Discover the intricacies of spontaneous alternation and delve into the functions of the basal forebrain, hippocampus, prefrontal cortex, and septum with our Y-Maze. A classical assessment in rodents for over 80 years, pioneered by Dennis & Sollenberger in 1934.

The Y-Maze test is a well-established behavioral assay designed to assess spontaneous alternation behavior in rodents, providing insights into the functions of the basal forebrain, hippocampus, prefrontal cortex, and septum. The maze consists of three arms of equal length, forming a Y shape at an angle of 120º from one another.


  • Animals: Mice, rats, honey bees, zebrafish
  • Sample Size:  n ≥ 12+ animals
  • Lighting: Lamps
  • Video: Video camera mounted on our portable camera stand with articulating arm.
  • Cleaning: SporKlenz, 10% Bleach, or 70% Ethanol
  • Optional: Dark box for the ends of the arms is sold separately as an add-on modification of the Y-maze

Testing Conditions

  • Lights: Lamps
  • Acclimation: There is no required acclimation period prior to examination.
  • Privacy: The examiner needs to be at least 5ft (1.5m) away from the testing apparatus.
  • Visual Cues: Pictures can be placed above the apparatus in the direction of the choice arms.
  • Photoelectric beam: All mazes with arm entry can be equipped with photoelectric beams.

Y-Maze Protocol



  • Allow rodents to acclimate to the experimental room for at least 30 minutes before the start of each session.
  • Place the animal in the center of the Y-Maze and allow free exploration for a few minutes to habituate to the apparatus.

Spatial Learning Training:

  • During the training phase, block one arm (chosen randomly) to force the rodent to explore the other two arms.
  • Allow the rodent to explore the maze for a predetermined time (e.g., 5 minutes).
  • Record the sequence of arm entries.


Choice Test:

  • Remove the block, allowing access to all three arms.
  • Allow the rodent to explore freely for a set period (e.g., 5 minutes).
  • Record the sequence of arm entries.


  • If using a video tracking system, record the sessions for subsequent analysis.
  • Alternatively, manually record the rodent’s behavior, noting the sequence and duration of arm entries.
  • Calculate and analyze the following parameters:
    • Alternation Rate: (Number of alternations / Total number of arm entries) x 100.
    • Arm Entry Sequences: Analyze specific patterns of arm choices.
    • Time Spent in Each Arm: Assess duration in each arm.
    • Latency to Make a Choice: Measure time from the start to the first arm entry.
    • Total Arm Entries: Sum of entries into all arms.
    • Re-entry Errors: Count instances of re-entering the same arm consecutively.
    • Zone Analysis: Divide the maze into specific zones and analyze parameters within each zone.
    • Novel Arm Entries: Count entries into the previously blocked arm.
    • Velocity: Calculate speed of rodent movement.
    • Learning Curves: Plot performance metrics across multiple trials.

Repeat Testing:

  • Conduct multiple trials with intervals between tests to assess spatial memory retention and learning over time.

Data Analysis for Y-Maze

Alternation Rate:

  • Specification: The percentage of alternation is a measure of spatial memory and refers to consecutive entries into three different arms.
  • Calculation: (Number of alternations / Total number of arm entries) x 100.
  • Interpretation: Higher alternation rates indicate better spatial working memory.

Arm Entry Sequences:

  • Specification: The specific sequence in which the rodent enters the arms during exploration.
  • Analysis: Identify and analyze the sequences to understand patterns in arm choices.
  • Interpretation: Patterns may reveal preferences or biases in arm exploration.

Time Spent in Each Arm:

  • Specification: The duration of time the rodent spends in each arm.
  • Calculation: Total time spent in each arm.
  • Analysis: Compare the time spent in different arms to assess spatial preferences.
  • Interpretation: Longer durations may indicate spatial preference or increased exploration.


  • Specification: The time taken by the rodent to make the first arm entry.
  • Calculation: Time from the start of the session until the first arm entry.
  • Analysis: Assess the speed of decision-making.
  • Interpretation: Shorter latencies may indicate better spatial learning or memory.

