Light and dark box model 1

A model to screen the anxiolytics has been brought to all exclusive.

1. H.K.E.s COLLEGE OF PHARMACY
DEPARTMENT OF PHARMACOLOGY

LIGHT AND DARK BOX MODEL
FOR TESTING ANXIOLYTIC AND ANXIOGENIC ACTIVITY IN RODENTS

INTRODUCTION: The LIGHT and DARK BOX test is a popular animal model used in
pharmacology to assay unconditioned anxiety responses in rodents .The conflict in between
the tendency to explore and the initial tendency to avoid the unfamiliar plays an important
role in this test. The exploratory activity reflects the combined result of these tendencies in
novel situation.
PRINCIPLE: The light and dark box test is based on the aversion of rodent of brightly illuminated
areas and the spontaneous exploratory behavior of rodents in response to mild stressors that is
mild environment and light. A natural conflict situation occurs when an animal is subjected to
an unfamiliar environment or novel objects.
Thus in light dark box test, drug induced increase in behaviors in the white part
of a two compartment box in which a large white compartment is illuminated and a small black
compartment is darkened is suggested as an index of anxiolytic activity.
An increase in transitions without an increase in spontaneous locomotion is
considered to reflect anxiolytic activity. However the effect is observed in case of certain strains
of mice or with certain drugs. This model provides consistent result when used to study
anxiolytic activity of benzodiazepines.
The test apparatus consists of a box divided into a small(one-third) dark
chamber and large (two-thirds) brightly illuminated chamber. Mice are placed into the
compartment lit with an artificial light and allowed to move freely between chambers .The first
latency to enter the dark compartment and the total time spent in lit compartment are indices
for the bright space anxiety in mice .Transitions are index of activity, exploration because of
habituation over time. LDT is quick and easy to use without requiring prior training in animals.
MATERIALS AND REAGENT:
 Paper Towel
 Laboratory bred mice [Mice housed in groups of 3-5 kept in a room with controlled
temperature with humidity under control with a light and dark cycle with ad libitum
access to food and water.]
 70% ethanol.
EQUIPMENT: The apparatus for the light dark transition test consists of a box (
42*21*25) divided into small compartments .A restricted opening of 3-7 cm is
made to connect the two chambers .Indirect white light source is provided to the
experiment model.

2. H.K.E.s COLLEGE OF PHARMACY
DEPARTMENT OF PHARMACOLOGY

PROCEDURE:
 The apparatus is placed in an isolated room away from any external
interferences and noises with a low intensity light source.
 The experimenter must restrict from making any excessive noise during the
test and from wearing odorant products with strong smell since it would act
as an anxiogenic stimulus for the mice.
 The illumination in the lit chamber should be around 200-400 lux or more
while in the dark compartment it should be 5 lux or less.
 The mice are transferred to the home cages to the behavioral testing room
for at least 30 minute before the experiment.
 Clean both the compartments with 70% ethanol.
 The mouse is placed in the middle of the brightly illuminated chamber.
 The experimenter should stay as far as possible from the box or out of sight
of the test animal.
 The mouse is allowed to run freely between chambers for 5 minutes.
 The following parameters should be recorded:
a) The latency time to enter dark compartment.
b) The time spent in the lit/dark experiment chamber.
c) Distance travelled in the lit chamber.
d) The no. of transitions.

NOTE:
1. The test was originally developed by CRAWLEY and colleagues .Difference in between
our original version and our test is the difference in the time as the original was carried
for 10 minutes whereas we went for 5 minutes.
2. Typically male mice are used for experiment as the variations in estrous cycle might
influence the performance in female mice.
3. Baseline may vary according to mouse strain and age.
4. In order to ensure uniform luminosity indirect light should be used to avoid the
production of shadows which can be a place of preference for the mice.
5. An entry into the lit and dark compartment is only considered when the animal places
frontal paws inside it, if it returns without this no entry is registered.
Sl.No. Body wt.
(gm)
Treatment Dose
(Mg/ml)
Latency time
(sec)
Distance
travelled(cm)
Time
spent(secs)
No of
transitions
light Dark
01. 50 Control - 12 15 45 25 3
02. 42 Control

- 15 23 74 14 2
03. 46 Control - 6 28 46 15 3

3. H.K.E.s COLLEGE OF PHARMACY
DEPARTMENT OF PHARMACOLOGY

04. 40 Control - 18 13,24 45 9 2
05. 44 Lorazepam 0.01 55 4
06. 38 Lorazepam 0.01 50 4
07. 44 Lorazepam 0.01 22 5
08. 40 Lorazepam 0.01 74 4

REPORT: From the above experiment drug induced anxiolytic activity can be understood from
the increase in latency period and total time spent in the light media.