Journal of Pharmacological and Toxicological Methods 68 (2013) 175183

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Journal of Pharmacological and Toxicological Methods

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Review

Animal models of anxiety: A comprehensive review

Vijender Kumar a, Zulqar Ali Bhat a,, Dinesh Kumar b
a
Department of Pharmaceutical Sciences, University of Kashmir, Srinagar 190006, India
b
Department of Pharmaceutical Sciences, Tshwane University of Technology, Arcadia Campus, Pretoria 0001, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: Animal models can be used to contribute to understanding the information about molecular mechanisms
Received 29 October 2012 involved in anxiety and for screening and developing new medications for their treatment that would be
Accepted 9 May 2013 impossible in humans. The human studies have established the genetic basis of anxiety and animal studies
have been used to attempt to further clarify its genetic determinants. In the eld of anxiety research, animal
Keywords:
models can be grouped into two main classes. The rst involves the animal's conditioned responses to stressful
Anxiety
Animal model
and often painful events (e.g. exposure to electric foot shock) and the second includes ethologically based
Conditioned responses paradigms and involves the animal's spontaneous or natural reactions (e.g. ight, avoidance and freezing)
Stress to stress stimuli that do not explicitly involve pain or discomfort (e.g. exposure to a novel highly illuminated
test chamber or to a predator). The current review enlightens the various aspects of animal model of anxiety,
which may be used for research purpose.
2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
2. Validity of animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3. Classication of animal models of anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
4. Models for anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
4.1. Elevated plus-maze (Bourin et al., 2007; Pellow, Chopin, File, & Briley, 1985) . . . . . . . . . . . . . . . . . . . . . . . . . . 176
4.2. Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
4.3. Elevated T-maze (Dutt, Dhar, Sharma, & Dutt, 2011; Onusic, Nogueira, Pereira, Flausino Junior, & Viana, 2003) . . . . . . . . . . . . . . 177
4.4. Elevated Zero-Maze model (Kulkarni, Singh, & Bishnoi, 2007; Kumar, Jaiswal, Singh, & Bhattacharya, 2000; Shepherd & Grewal, 1994) . . . 177
4.5. Unstable elevated exposed plus maze (Jones, Duxon, & King, 2002) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.6. Y-maze model (Monique et al., 1997) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.7. Open eld test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.8. Hole board test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.9. Hole cross test (Takagi, Watanabe, & Saito, 1971) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.10. Digital hole-board apparatus (Pathak et al., 2011; Takeda, Tsuji, & Matsumiya, 1998) . . . . . . . . . . . . . . . . . . . . . . . . 178
4.11. Hole-board apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.12. Social interaction test (File and Pellow 1985, Min et al., 2005) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.13. Apparatus and procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4.14. Acoustic startle response in rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4.15. Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4.16. Vogel thirsty rat conict test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4.17. Mirrored chamber test (Dutt et al., 2011, Kulkarni S.K. 1996) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4.18. Lightdark test (Crawley, 1981; Hossain et al., 2010) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
4.19. Suok test and modied lightdark Suok test (Kalueff & Tuohimaa, 2005) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
4.20. Suok test (ST) apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
4.21. Testing protocol of Suok test (Bourin & Hascoet, 2003; Crawley, 1999; Kalueff & Tuohimaa, 2005) . . . . . . . . . . . . . . . . . . 180
4.22. Isolation-induced aggression test (Abramov et al., 2004) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
4.23. Locomotor activity (Rabbani, Wright, & Little, 1995) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Corresponding author at: Department of Pharmaceutical Sciences, University of Kashmir, Srinagar (J & K) 190006, India. Tel.: +91 9419077701.
E-mail addresses: vijenderpareek24@yahoo.com (V. Kumar), zabhat2000@gmail.com (Z.A. Bhat), sharmadinesh82@gmail.com, kumard@tut.ac.za (D. Kumar).

