6 min read

The Barnes Maze is a widely recognized and extensively used experimental procedure offers a systematic approach to assess cognitive abilities in rodents.

We utilize this assay at our CRO for efficacy assessments of pharmaceutical drugs and devices for the following therapeutic areas:

  • Alzheimer’s Disease
  • Traumatic brain Injury
  • Neurodevelopmental Disorders

Purpose and Significance of the Barnes Maze Protocol

The Barnes Maze Protocol serves as an invaluable tool for researchers in the field of neuroscience. Its primary purpose is to evaluate the spatial learning and memory capabilities of animals, particularly rodents. By observing their behavior in a controlled environment, researchers can gain valuable insights into their cognitive abilities, such as navigation, memory retention, and problem-solving skills. As a result, the assay is commonly used to assess neurodegenerative and neurodevelopmental disorders that impair cognition. In drug discovery, these assays provide efficacy evaluations of pharmaceuticals such as cholinesterase inhibitors or memory-improving drugs.

Theoretical Background and Principles of the Barnes Maze Protocol

The Barnes Maze Protocol was developed by Carol Barnes in in 1979 and is built upon the principles of spatial reference memory and the Morris water maze, which is another widely used tool for studying spatial learning and memory in animals. It differs from the Morris Water Maze because it is a “dry” apparatus while water mazes can challenge animals with the stress of swimming. The Barnes Maze consists of a circular platform with 40 equally spaced holes along the periphery, with only one or few leading to an escape tunnel or target hole.

The underlying theory behind the Barnes Maze Protocol is that animals naturally seek a safe and enclosed environment. By introducing a mild stressor, such as bright lights or an open space, researchers can observe the animal’s instinct to find the escape tunnel. This stress-free and non-invasive testing method ensures that the animal’s behavior is not compromised, allowing for accurate and consistent results.

Components and Setup of the Barnes Maze

The Barnes Maze consists of several key components that facilitate the assessment of spatial learning and memory in animals.

The Barnes Maze Platform: Our Barnes Maze is a circular platform with 40 equally spaced holes, made of acrylic and placed on adjustable legs at least 20-24  inches (60 centimeters) above the ground.  The platform can be modified to include one or multiple escape tunnels to provide multiple escape routes for the subject to choose. The holes that lead to an escape tunnel serves as the target hole.

To ensure accurate results, the Barnes Maze is designed to minimize external cues that may influence the animal’s behavior. Therefore, our apparatus is of uniform surface texture, and it is kept in our behavioral suite with consistent lighting conditions and soundproofing to reduce distractions. Additionally, we utilize visual cues that are strategically placed to aid the animal in locating the target hole.

Procedure and Steps of the Barnes Maze Protocol

The Barnes Maze Protocol follows a systematic approach to evaluate spatial learning and memory in animals. This procedure involves habituation, acquisition, and probe trials that are carried out over the course of 10-14 days depending on the capabilities of the test subjects:

  • Habituation phase: The animal is introduced to the Barnes Maze and allowed to explore the platform without any time constraints. This step familiarizes the animal with the maze and reduces any potential neophobic or anxiety-related behaviors.
  • Acquisition phase: The animal is trained to locate the target hole. The animal is placed in the maze and allowed to navigate towards the escape tunnel. To encourage learning, researchers may use positive reinforcement, such as food rewards, to motivate the animal to find the target hole. Once the animal consistently locates the target hole during the acquisition phase, probe trials can be conducted.
  • Probe Trials: These trials involve removing the target hole and observing the animal’s behavior to assess memory retention. The time spent searching for the target hole and the number of errors made are recorded and analyzed to evaluate the animal’s spatial memory.

The procedure steps for the Barnes Maze Protocol are listed below along with the approximate duration of each step:

  1. Habituation (2-3 days): Allow the animals to acclimate to the testing room and handling procedures without exposure to the maze. This helps reduce stress and anxiety.
  2. Pre-training (1-2 days): Familiarize the animals with the maze and the escape box. Initially, place the animal in the escape box and then gradually introduce the maze. This step helps them understand the concept of the maze and the escape route.
  3. Training (3-5 days): Conduct daily training sessions, during which the animal is placed in the center of the maze and allowed to explore. The escape box is placed under one of the holes. The animal must learn to find the escape box based on spatial cues.
  4. Probe trials (1-2 days): After training, conduct probe trials to assess memory retention. In these trials, the escape box is removed, and the animal’s ability to locate the target hole is evaluated. This step helps determine if the animal has learned the spatial layout of the maze.
  5. Reversal training (3-5 days): In this phase, the location of the escape box is changed to the opposite side of the maze. This forces the animal to adapt and learn a new escape route based on the updated spatial cues.
  6. Retention Test (1 day): After the reversal training, conduct a retention test to assess the animal’s ability to retain the new spatial information.
  7. Data analysis: Analyze the data collected during the training and probe trials to measure the animal’s performance, such as the latency to find the escape box and the number of errors made.

