First introduced by the researchers Crawley and Goodwin in 1980,[1] the light/dark box consists of two chambers: a small, dark chamber which comprises one-third of the total space and a larger, illuminated chamber which comprises the remaining two-thirds of the box. These two chambers are connected by a small tunnel through which the subject animal may move from one area to another.[2] The use of the light/dark box is predicated on the assumption that rodents find highly-illuminated open areas to be aversive as they represent areas of potential danger, while dark areas imply a degree of safety (see: “Safety in the Shadows”). Thus, the light/dark box is commonly used to assess levels of stress, anxiety, and depression in mice and rats.
The most common uses of the light/dark box are the assessment of various models of stress and anxiety, as well as the efficacy of anxiolytic and antidepressant drugs. Multiple models of anxiety-inducing procedures have been shown to decrease time spent in the lit area of the apparatus in addition to reducing the overall number of crossings, including mild traumatic brain injury[3] and anxiogenic drugs.[4] In both cases, traditional anxiolytic drugs such as benzodiazepines effectively reverse this effect. However, the conditions of the test itself are anxiogenic enough that induction methods of this type (i.e. behaviorally- or pharmacologically-induced stress) do not need to be used to assess the efficacy of antidepressant and anxiolytic drugs. For instance, the now commonly used antidepressants moclobemide and selegiline were tested in the light/dark box during their early development.[5] Thus, with or without the induced anxiety-like behaviors the light/dark box provides an ample testing ground for pharmacological or behavioral intervention therapies which aim to reduce levels of stress and anxiety in rodents.
Procedures and notes
In order to perform an assay in the light/dark box, animals should first be habituated to the room in which the test will take place. Specifically, animals should be left for at least one hour in this room in order to habituate to the environment. The box may either be cleaned and sterilized between animals or left in a constant “soiled” state (i.e. not cleaned between animals) in order to prevent neophobic responding.
For the test, the mouse or rat is placed in the center of the well-lit compartment of the apparatus facing away from the connecting tunnel. Tests may last between 5 and 10 minutes during which multiple measurements of time-spent in the two compartments and exploratory behavior may be collected.[6]
The most common measurements for the light/dark box are time-spent in the light compartment versus the dark compartment and the number of crossings made between the two compartments. The former is thought to represent the level of anxiety. An additional, less-common measure that may be used in the light/dark box is the “latency to first crossing.” Simply, this is the time between the commencement of the experiment and the first crossing into the dark or light area of the box (i.e. the area opposite to the starting position). While this measurement may often be similar to the total time spent in the two compartments, it may also reveal strategic differences between animals or conditions.
Animals should not be habituated to the light/dark box itself, as the basis of the test relies on environmental novelty and exploration. Indeed, previous exposure to the light/dark box has been shown to preclude the effects of both anxiolytic and anxiogenic drugs, since the animals are already familiar with the environment and therefore do not perceive a significant difference of safety between the two compartments.[7][8] Thus, animals may only be tested in the light/dark box once, making this apparatus useful for between-group, but not within-group comparisons.
Mouse and rat strain may play a role in the light/dark box results, as previous studies have identified baseline differences between two commonly used strains of mice, the BALB/c and C57BL/6j strains. Specifically, BALB/cj mice appear to exhibit higher levels of baseline anxiety as compared to C57BL/6j mice.[9] Not surprisingly, the C57BL/cj strain which exhibits higher levels of baseline anxiety also proved to be more sensitive to the anxiolytic drug diazepam. This is an important caveat to researchers who use multiple strains of mice in their experiments.
Similarly, a comparison of Sprague Dawley and Dark Agouti rats revealed baseline differences in light/dark box performance. Specifically, Dark Agouti rats spend less time in the open, lit area of the apparatus as compared to Sprague Dawley.[10] While both of the animals respond to the anxiolytic drug diazepam by exhibiting increased time in this compartment under the influence of the drug, this baseline difference is of importance to researchers seeking to optimize their experiments.