1056-8719/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.vascn.2013.05.003
176 V. Kumar et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 175183

4.24. Forced swimming test (FST) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

4.25. Learned helplessness test in rats (Bhattacharya, 1994; Katz, 1981; Seligman & Beagley, 1975) . . . . . . . . . . . . . . . . . . . . 180
4.26. Muricidal behavior (Bhattacharya, 1994) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
4.27. Geller type conict test (Dutt et al., 2011; Umezu, 2000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
4.28. Suppression of feeding by novelty (Bhattacharya, Satyam, & Ramanathan, 1999; Dutt et al., 2011) . . . . . . . . . . . . . . . . 181
4.29. Social separation (Dutt et al., 2011) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
4.30. Chick social separation-stress test (Dutt et al., 2011; Sufka, Roach, & Chambliss, 2001) . . . . . . . . . . . . . . . . . . . . . . . . 181
4.31. Cat odor exposure (Dutt et al., 2011; Johnston & File, 1988) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
4.32. Staircase test (Vogel & Vogel, 1997) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
4.33. Foot shock induced aggression test (Tedeschi et al., 1959) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
4.34. Foot shock-induced freezing behavior in rats (Dutt et al., 2011; Vogel & Vogel, 1997) . . . . . . . . . . . . . . . . . . . . . . . . 181
4.35. Exploratory rearings (Hiller & Zetler, 1996; Oliva, Gonzalez-Trujano, Arrieta, Enciso-Rodrguez, & Navarrete, 2004; Ugalde et al., 2005) . . . 182
4.36. Four plate test in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5. Anxiogenic agent-using model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.1. Antagonism of discrimination stimuli produced by anxiogenic drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
6. -Carboline-induced behavioral syndrome in monkeys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
7. Limitation of animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

1. Introduction
Anxiety is affecting one-eighth of the total population of the world
and has become a very important area of research interest in psycho-
pharmacology in the past decade (Rabbani, Sajjadi, & Zarei, 2003). Phys-
ical symptoms of anxiety disorders are due to released stress hormones
like adrenaline and cortisol, which have an effect on almost every organ
in the body. Untreated anxiety disorders can lead to depression
which may result in increased blood pressure, heart palpitations,
chest pain, rapid breathing or breathlessness, sweating, increased
muscle tension or irritability, and decreased intestinal blood ow
resulting in nausea or diarrhea. There is often a decreased sex
drive. Children with anxiety may also have a fear of being away
from the family, a refusal to go to school, a fear of strangers, a fear
of falling asleep or have recurrent nightmares (Jain, Sharma, &
Gahalain, 2010). Animal models for psychopathology have become
an invaluable tool in the analysis of the multitude of causes, genetic,
environmental or pharmacological, that can bring about symptoms
homologous to those of patients with a specic disorder (Shekhar,
McCann, & Meaney, 2001) (Table 3).
2. Validity of animal models
An important criterion for developing animal models to study
psychopathology involves establishing the validity of the model as
a true representation of the process being studied. Generally, three
types of validity parameters are applied to animal models: face validity,
construct validity, and predictive validity (Bourin, Petit-Demouliere,
Dhonnchadha, & Hascoet, 2007):
Face validity, where the model is phenotypically similar and
implies that the response observed in the animal model should be
identical to the behavioral and physiological responses observed in
humans.
Predictive validity entails that the model should be sensitive to
clinically effective pharmacological agents and conversely anxiogenic
compounds should elicit opposite effects, while agents that have no
effect in the clinic should have no effect in these tests.
Construct validity relates to the similarity between the theoretical
rationale underlying the animal model and human behavior. It should
be noted that the different types of validity can be independent of
each other; an animal model can possess predictive and construct
validity without possessing face validity. Ideally, an animal model
should possess both construct and predictive validity so that it may
be used to understand the mechanisms and etiology of the behavioral
and biological factors underlying the disorder that may be similar
in animals and humans. The animal models use experimental pre-
parations developed in one species for the purposes of studying
phenomena occurring in another species and it helps to identify
promising treatments for the disorder (Belzung, 1999; Pathak,
Kasture, Bhatt, & Patel, 2011; Raison & Miller, 2003). Mice and
humans share more than 90% of their genes, and animal models
seem to be a useful tool in biomedical sciences, as evidenced by a
notable increase in the number of active laboratories working in
the eld (Belzung, 1999; Belzung & Griebel, 2001; Pathak et al.,
2011). Furthermore, animal models are particularly of help in situ-
ations when the impact of stress cannot be studied in humans be-
cause of ethical and other like reasons. However, the choice of
which biological phenomenon correlates to study is not easy,
since problems with animal models of human psychic disorders in-
clude: (i) the difference between humans and nonhuman nervous
systems; (ii) the difculty in determining analogous behaviors
among species; and (iii) the need of extrapolation of results from
animals to humans. Such problems most likely reect a signicant
difference in etiology and complexity of anxious behaviors. In addi-
tion, it is important to know that the data derived from animal
models are of value only to the extent that the models are valid,
and that the level (severity) of the disorder evoked in animals
may not be the level of human disorder, thus the need for a new
model (Pathak et al., 2011).
3. Classication of animal models of anxiety
Animal models of anxiety can be classied as conditioned
unconditioned responses (Table 1, Bourin et al., 2007) and as
exteroceptiveinteroceptive stimuli models (Table 2, Divekar, Oswal,
Bagul, & Antre, 2011). The rst involves the animal's conditioned re-
sponses to stressful and often painful events and the second includes
ethologically based paradigms and involves the animal's spontaneous
or natural reactions to stress stimuli that do not explicitly involve pain
or discomfort (Bourin et al., 2007).
4. Models for anxiety
4.1. Elevated plus-maze (Bourin et al., 2007; Pellow, Chopin, File,
& Briley, 1985)
Elevated plus-maze (EPM) is one of the most popular behavioral
tests for research on anxiety, initially developed for rats (Pellow
 V. Kumar et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 175183 177

