The 4-box maze is used for analyzing cognitive mapping, spatial learning, and memory recall in rodents. The apparatus tests subjects’ ability to memorize the path based on the environmental cues and use the spatial information for shortcutting and detouring. Spatial learning is necessary for effective foraging, survival instincts, and path integration (Singer, Abroms, & Zentall, 2006). The 4-box maze is designed to investigate the learning behavior of rodents and their capability to adapt to changes in paths and dimensions in a specific location.

Tolman (1948) suggested that rats develop a field map of the environment in their brains, called a cognitive map. This internal representation of surroundings could control their flexible spatial responses like taking a shortcut or alternate routes. However, the spatial recognition of the environment is dependent upon the presence of intrinsic and extrinsic environmental cues (Roberts, Cruz, & Tremblay, 2007). The 4 -boxes apparatus provide different environmental cues for testing spatial inference capacity in rodents. It can also be used for evaluating a wide range of cognitive behaviors and memory dysfunction in different spatial settings.

The 4-box maze consists of four open-top square boxes. Each box is linked with the other through transparent alleyways. The configuration of the boxes and the alleyways can be changed according to the experimental requirements. Other apparatus used to explore cognitive behavior and spatial learning in rodents include the Circular Central Platform T Maze, Hebb Williams, T-maze, Y maze, and the Radial Arm Maze.

The apparatus consists of four open-topped square boxes (A, B, C, D) linked with each other through transparent, enclosed alleyways. Box A serves as the start box, and box D acts as a goal box. Each box measures 510 mm x 510 mm x 510 mm in dimensions. The wall thickness of each box is 10 mm. Each box is provided with 4 doors allowing the passage to the alleyways. The alleyways are 320 mm high, 260 mm deep and 800 mm long. The walls of the alleyways are transparent with opaque floor and roof. The goal box contains a bowl to hold a food reward.

Clean the apparatus between trials to remove any olfactory stimuli and leftover food rewards. An external tracking and recording systems such as Noldus Ethovision XT can be used with the 4 box maze to record the subject behavior.

Pretraining

Connect the boxes such that the subject has to navigate from start box (A) to goal box (D) via intermediate boxes (B and C) and alleyways. Fill the bowl in the goal box with the food reward. Place the subject in the start box and observe its movements till it retrieves the reward. Remove the subject from the goal box and put it back in the start box for another run. Perform 15 runs of 10 minutes each per day for each subject. Overall conduct 110 runs per subject in 18 days.

Shortcut probe

Connect the goal box (D) and two novel boxes (E and F) via the alleyways to the start box (A) such that they form a T-shaped maze. Place the subject in the start box and observe its movement. Conduct five runs. Refill the food reward bowl present in the goal box before every run.

Start-box probe

Rotate the layout used in the shortcut probe by 90-degree angle. Place the subject in the start-box and record its movements. Do not provide any food reward to the subjects during the start-box probe.

Evaluation of the capacity of spatial inference and cognitive mapping in rats

Grieves and Dudchenko (2013) analyzed the capacity of rats to form a spatial inference using different intrinsic and extrinsic environmental cues in a 4-box maze. Lister-Hooded rats underwent a training session before being evaluated in the probe trials. The subjects were first trained using the standard configuration of boxes (start box A to box B to box C to goal box D). Results showed a significant decrease in time while moving from the start box to the goal box; indicating rapid learning behavior in subjects. However, when evaluated in the probe session using two novel routes and shortcut route, only 25% of subjects attempted the shortcut route while 33% of subjects attempted the novel route. Similarly, when assessed using a 3-platform set-up following the same training and probe protocols, rats failed to select the shortcut route more than the alternative routes. In order to assess if knowledge of the shortcut route would influence performance, naive subjects were allowed to explore the shortcut route before the probe trial. In comparison to the naïve subjects that did not explore the shortcut route, the exposed group opted for the shortcut significantly faster and more frequently. Thus, the results indicated that pre-exposure to a shortcut is a pre-requisite for attempting it.

Following parameters can be recorded by using 4-box maze

• Time taken in reaching the goal box
• Number of subjects choosing each doorway
• Frequency of visits in each box
• Number of subjects reaching the goal box
• Number of subjects choosing the shortcut
• Number of subjects choosing alternate routes
• Percentage of times each box is chosen
• Percentage of times each alleyway is chosen

Strengths

The 4-box apparatus is a useful tool to observe spatial learning and memory traits in rodents.  It is easy to operate and produces reproducible results. The apparatus can be configured easily according to the task demands. It allows the introduction of shortcuts and novel routes for challenging the subjects. Use of positive or negative reinforcements in the 4-box apparatus can help in evaluating more complex behavioral responses in rodents.

Limitations

Subject training is a critical factor while using the 4-box apparatus for the assessment of spatial learning behavior in rodents. Presence of appropriate environmental cues also directly impact spatial recognition in subjects during the experiment. Any changes in the environment may mislead subjects navigating ability. Mishandling or overtraining may induce fear or anxiety in subjects that can also influence task performance. Presence of unintentional auditory, olfactory or visual stimuli, can also affect the subject’s task performances. Factors such as age, gender, and strain, among others, may also contribute to the subject behavior.

• The 4-box maze is used to analyze the capacity of spatial orientation and memory traits in rodents.
• The apparatus consists of four boxes and alleyways that can be configured as required.
• The apparatus helps in assessing the ability of rodents to apprehend changes in the environment and adapt to novel situations.
• The use of extrinsic and intrinsic environmental cues, as well as reinforcements, can add further complexity to the 4-box maze task.

1. Grieves, R. M., & Dudchenko, P. A. (2013). Cognitive maps and spatial inference in animals: Rats fail to take a novel shortcut, but can take a previously experienced one. Learning and Motivation, 44(2), 81-92. doi:10.1016/j.lmot.2012.08.001
2. Roberts, W. A., Cruz, C., & Tremblay, J. (2007). Rats Take Correct Novel Routes and Shortcuts in an Enclosed Maze. Journal of Experimental Psychology: Animal Behavior Processes, 33(2), 79-91. doi:10.1037/0097-7403.33.2.79
3. Singer, R. A., Abroms, B. D., & Zentall, T. R. (2006). Formation of a Simple Cognitive Map by Rats. International Journal of Comparative Psychology, 19(4), 417-425.
4. Tolman, E. C. (1948). Cognitive maps in rats and men. Psychological Review, 55(4), 189–208.