The duration of each step may vary depending on the specific experimental design, the animal species, and individual learning capabilities.

Data Collection and Analysis in the Barnes Maze Protocol

Accurate data collection and analysis are essential for drawing meaningful conclusions from the Barnes Maze Protocol. During the experiment, various parameters are video recorded and when the study is conducted with Anilocus, we will make all data is available for your review including video recorded sessions. In addition to the video recordings, we include the time taken to reach the target hole, the number of errors made, and the total distance traveled.

To assess spatial memory, researchers analyze these parameters across different trials and compare them to baseline measurements. Statistical methods, such as analysis of variance (ANOVA) or t-tests, may be employed to determine significant differences between groups or treatment conditions.

Advantages and Limitations of the Barnes Maze Protocol

The Barnes Maze Protocol offers several advantages that make it a preferred choice for studying spatial learning and memory in animals. Firstly, it provides a stress-free and non-invasive testing environment, ensuring that the animal’s behavior remains unaffected. Additionally, the maze design allows for easy customization and adaptation to suit different research needs.

However, like any experimental protocol, the Barnes Maze has its limitations. One limitation is that it primarily focuses on spatial memory and may not capture other cognitive domains. Furthermore, the learning process may be influenced by external factors, such as olfactory cues or noise, which may introduce potential confounding variables.

Applications and Research Areas Where the Barnes Maze Protocol Is Used

The Barnes Maze Protocol finds applications in various research areas, particularly in neuroscience and the study of cognitive function. It is commonly used to investigate neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, and traumatic brain injuries. By evaluating spatial learning and memory impairments in animal models, researchers can gain insights into the underlying mechanisms and potential therapeutic interventions.

Additionally, the Barnes Maze Protocol is utilized in the evaluation of pharmacological treatments and interventions aimed at improving cognitive function. The ability to accurately measure spatial learning and memory in animals allows researchers to assess the efficacy of new drugs or therapies and guide the development of potential treatments.

Alternative Protocols for Studying Spatial Learning and Memory

While the Barnes Maze Protocol is widely used, alternative protocols exist for studying spatial learning and memory in animals. One such protocol is the Morris Water Maze, which involves placing the animal in a pool of water and requiring it to find a hidden platform. This protocol emphasizes the use of spatial cues and allocentric navigation, providing a different perspective on spatial cognition. You can review the Morris Water Maze apparatus and our comprehensive protocol is available for free download.

Other alternatives include the radial arm maze and the T-maze, each with their own advantages and limitations. The choice of protocol depends on the specific research question, the animal model being used, and the desired outcome measures.


  1. Pitts M. W. (2018). Barnes Maze Procedure for Spatial Learning and Memory in Mice. Bio-protocol, 8(5), e2744. doi:10.21769/bioprotoc.2744.
  2. Van Den Herrewegen, Y., Denewet, L., Buckinx, A. et al. The Barnes Maze Task Reveals Specific Impairment of Spatial Learning Strategy in the Intrahippocampal Kainic Acid Model for Temporal Lobe Epilepsy. Neurochem Res (2019) 44: 600. https://doi.org/10.1007/s11064-018-2610-z.
  3. Gawel, K., Gibula, E., Marszalek-Grabska, M., Filarowska, J., & Kotlinska, J. H. (2019). Assessment of spatial learning and memory in the Barnes maze task in rodents-methodological consideration. Naunyn-Schmiedeberg’s archives of pharmacology, 392(1), 1–18. doi:10.1007/s00210-018-1589-y.
  4. Gee CC, Steffen R, Kievit FM. An updated Barnes maze protocol for assessing the outcome of controlled cortical impact mouse models of traumatic brain injury. J Neurosci Methods. 2023;392:109866. doi:10.1016/j.jneumeth.2023.109866.
  5. Attar A, Liu T, Chan WT, et al. A shortened Barnes maze protocol reveals memory deficits at 4-months of age in the triple-transgenic mouse model of Alzheimer’s disease. PLoS One. 2013;8(11):e80355. Published 2013 Nov 13. doi:10.1371/journal.pone.0080355.