Developed by Wood, Glynn, and Morton, the Rodent OX-Maze is used to assess learning and memory in rodents. It gets its name from the ‘O’ and ‘X’ symbols drawn onto black boxes placed within the maze.
The maze is essentially an open arena with grid lines that produce equal-sized squares on the arena’s floor. The black boxes placed within the arena contain holes on each block side, which are used for placing food rewards.
Rodents are frequently used as a model for examining learning and memory for diseases characterized by cognitive dysfunction, including, Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease (Morellini, 2013). Common mazes used for studying spatial learning include the Y-Maze, the Radial Arm Maze, and the Morris-Water Maze. The OX-Maze is a newer spatial and learning maze developed by Wood, Glynn, and Morton in 2011.
Compared to the Y-Maze, which only utilizes a two-choice paradigm, the OX-Maze challenges rodents by tasking them to find reward pellets hidden on each side of the rewarded block. Each of the four blocks contains a different symbol (O, X, =, and _) and the subject must utilize these symbols as spatial cues to perform the task. The position of the blocks can be moved across trials or the reward can be placed on a different symbol to challenge the subject further.
Additionally, the OX-Maze also analyzes the subject’s working and reference memory similar to the Hole Board apparatus by evaluating the number of visits and revisits the subject makes to each reward hole. Apart from assessing learning and memory, the OX-Maze can also be used as environmental enrichment for rodents.
The OX-Maze consists of a square acrylic box. It is essentially an open arena with grid lines and four white boxess placed in the squares near the center of the arena. One of four symbols (O, X, =, and _) is attached to each block. A hole is present on each block side.
Rodent location can be tracked using a video tracking software package such as Noldus EthoVision, ANY-Maze, or BehaviorCloud.
Clean the maze and the blocks with 20% ethyl alcohol between trials to minimize olfactory cues from previous trials. Ensure that the experimenters conducting the test are blind to the experimental conditions.
Habituation
Feed the subjects the food reward used during testing along with their regular diet for two days before starting trials. Reduce the subjects’ diet to about 90% of their normal amount a day before testing for food restriction.
OX-Maze Task
Place the reward in each reward hole of only one symbol, which serves as the reward symbol. Place a subject in the center of the maze and allow it to explore it. Allow the subject to find all four rewards within 10 minutes. Change the positions of the blocks clockwise every day. Test the subjects over 10 days.
The following parameters can be observed using the OX-Maze:
Investigation of the effect of environmental enrichment on rodents performance of a maze task
Rojas et al. (2015) investigated the effect of environmental enrichment on the behavioral performance of rodents that underwent neonatal hypoxia–ischemia (HI). The OX-Maze was used to examine the subjects’ behavioral performances. Forty-six rats were used in the study and were divided into four groups: a control group maintained in a standard environment (CTSE), a control group exposed to environmental enrichment (CTEE), a group submitted to HI and maintained in a standard environment (HISE), and a group submitted to HI and exposed to environmental enrichment (HIEE).
Behavioral testing consisted of evaluating the subject’s spatial learning and memory to retrieve a food reward hidden in a block on the OX-Maze. The maze contained four blocks with each block containing one of four symbols (O, X, =, and _). During testing, the subject was allowed to explore the arena and find the reward within 10 minutes. The number of correct nose pokes into the rewarded boxes, incorrect nose pokes in the other boxes, revisits to each box, the time it took to find the first reward, and the time it took to complete the trials were used to evaluate its performance.
The results from the number of correct nose pokes indicated that the HISE group had fewer correct nose pokes compared to all other groups. Similarly, analysis of the time it took the subjects to find the first reward revealed that the HISE group should have impaired performance compared to the other groups. Lastly, evaluation of the time the subjects took to complete the task revealed that the HISE group took a longer time to complete the task than all other groups. Overall results indicated that neonatal HI impaired rodents’ performance in the OX-Maze; however, environmental stimulation inversed this impairment. This is supported by the evidence of the HIEE group showing similar performance to the CT groups.