Total Arm Entries:

  • Specification: The total number of entries into any arm during the session.
  • Calculation: Sum of entries into all arms.
  • Analysis: Assess overall exploration activity.
  • Interpretation: Changes in total entries may indicate alterations in general locomotor activity.

Re-entry Errors:

  • Specification: The number of times the rodent re-enters the same arm consecutively.
  • Calculation: Count the instances of re-entering the same arm.
  • Analysis: Assess the tendency for repetitive choices.
  • Interpretation: Higher re-entry errors may indicate impaired spatial memory.

Zone Analysis:

  • Specification: Dividing the maze into specific zones to analyze behavior in different spatial regions.
  • Analysis: Define zones (e.g., arms, center, junction) and analyze parameters within each zone.
  • Interpretation: Differential behavior in specific zones may provide insights into spatial preferences.

Novel Arm Entries:

  • Specification: The number of entries into the arm that was blocked during training.
  • Calculation: Count entries into the previously blocked arm.
  • Analysis: Assess memory recall by examining exploration in the novel arm.
  • Interpretation: Increased entries into the novel arm suggest memory retention.


  • Specification: The speed of the rodent’s movement within the maze.
  • Calculation: Distance traveled divided by time.
  • Analysis: Assess the effect of experimental conditions on locomotor activity.
  • Interpretation: Changes in velocity may indicate alterations in general activity levels.

Learning Curves:

  • Specification: Graphical representation of performance across multiple trials.
  • Analysis: Plot performance metrics (e.g., alternation rate) over consecutive trials.
  • Interpretation: Learning curves help visualize improvements or changes in behavior over time.

Statistical Tests:

  • Specification: Use appropriate statistical tests (e.g., t-tests, ANOVA) to compare groups or conditions.
  • Analysis: Apply statistical tests to assess the significance of differences in the measured parameters.
  • Interpretation: Identify significant effects and draw conclusions based on statistical significance.


  1. Wolf, A., Bauer, B., Abner, E. L., Ashkenazy-Frolinger, T., & Hartz, A. M. (2016). A Comprehensive Behavioral Test Battery to Assess Learning and Memory in 129S6/Tg2576 MicePloS one11(1), e0147733. doi:10.1371/journal.pone.0147733.
  2. Lennartz, R.C. The role of extramaze cues in spontaneous alternation in a plus-mazeLearning & Behavior 36, 138–144 (2008) doi:10.3758/LB.36.2.138.
  3. Kraeuter AK., Guest P.C., Sarnyai Z. (2019) The Y-Maze for Assessment of Spatial Working and Reference Memory in Mice. In: Guest P. (eds) Pre-Clinical Models. Methods in Molecular Biology, vol 1916. Humana Press, New York, NY.
  4. Conrad, C. D., Galea, L. A. M., Kuroda, Y., & McEwen, B. S. (1996). Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine treatment. Behavioral Neuroscience, 110(6), 1321–1334. https://doi.org/10.1037/0735-7044.110.6.1321
  5. Cheryl D Conrad, Sonia J Lupien, Leila C Thanasoulis, Bruce S McEwen. The effects of Type I and Type II corticosteroid receptor agonists on exploratory behavior and spatial memory in the Y-maze. Brain Research. Volume 759. Issue 1. 1997. Pages 76-83. ISSN 0006-8993. https://doi.org/10.1016/S0006-8993(97)00236-9.
  6. Miedel, C. J., Patton, J. M., Miedel, A. N., Miedel, E. S., Levenson, J. M. Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau NeuropathologyJ. Vis. Exp. (123), e55523, doi:10.3791/55523 (2017).
  7. Prieur, E. A. and Jadavji, N. M. (2019). Assessing Spatial Working Memory Using the Spontaneous Alternation Y-maze Test in Aged Male Mice. Bio-protocol 9(3): e3162. DOI: 10.21769/BioProtoc.3162.

Additional information

Weight 21.87 lbs
Dimensions 15 × 3 × 9 in

Mouse, Rat



Maze Type

Elevated, Enclosed


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