See JAX Pipeline on the IMPC website for order of tasks for all mice tested

Project protocol: Light-dark box test

• Introduction
• Study design
• Workflow
• Equipment, software and supplies
• Reagents and solutions
• Procedure
• Measurements
• Experimental and technical parameters

Introduction

The light-dark box test measures the anxiety-related behavior of mice in a two-chambered light-dark arena.

Study design

Paradigm: Baseline study

Panel: Collaborative Cross

Number of strains: 18

Sex: Both

Age: 8-10 wks

Sample size: Seven mice per sex from at least two litters

Number of cohorts: 1

Workflow

Step	Description	Equipment	Data collected
1	Mice are acclimated to test room for at least 3 days	-	-
2	Each mouse is placed in the light side of the arena	Light-dark box	-
3	Behavior is recorded for 20 min	Light-dark box	Latency to enter dark zone, time mobile in each zone, time spent in each zone, percentage of time in each zone, number of transitions between zones, number of fecal boli

Equipment, software and supplies

• Light-dark box: Accuscan Versamax with Light-Dark Insert [See Experimental and technical parameters]

Reagents and solutions

• 1% Virkon S solution

 

Procedure

1. Mice are allowed to acclimate to the testing room for a minimum of 3 days.
2. Light levels within the room are set so that the light intensity in the light arena is between 100-200 lux and 4-7 lux in the dark arena. Light metering is performed at the start of each test day.
3. Photobeam sensor check is performed using the built-in diagnostic function at the beginning and end of every trial.
4. Each mouse is placed in the light side of an arena.
5. Behavior is recorded for 20 min.
6. At the end of the 20 min test, mice are removed from the arenas and returned to their home cages.
7. Arenas are cleaned with 1% Virkon S solution to disinfect and remove scent cues from previously tested mice.
8. Analysis of the recording is done to measure the activity of each mouse in each of the two zones and the number of transitions between zones.

Data collection: The Accuscan Versamax system in configured to report parameters for each mouse. Versamax files are converted into text data files using Versadat. It outputs a .DPP file containing light-dark information. Raw data is stored until outlier detection and other QA/QC procedures are complete. Local data processing is implemented to calculate derived measures from this data and a table of the derived measures and raw time per bin per zone is reported per mouse in the LIMS system.

Data QC: Verify that total time of each run is 20 min.

Formulas and derivations: Local data processing is implemented to calculate derived measures from these data, and a table of the derived measures and raw time per zone are reported per mouse in JaxLIMS.

 

Measurements

LIMS field name	MPD variable name	Description	Units	Series	Primary measurement	Series order	MPD computed	Calculation	Method	Data type
Side changes	transitions_LD	number of transitions between light and dark zones, 20 min test	n						light-dark box test	Integer
Left side time spent	light_dur_LD	time spent in light zone, 20 min test	s						light-dark box test	Float
Time mobile left side	time_mobile_LD	time mobile in light zone, 20 min test	s						light-dark box test	Float
Right side time spent	dark_dur_LD	time spent in dark zone, 20 min test	s						light-dark box test	Float
Time spent mobile right side	time_mobile_LD	time mobile in dark zone, 20 min test	s						light-dark box test	Float
Fecal boli	defecation_LD	fecal boli count, 20 min test	n						light-dark box test	Integer
Reaction time	reaction_time_LD	latency to enter dark zone, 20 min test	s						light-dark box test	Float
Percent time in dark	pct_dark_LD	percentage of total time in dark zone, 20 min test	%						light-dark box test	Float
Percent time in light	pct_light_LD	percentage of total time in light zone, 20 min test	%						light-dark box test	Float

Experimental and technical parameters

Light level in light zone	100-200 lux
Light level in dark zone	4-7 lux
Equipment name	Light-Dark Insert
Equipment manufacturer	Accuscan
Equipment model	Accuscan Versamax
Start time	0700 - 1700 EST
Disinfectant	1% Virkon Solution