Evaluation of the effect of energy drinks and their constituents on the learning and memory of rodents
Valle et al. (2018) evaluated the effect of energy drinks and their constituents (caffeine and taurine) on the learning and memory of male Wistar rats. The rats were divided into the following groups: a control group that was given 1 mL/kg distilled water (control), and energy drink groups with different doses of energy drink (ED) 5, ED 7.5, ED 10, 3.2-mg/kg caffeine, 40-mg/kg taurine, and a combination 3.2-mg/kg caffeine + 40-mg/kg taurine (caffeine + taurine group). During testing, a food pellet was always placed in the block symbol O. The time it took for the subject to find the first reward, the number of times the subject correctly smelled the holes of each block, and the time it took to complete the trial was used to evaluate its learning and memory.
The results indicated that the groups treated with ED10, caffeine, taurine, and a combination of caffeine and taurine took a lesser amount of time to find the first reward compared with the control group. Similarly, all groups treated with caffeine, taurine, and caffeine + taurine completed the task in a lesser amount of time than the control group. Less time was spent at the block for the caffeine and taurine-treated groups than for the control group. Additionally, results from the object recognition memory test demonstrated that treatment of caffeine and taurine improved short-term memory in comparison to controls.
Investigation of the effect of environmental enrichment in rodents with cognitive dysfunction
Wood, Glynn, and Morton (2011) utilized the OX-MAZE for environment enrichment and observed the effect it had on rodents with cognitive dysfunction. Male and female R6/2 transgenic and wild-type mice were used in the study. The subjects were divided into control and environmental enrichment groups. The control group remained in their home cage throughout training, while the enrichment group was placed in the OX-Maze for training.
The OX-Maze was marked with grid lines that produced twenty-five 12 × 12 squares. Six white blocks were placed around the maze floor. The blocks had two drilled holes, one on each side of each block. Each hole was located in the middle of one of four symbols (O, X, =, II), which was drawn on the box using a black marker. One of the four symbol-denoted holes in each block contained a pellet as a reward. During testing, a subject was placed in the center of the maze and could explore the maze for ten minutes. The number of correct and incorrect nose pokes into each hole of the six blocks was recorded. After training, the subjects were tested on the Lashley III Maze and the Rotarod. The subjects returned to training and were re-tested on the apparatuses.
The results from the OX-Maze task indicated no difference in learning between R6/2 and wild-type mice of either sex. The R6/2 mice learning and memory on the maze task increased over time. The results on the OX-Maze after the first Lashley maze and Rotarod testing session revealed no differences between genotypes. However, reversal training revealed a significant effect of genotype in which both male and female R6/2 mice displayed impaired learning when the reward position was shifted.
The OX-Maze can assess the spatial learning and memory, and the working and reference memory of rodents. In addition, it can also be used for environmental enrichment. The maze easily analyzes spatial learning and memory by observing how quickly the subject takes to find the food reward from each reward hole present on the reward block. Working and reference memory are also easily assessed by observing the number of correct and incorrect nose pokes into each reward hole and the number of revisits. The position of the blocks within the maze can be moved to any configuration. Since the maze is essentially an open arena, the experimenter can also utilize the maze for open arena experiments used for assessing anxiety-related behaviors by removing the blocks from the maze.
The OX-Maze is used to assess learning and memory in rodents.
Rojas, J. J., Deniz, B. F., Schuch, C. P., Carletti, J. V., Deckmann, I., Diaz, R., Matté, C., dos Santos, T. M., Wyse, A. T., Netto, C. A., & Pereira, L. O. (2015). Environmental stimulation improves performance in the ox-maze task and recovers Na+,K+-ATPase activity in the hippocampus of hypoxic-ischemic rats. Neuroscience, 291, 118–127. https://doi.org/10.1016/j.neuroscience.2015.01.017
Morellini F. (2013). Spatial memory tasks in rodents: what do they model?. Cell and tissue research, 354(1), 273–286. https://doi.org/10.1007/s00441-013-1668-9
Valle, M. T. C., Couto-Pereira, N. S., Lampert, C., Arcego, D. M., Toniazzo, A. P., Limberger, R. P., Dallegrave, E., Dalmaz, C., Arbo, M. D., & Leal, M. B. (2018). Energy drinks and their component modulate attention, memory, and antioxidant defences in rats. European journal of nutrition, 57(7), 2501–2511. https://doi.org/10.1007/s00394-017-1522-z
Wood, N. I., Glynn, D., & Morton, A. J. (2011). “Brain training” improves cognitive performance and survival in a transgenic mouse model of Huntington’s disease. Neurobiology of disease, 42(3), 427–437. https://doi.org/10.1016/j.nbd.2011.02.005
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