Table 1 Table 3
Classication of animal models of anxiety (Bourin et al., 2007). Summary of a National Institute of Mental Health workshop: developing animal
models of anxiety disorders (Shekhar et al., 2001).
Conditioned responses Unconditioned responses
Category Example of animal model
GellerSeifter conict (GS) Elevated plus maze (zero/T maze)
Vogel conict Light/dark exploration (L/D) 1. Punishment-induced conict GellerSeifter test
Four-plate test (FPT) Social interaction Vogel test
Conditioned emotional Open eld Conditioned suppression test
response (CER) Ultrasonic vocalization (pain or separation) Ethological conict Open eld test
Conditioned taste aversion (CTA) Fear/anxiety-defense test batteries Elevated plus-maze
Fear-potentiated startle Staircase test Light-dark compartment test
Defensive burying Hole board Social interaction test
Active/passive avoidance Predator Aversive tests Defensive probe-burying
Electrical brain stimulation (dPAG) PAG stimulation
Exposure to predator
Drug discrimination tests Pentylenetetrazol administration
et al., 1985) and more recently for other species such as mice, guinea Other anxiogenic drugs
pigs, voles, hamsters and gerbils. There has also been the develop- Conditioned fear tests Fear-potentiated startle
Conditioned place-aversion
ment of several derivatives of the EPM including the elevated Developmental models Maternal separation model
T-maze, zero mazes and the unstable elevated exposed plus maze Neonatal grooming model
(Bourin et al., 2007). The EPM is in the form of a plus with two Neonatal sepsis model
open elevated arms facing opposite to each other and separated by Pathophysiological models Hypothalamus dysfunction model
Amygdala priming model of anxiety
a central square and two arms of the same dimensions, but enclosed
Trauma and HPA axis sensitization-models
by walls. The maze is raised off the ground so that the open arms of PTSD
combine elements of unfamiliarity, openness and elevation. The Transgenic models Serotonin receptor knockout mice
EPM is based on the natural aversion of rodents for open spaces and CRF receptor knockdown models
uses conict between exploration and aversion to elevated open GABA system mutant mice

