Dedicated to your research needs.

Subscribe

Please enter subscribe form shortcode

Photograph of a laboratory mouse in the hands of a technician. At Anilocus, we are a preclinical CNS disorders CRO that conducts in vivo safety and efficacy evaluations for drug discovery and pharmaceutical drug development. Learn more.

Protocol: Light Dark Box for Assessing Anxiety-Like Behaviors in Mice

Discover the highly effective Light Dark Box Protocol for evaluating anxiety-like behaviors in mice. Our comprehensive guide provides step-by-step instructions on implementing the protocol, its significance in behavioral studies, and the benefits it offers to researchers investigating anxiety and efficacy of anxiolytics. Here, we will share the key considerations, best practices, and potential applications of the Light Dark Box protocol to enhance your understanding of anxiety-related behaviors in mice. Optimize your research with this valuable resource today and contact us to build a customizable Light Dark Box apparatus.

Discover the highly effective Light Dark Box protocol for evaluating anxiety-like behaviors in mice.  Our comprehensive guide provides step-by-step instructions on implementing the protocol, its significance in behavioral studies, and the benefits it offers to researchers investigating anxiety and efficacy of anxiolytics. Explore the key considerations, best practices, and potential applications of the Light Dark Box protocol to enhance your understanding of anxiety-related behaviors in mice. Optimize your research with this valuable resource today.

The Light Dark Box Protocol

The Light Dark Box protocol involves several steps to assess anxiety-like behavior in rodents. Remember that specific protocols may have slight variations depending on the research objectives and the species of rodents used. Here are the typical steps involved:

Preparation: Set up the Light Dark Box apparatus, which consists of a two-compartment box with one brightly illuminated compartment (light compartment) and one dark compartment. Ensure that the lighting conditions and other environmental factors are consistent.

Habituation: Allow the rodents to acclimate to the testing room between 30 minutes to 1 hour, to reduce stress caused by novel surroundings. Behavioral testing should take place during lights on phase.

Rodent Placement in Apparatus: Gently introduce the rodent into the dark compartment of the box. Our apparatus allows you to transfer the animal to the dark region without issue. You may use a transfer cage or other suitable means. Ask us about our apparatus customization if you have specific requests.

Light Dark Box Test Duration: Record the behavior of the rodent during a predetermined testing period, typically 5-10 minutes. With an Anilocus Light-Dark Box apparatus, your equipment comes with a digital camera to record rodent activity throughout the assay. You may use video acquired in combination with a video tracking software. Download our comprehensive protocol for a complete list of suggested animal behavioral tracking software for analysis!

Data Analysis for Light Dark Box Transition Test: Analyze the recorded behaviors to assess anxiety-like behavior. Suggested parameters for measurement:

  • Time spent in each compartment
  • The number of transitions between compartments
  • Total distance traveled during test session
  • Latency to enter light chamber

Cleaning & Sanitization: Properly clean and sanitize the box with a 10% bleach solution or other hypochlorous solution and any equipment used to avoid potential cross-contamination between animals and to eliminate olfactory cues.

What are the potential confounding factors in the Light Dark Box protocol?

While the light dark box transition test is a valuable assay for evaluation anxiety-like behaviors and efficacy of pharmaceutical anti-anxiety (anxiolytic) drugs, there are potential confounding factors that researchers should be aware of when conducting tests:

  1. Subject Variability: Like humans, different animals may exhibit varying levels of anxiety, which will be reflected in the results. This is why we conduct behavioral studies with a large number of test subjects after a power analysis (n=12-15/group). It is essential to have a sufficient sample size and randomize the animals.
  2. Time of testing: The time of day when the experiment is conducted can influence the behavior of rodents. Nocturnal animals might display different responses during the light phase compared to the dark phase of their circadian rhythm.
  3. Environmental factors: The physical environment in which the experiment takes place can impact the behavior of animals. Variables such as noise, temperature, and odors should be controlled and kept consistent across all experimental conditions.
  4. Prior Exposure: Animals with prior exposure to similar behavioral tests or stressful situations may display altered responses. It is important to consider the history of the animals and control for any potential confounds related to prior experience.
  5. Animal Handling Stress: The process of handling and transferring animals to the testing apparatus can induce stress, which may influence subsequent behavior. Minimizing handling stress and allowing for sufficient habituation time can help mitigate this confounding factor.
  6. Observer bias: The behavior of animals in the Light Dark Box is typically scored by an observer. However, at Anilocus, we remove this confounding bias by blinding the observer and automating data analysis with animal behavior video tracking software to reduce observer bias.
  7. Pharmacological interference: If the experiment involves the administration of drugs or substances, it is important to consider potential interactions between the pharmacological agent and the behavior being measured. Some substances may directly affect anxiety-like behavior and confound the results.
  8. Genetic factors: Different strains or genetically modified animals may have inherent differences in anxiety-related behaviors. Considering the genetic background of the animals and controlling for genetic factors can help ensure accurate interpretation of the results.

Reliability and validity of the results can be ensured when researchers are mindful of these potential confounding factors including implementing appropriate controls.

Can the Light Dark Box protocol be used to study other behaviors besides anxiety?

The Light Dark Box protocol is primarily designed and widely used for measuring anxiety-like behavior in preclinical research. While it is a reliable tool for assessing anxiety, its applicability to other behaviors may be limited. Other behavioral apparatuses such as the elevated plus maze and open-field test are available at Anilocus to study a broader range of behaviors in rodents including depression-like behaviors and locomotion disorders (Parkinson’s Disease, Huntington’s Disease, neurodevelopmental disorders, traumatic brain injuries, etc.).

To request our comprehensive guides for any of these assays view our complete list of protocols available for download.

References

  1. Bourin M, Hascoët M. The mouse light/dark box test. Eur J Pharmacol. 2003;463(1-3):55-65. doi:10.1016/s0014-2999(03)01274-3.
  2. Han YY, Zhou JW, Guo ZW, et al. Multiple brain regions are involved in reaction to acute restraint stress in CYLD-knockout mice. Stress. 2023;26(1):2228925. doi:10.1080/10253890.2023.2228925.
  3. Medeiros Contini F, Seggio JA. Constant light and single housing alter novelty-induced locomotor activity and sociability in female Swiss Webster mice [published online ahead of print, 2023 Jun 14]. Neuro Endocrinol Lett. 2023;44(3):117-122.
  4. Moreira CVL, Faria ALG, Silva DPB, et al. Heteroaromatic salvinorin A analogue (P-3 l) elicits antinociceptive and anxiolytic-like effects. Fitoterapia. 2023;167:105488. doi:10.1016/j.fitote.2023.105488.
  5. Cabral IB, de Lima Moreira CV, Rodrigues ACC, et al. Preclinical data on morpholine (3,5-di-tertbutyl-4-hydroxyphenyl) methanone induced anxiolysis [published online ahead of print, 2023 Apr 25]. Naunyn Schmiedebergs Arch Pharmacol. 2023;10.1007/s00210-023-02502-9. doi:10.1007/s00210-023-02502-9.

Check out our fully customizable Barnes Maze apparatus protocols for spatial learning and memory.

    Post Tags :

    Dr. Shermel Sherman is an academic entrepreneur, neuroscientist, and the founder of AniLocus Inc. Established in June 2021 and incorporated in California and Maryland, AniLocus provides comprehensive research tools for the development of neurotherapeutics.