo, J. D., Murphy, E., Boada-Saña, M., Varholick, J. A., Hintze, S., Baussière, C., … Würbel, H. (2018). Effects of Cage Enrichment on Behavior, Welfare and Outcome Variability in Female Mice. Frontiers in Behavioral Neuroscience, 12. doi:10.3389/fnbeh.2018.00232
4. He, C., Tsipis, C. P., LaManna, J. C., & Xu, K. (2017). Environmental Enrichment Induces Increased Cerebral Capillary Density and Improved Cognitive Function in Mice. Oxygen Transport to Tissue XXXIX, 175–181. doi:10.1007/978-3-319-55231-6_24
5. Peck, J. A., Galaj, E., Eshak, S., Newman, K. L., & Ranaldi, R. (2015). Environmental enrichment induces early heroin abstinence in an animal conflict model. Pharmacology Biochemistry and Behavior, 138, 20–25. doi:10.1016/j.pbb.2015.09.009