Additionally, some debate exists as to whether or not there are differences between male and female rats in the light/dark box. For instance, in one study, while female rats were found to exhibit less baseline anxiety than males in several tests designed around stressful environments including the elevated plus maze and the open field maze, measurements in the light/dark box revealed no difference between the two sexes.[11] On the other hand, females in other studies have been shown to spend more time in the light side of the box, suggesting lower levels of baseline anxiety.[12] Additionally, delivery of the anxiogenic drug scopolamine significantly reduced time spent in the light compartment in females only in this same study, suggesting that they may be more sensitive to certain anxiogenic conditions. In any case, it is important that investigators take note of their animals’ sexes in order to identify such potential differences.
Conclusions
The light/dark box is a simple test used to measure levels of anxiety in mice and rats. The test requires no prior training and very little environmental habituation. Assays in the light/dark box can be performed with a variety of ages and strains of rodents with potential small differences existing between both sexes and strains.
References
- Crawley, J., & Goodwin, F. K. (1980). Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacology, Biochemistry, and Behavior, 13(2), 167–170.
- Bourin, M., & Hascoët, M. (2003). The mouse light/dark box test. European Journal of Pharmacology, 463(1–3), 55–65.
- Kosari-Nasab, M., Shokouhi, G., Ghorbanihaghjo, A., Abbasi, M. M., & Salari, A.-A. (2018). Anxiolytic- and antidepressant-like effects of Silymarin compared to diazepam and fluoxetine in a mouse model of mild traumatic brain injury. Toxicology and Applied Pharmacology, 338, 159–173.
- Bilkei-Gorzó, A., Gyertyán, I., & Lévay, G. (1998). mCPP-induced anxiety in the light-dark box in rats–a new method for screening anxiolytic activity. Psychopharmacology, 136(3), 291–298.
- De Angelis, L., & Furlan, C. (2000). The anxiolytic-like properties of two selective MAOIs, moclobemide and selegiline, in a standard and an enhanced light/dark aversion test. Pharmacology, Biochemistry, and Behavior, 65(4), 649–653.
- Hascoë T, M. (1998). A New Approach to the Light/Dark Test Procedure in Mice. Pharmacology Biochemistry and Behavior, 60(3), 645–653.
- Holmes, A., Iles, J. P., Mayell, S. J., & Rodgers, R. J. (2001). Prior test experience compromises the anxiolytic efficacy of chlordiazepoxide in the mouse light/dark exploration test. Behavioural Brain Research, 122(2), 159–167.
- Rodgers, R. J., & Shepherd, J. K. (1993). Influence of prior maze experience on behaviour and response to diazepam in the elevated plus-maze and light/dark tests of anxiety in mice. Psychopharmacology, 113(2), 237–242.
- Lepicard, E. M., Joubert, C., Hagneau, I., Perez-Diaz, F., & Chapouthier, G. (2000). Differences in anxiety-related behavior and response to diazepam in BALB/cByJ and C57BL/6J strains of mice. Pharmacology, Biochemistry, and Behavior, 67(4), 739–748.
- Mechan, A. O., Moran, P. M., Elliott, M., Young, A. J., Joseph, M. H., & Green, R. (2002). A comparison between Dark Agouti and Sprague-Dawley rats in their behaviour on the elevated plus-maze, open-field apparatus and activity meters, and their response to diazepam. Psychopharmacology, 159(2), 188–195.
- Domonkos, E., Borbélyová, V., Csongová, M., Bosý, M., Kačmárová, M., Ostatníková, D., … Celec, P. (2017). Sex differences and sex hormones in anxiety-like behavior of aging rats. Hormones and Behavior, 93, 159–165.
- Hughes, R. N., Desmond, C. S., & Fisher, L. C. E. (2004). Room novelty, sex, scopolamine and their interactions as determinants of general activity and rearing, and light-dark preferences in rats. Behavioural Processes, 67(2), 173–181.