History: The Light Dark Transition Test

The Light Dark Box Transition Test (Light-Dark, Light/Dark) was developed by Crawley & Goodwin, 1980. The transition test of two compartments assesses unconditioned, anxiety-like behaviors of rodents. Rodents have an innate aversion to light and exploration in the light of an open field can elicit a stress response. Rodent preference for dark, enclosed spaces draws them into the darker side of the box.

When assessing the transgenic or pharmaceutical effects that can be anxiolytic or anxiogenic, researchers make use of the Light Dark Box transition test in the experimental battery of behavioral assays.

Apparatus: The Light Dark Transition Test

The Light Dark Box apparatus is (length x width x height): Centimeters: 45 x 91 x 38; Inches: 18 x 36 x 15. The entire box is separated into thirds; the light area is two-thirds of the box while the remaining one-third is the dark side. The transition door from the dark to the light side is 10cm x 10cm (4 x 4 inches).

Testing Protocol: Light Dark Transition Test

Acclimation time: Animals can acclimate to the testing room prior to testing for one (1) to three (3) hours.

Animal placement: The animal is placed into the center of the white, light side of the box. At that time, latency to enter into the dark area is measured (time; minutes/seconds).  An animal that is placed into the dark side will allow the user to measure latency to enter into the light area (time; minutes/seconds).

Cleaning: the apparatus should be cleaned with 70% ethanol or 10% bleach before use and after each session.

Assessment of general locomotion: some drug manipulations can modulate locomotion by increasing and decreasing motion in a non-specific manner. Assessing locomotion in the horizontal and vertical directions can determine if the drug is a general motor stimulant and not an anxiolytic.

Important Considerations for Testing

Circadian rhythm: The circadian rhythm of the testing animal must be considered due to changes in activity during light and dark cycles.

Data Analysis: Light Dark Transition Test

When all four paws enter either the light or dark areas, this is considered entrance into that arena with total testing time considered as the limitation by which analysis is performed, the following can be assessed:

• Number of light-dark transitions (Counts)
• Duration in light area (time; seconds, s)
• Duration in dark area (time; seconds, s)
• Latency to enter dark area (time; seconds/minutes)
• Latency to enter the light area (time; seconds/minutes)
• Light to dark ratio (Total Light Time/Total Dark Time)
• Exploratory rearing: directed sniffing, standing on hindpaws, forepaws extended upwards or on sides of the chamber (Counts)
• Leaning/Peeking Out: checking out the light space while remaining inside the dark space (Counts)
• Stretch attend: stretching forward and retreating to original position

References

1. Jacqueline Crawley, Frederick K. Goodwin. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacology Biochemistry and Behavior. Volume 13, Issue 2. 1980. Pages 167-170. ISSN 0091-3057. < https://doi.org/10.1016/0091-3057(80)90067-2. >
2. Takao, K., & Miyakawa, T. (2006). Light/dark transition test for mice. Journal of visualized experiments: JoVE, (1), 104. doi:10.3791/104
3. Serchov, T., van Calker, D. and Biber, K. (2016). Light/Dark Transition Test to Assess Anxiety-like Behavior in Mice. Bio-protocol 6(19): e1957. DOI: 10.21769/BioProtoc.1957.
4. Hascoët M., Bourin M. (2009) The Mouse Light–Dark Box Test. In: Gould T. (eds) Mood and Anxiety Related Phenotypes in Mice. Neuromethods, vol 42. Humana Press, Totowa, NJ.
5. Miller, S. M., Piasecki, C. C., & Lonstein, J. S. (2011). Use of the light-dark box to compare the anxiety-related behavior of virgin and postpartum female rats. Pharmacology, biochemistry, and behavior, 100(1), 130–137. doi:10.1016/j.pbb.2011.08.002.