Project protocol — Contents
Workflow, sampling, and experimental treatment
Equipment for all procedures
Procedure: Open Field Test (OFT)
Procedure: Light – Dark Box (L-D)
Procedure: Elevated Plus Maze (EPM)
Time/ Steps Procedure performed Equipment/ Reagent Procedure duration Age (wk) Data collected -6 wks Mice arrived at the animal facility, housed 2-4 per cage, separated according to sex and strain - - 6-8 - -1 wk Mice are habituated to the main colony holding room for at least one week prior to behavioral testing - - 12-14 - day-1a On the day of testing, mice are transported to a quiet anteroom adjacent to the testing room and acclimated for at least one to two hours prior to the start of testing transport cages 2 hr 12-14 - day-1b A cohort of naive mice are tested in the open field Open Field 10 min 12-14 distance traveled, rearing event day-1c Another cohort of mice are tested in the elevated plus maze (EPM) and the light-dark (LD) box Elevated Plus Maze, Light-Dark Box 5 min, 10 min 12-14 distance traveled in the EPM and % duration in the light zone of LD box
- Transport cages
- Open Field Test (OFT): The open field is a 17" × 17" × 13" arena with a white Plexiglas floor and clear Plexiglas walls (ENV-515-16; Med Associates, St Albans, VT, USA), which is surrounded by infrared detection beams on the x-, y- and z-axes that track the mice position and activity over the course of the experiment. The apparatus is isolated within a 73.5 × 59 × 59 cm testing chamber fitted with overhead fluorescent lighting (lux level 14).
- Elevated Plus Maze (EPM): The elevated plus maze (7001-0336; San Diego Instruments, San Diego, CA, USA) consists of two open arms and two closed arms [26.5" × 2.5”] that are directly opposing each other. The walls of the enclosed arms completely surround the end of the runway and are 6” high. The top of the enclosed arms is open to the testing room. The entire apparatus is 15” high and is placed on the floor for testing. A video camera above the maze captures the mice location in the maze. Data is collected and analyzed with Actimetrics LimeLight software (Actimetrics, Wilmette, IL).
- Light/Dark Box (L-D): The light/dark enclosure (ENV-511; Med Associates) inserts into the Med-Associates open field apparatus and is a light-impermeable box that covers one third of the area of the open field.
- Computer system
- Light bleach solution (0.1%)
- Paper towels
a. Mice 6-8 wks-of-age are acclimated to housing conditions for 5-6 wks. Initially they are grouped housed at 2-4 mice per cage.
b. Mice are habituated to the main colony holding room for at least one week prior to behavioral testing.
c. On the day of testing, mice are transported to a quiet anteroom adjacent to the testing room and acclimated for at least 1-2 hrs prior to the start of testing.
d. All testing equipment is cleaned with a light bleach solution (0.1%) after each use.
e. Mice are tested in two behavioral cohorts that each go through a separate series of tests. First group of mice are tested in the open field. Mice are experimentally naïve for both open field and elevated plus maze testing.
f. Second group of mice are tested on the elevated plus maze, followed by light/dark test.
OFT: open field test
a. Mice are removed from their home cage, immediately placed in the corner of the open field arena and allowed to freely explore the apparatus for a test interval of 10 min.
b. Mice are scored for a number of behaviors in the open field including:
- total distance traveled (in cm)
- ambulatory episodes (number of times a mouse breaks user-defined number of beams before coming to rest)
- percent time resting, average velocity (in cm per second)
- number of rearings and percent time spent in the center of arena (defined as nine square-inch central part of arena)
L-D: light and dark test
a. The mice are placed in the center of the open field facing the dark enclosure and allowed to explore the arena for 10 min.
b. Mice are scored for the same behaviors as measured in the open field but analysis is divided into light and dark quadrants of the arena.
c. Transitions between light and dark quadrants are also analyzed.
EPM: elevated plus maze
a. A video camera above the maze captures the mice location in the maze.
b. Data are collected and analyzed with Actimetrics LimeLight software.
c. The mice are placed in the center of the maze and allowed to investigate the maze for 5 min.
d. Data recorded include distance traveled in each region, time spent in each region and crossings into each region.
Data collected by investigator
Open Field Test: total distance traveled and rearing events.
Light-Dark Box: percent duration in the light zone.
Elevated Plus Maze: total distance traveled.