Jan 10, 2025
Elevated Zero Maze Protocol
- 1University of Alabama at Birmingham
Protocol Citation: Ashley Adamson, Allie Smith, Briana R De Miranda 2025. Elevated Zero Maze Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mpe8gwz/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: December 07, 2024
Last Modified: January 10, 2025
Protocol Integer ID: 114531
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 anxiety-related behaviors using the Elevated Plus Maze 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.
Before start
You will need a test mouse whose behavior is not included in the experiment to calibrate detection settings
Set Up
Set Up
Bring animals into testing room for habituation (at least 30 minutes prior to start of experiment)
Set up maze under camera
Clean the maze with chlorohexidine
Make sure the EthoVision XT Version 17 software dongle is 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
- select the camera to 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 analysis
- mark placement with tape so the maze can be returned to the exact same lactaion if accidentally moved between trials
Draw scale
- measure diameter of the whole maze
- draw a scale bar over the area that you measured and indicate its length
Click first arena in the list on the right
- select the ellipse drawing tool
- trace the outer circle of the maze
- trace the inner circle of the maze
- drag the arena label inside the middle of the two circles
Draw zones
Select zone 1
- use closed polygon tool to trace one of the closed regions
Add a zone
- use closed polygon tool to trace the other closed region
Add a zone
- use closed polygon tool to trace one of the open regions
Add a zone
- use closed polygon tool to trace the other open region
Create 2 cumulative zones
- Closed - the two closed regions
- Open - the two open regions
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 zone
Stop track with the condition being after 5 minutes
Detection Settings
Click advanced settings
Dynamic response
Select appropriate setting to 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 the desired number of trials
Running the Experiment
Running the Experiment
Acquisition
Software will load the first trial
Put a subject in starting area
- use one of the enclosed 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
Results and Analysis
Results and Analysis
Data Profile
Ensure that it is pulling data from all trails
Analysis Profile
Parameters
Distance traveled
- arena (statistics - total only)
Velocity
- arena (avering interval - 1; statistics - mean)
In zone (open and closed cumulative zones)
- frequency
- latency to first
- cumulative duration
Results
Statistics and charts
Calculate to generate a file with the analysis parameters chosen
Export and save
Ensure the video files are exported and save.
- analysis can be repeated on video files if a problem occurs
Acknowledgements
We would like to thank the UAB Neuroscience Behavioral Assessment Core for use of their facilities.