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Maze Basics

The Light Dark Box Test

By September 10, 2018February 4th, 2023No Comments

Hi everyone and welcome back to the next installment of Maze Basic articles! Today we’ll be talking about the Light Dark Box test. This test consists of exactly what it sounds like: a dark black chamber connected to a brightly illuminated white chamber by a restricted opening. The light/dark box takes advantage of a rodent’s natural preference for the dark and an aversion to bright lights, making this a good test of anxiety.

There are several measures that you can use to measure anxiety with the light/dark box, for example the distance travelled in each chamber, the number of transitions between the chambers, but the most common is probably the time spent in each chamber. The more time the mouse or rat spends in the light chamber, the less anxious it is. This makes this test a streamlined and easy way to test the effect of anxiolytic (anxiety reducing) and anxiogenic (anxiety producing) drugs or other anxiety-inducing procedures. By simply comparing the amount of time spent in the light chamber between the control and the test case, you can see just how much of an effect your test condition has on the rodent’s anxiety.

Examples of Testing Anxiety-like Behavior with the Light/Dark Box

Miller et. al. (2011) in “Use of the light-dark box to compare the anxiety-related behavior of virgin and postpartum female rats” used the light/dark box to look at the anxiety levels of rats which had given birth to a litter and rats who had not.1 They found that the dams (postpartum rats) spent more time in the light chamber than the diestrous virgins. This is an interesting effect because it makes the dams better able to care and defend their pups.

A different study by Bilkei-Gorzó et. al. (1998) tested the effect of a variety of anxiolytic drugs, including diazepam, chlordiazepoxide, ritanserin, and deramciclane, among others.2 First, they used mCPP, an anxiogenic drug also known as ecstasy, to induce anxiety in the rats. The mCPP-treated rats spent less time exploring the light chamber of the light/dark box compared to the normal rats, meaning that there was a noticeable anxiogenic effect. The majority of anxiolytic drugs they tested reduced the effect of mCPP, meaning the rats spent more time in the light chamber compared to when they were only treated with mCPP. Interestingly, with the 2,3-benzodiazepine girisopam, there was a dose-dependent increase in the exploratory activity of the rats, meaning that the more anxiolytics they received, the less anxious they were.

Limitations and Other Models

With the light/dark box, there are some considerations that need to be taken. For example, the side that the rodent starts in can affect its exploratory or anxiety-like behaviors. One study found that placing a mouse in the light compartment first led to the mouse spending more time in the light chamber. This can artificially make it seem like the mouse is more exploratory and less anxious than other mice. But, it can also imply that the mouse may just be anxious and so “frozen,” as they also found a reduced latency to the first transition.3 To handle complications like this, it is important to keep the protocol consistent for all the animals and cohorts.

Other models of anxiety include the Elevated Plus maze and the Open Field test, which you can read about more in our Maze Basics section.


  1. Miller SM, Piasecki CC, Lonstein JS (2011) Use of the light-dark box to compare the anxiety-related behavior of virgin and postpartum female rats. Pharmacol Biochem Behav 100(1): 130-137.
  2. 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.
  3. Kulesskaya N, Voikar V (2014) Assessment of mouse anxiety-like behavior in the light-dark box and open-field arena: Role of equipment and procedure. Physiology & Behavior 133(22): 30-38.
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