Jan 15, 2025
Y Maze - Spontaneous Alternation
- 1University of Alabama at Birmingham
Protocol Citation: Ashley Adamson, Allie Smith, Briana R De Miranda 2025. Y Maze - Spontaneous Alternation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8dw1dg2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 14, 2025
Last Modified: January 15, 2025
Protocol Integer ID: 118352
Funders Acknowledgements:
Briana De Miranda
Grant ID: R01ES034846
Briana De Miranda
Grant ID: TX220241
Ashley Adamson
Grant ID: F31 ES036890
Abstract
The following protocol is used to assess spatial working memory using the Y Maze (spontaneous alternation) at the University of Alabama at Birmingham's Neuroscience Behavioral Assessment Core.
Guidelines
Use of this protocol needs approval by user's Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
- Chlorhexidine
- Paper towels
- Open field boxes (appropriately sized for test subject)
- Ethovision version 17 software
Before start
You will need a test mouse whose behavior is not included in the experiment to calibrate detection settings.
Set Up
Set Up
Set Up
Bring animals into testing room for habituation (at least 30 minutes prior to start of experiment)
Set up maze
Clean the maze
- Spray with chlorohexidine and wipe out
Plug the EthoVision XT Version 17 software dongle in the computer
- Shines a red light when connected
Open EthoVision XT Version 17 software
Start new experiment
Name the file
Indicate where file will be saved
Experiment Settings
Select live tracking
- Indicate which camera will be used
- Center the arena under the camera
Select center point detection
Select the number of arenas that you will be using
Units should be cm, s, and deg
Arena settings
Grab a background image from the camera
- Ensure the maze is in its intended area moving it after this step will affect the analysis
- Mark placement with tape so you can return it to the exact same location if it is moved while cleaning between trials
Draw scale
- Measure the bottom arm of the Y
- Draw a scale bar over the area that you measured and indicate its length (cm)
Click the arena in the list on the right
- Select the closed polygon tool
- Trace the outside barrier of the entire maze
Draw zones
Select zone
- Rename “Arm 1”
- Used closed polygon tool to trace one arm of the maze
Add a zone
- Rename “Arm 2”
- Used closed polygon tool to trace another arm
Add a zone
- Rename “Arm 3”
- Used closed polygon tool to trace the last arm
Validate setup
- If you have any overlapping zones, they will be considered invalid and must be redrawn
Trial Control Settings
Begin condition should be on the start button with the condition of center-point being in the arena
Stop track with the condition being after 5 minutes
Detection Settings
Click advanced settings
Dynamic response
Indicate if subject is darker or lighter than the background
Place test subject in arena
Click and drag to create a box around the subject
- The software will create an automatic threshold around the subject that moves with it
Fine-tune it with the scrub bar to make it fit around the subject perfectly
Trial list
Add enough trials for each subject
Acquisition
Software will load the first trial
Put a subject in starting area
- At the outside edge of one of the baseline arms
- Keep starting area consistent across subjects
Press record
- The program will run for 5 minutes and automatically stop
Remove subject
Clean enclosure with chlorhexidine
Repeat for all trials
Data Profile
Ensure that it is pulling data from all trails
Analysis Profile
Distance traveled
- Arena
- Statistics - total only
Velocity
- Arena
- Averaging interval – 1
- Statistics – mean
Zone alternation
- Select all 3 zones
- Alternations
- Max alternations
- Direct revisits
- Indirect revisits
- Trial statistics – frequency
Results
Statistics and charts
Calculate to generate a file with the analysis parameters chosen
Export and save
Ensure the video files are exported and saved. If there is a problem, the analysis can be repeated from the video file.
Acknowledgements
We would like to thank the UAB Neuroscience Behavioral Assessment Core for use of their facilities.