places. Provoked behavior proles in the EPM appear to include ele-

ments of neophobia, exploration and approach/avoidance conict; enclosed and open arms are recorded for a 5 min test. Entry into an
thus, the apparatus is often referred to as an unconditioned spontane- arm is dened as the animal placing all four paws onto the arm. During
ous behavioral conict model (Bourin et al., 2007). EPM is a widely the 5 min experiment, the behavior of the rats is recorded as: (i) prefer-
used test based on the natural aversion of rodents to height and ence of the rats for its rst entry into the open or closed arms, (ii) the
open spaces and is sensitive to both anxiolytics as well as anxiogenics number of entries into the open or closed arms, and (iii) average time
and has been validated for both rats and mice (Dawson & Tricklebank, spent by the rats in each of the arms (average time total time spent
1995; Lister, 1987). in open arms / number of entries in arms). All tests can be taped by
using a video camera. Similar studies are carried out on the 1st, 3rd
4.2. Apparatus and 7th day for acute, sub acute and chronic model. In acute study
60 min after the rst dose, in sub acute 60 min after the 3rd dose and
The apparatus is composed of two open arms (50 10 cm) and two in chronic study 60 min after the last dose on the 7th day of test drug
enclosed arms (50 10 cm) with 40 cm high wall arranged so that the or vehicle administration and 30 min after standard drugs on similar
arms of the same type are opposite to each other with a central square day of test drugs. After each test, the maze is carefully cleaned up with
of 10 cm to form a plus sign. The apparatus is elevated to a height of 10% ethanol (Kumar et al., 2012).
50 cm above oor level by a single central support (for rats). A slight
raised edge on the open arms (0.25 cm) provides additional grip for 4.3. Elevated T-maze (Dutt, Dhar, Sharma, & Dutt, 2011; Onusic, Nogueira,
the animals, whereas open arm activity can be further encouraged by Pereira, Flausino Junior, & Viana, 2003)
testing under dim red light (4 25 W). The experiment is mostly
conducted during the dark phase of the light cycle (914:00 h). To facil- The elevated T-maze (ETM) consists of three arms of equal dimen-
itate adaptation to new surroundings, animals are transported to the sion (50 12 cm). One arm is enclosed by 40 cm high walls and dis-
laboratory at least 1 h prior to testing. The pre experimental trial in- posed perpendicularly to the two opposing open arms. The entire
volves placing the individual animal on the central platform of the apparatus is elevated 50 cm above the oor. To avoid falls, the open
maze facing an open arm. Number of entries and the time spent in arms are surrounded by a Plexiglas rim 1 cm high. The animal is
placed at the distal end of the enclosed arm of the ETM, facing the in-
Table 2 tersection of the arms, 30 min after administration of drug. The time
Classication of behavioral model for determination of anxiolytic activity (Divekar
taken by the rat to leave this arm with four paws is recorded (baseline
et al., 2011; Sink et al., 2011).
latency). The same measurement is repeated in two subsequent trials
Exteroceptive stimuli models Interoceptive stimuli models (avoidance 1 and 2) at 30 s intervals. Following avoidance training,
1. Response-independent presentation 1. Electrical stimulation of brain rats are placed at the end of the right arm of the maze and the latency
of stimuli 2. Pharmacological manipulation to leave this arm with the four paws is recorded for three consecutive
Open eld test (drug discrimination tests) time intervals (escape 1, 2 and 3), each at 30 s.
Y-maze model FG-7142-induced anxiety
Elevated plus-maze model Caffeine induced anxiety
Head dipping test Yohimbine-induced anxiety 4.4. Elevated Zero-Maze model (Kulkarni, Singh, & Bishnoi, 2007; Kumar,
Black and white test box Flumazenil-induced anxiety Jaiswal, Singh, & Bhattacharya, 2000; Shepherd & Grewal, 1994)
2. Response-contingent presentation Amphetamine-induced anxiety
of stimuli 3. Aggression/anxiogenesis
Gellerseifter's test Foot shock-induced aggression Elevated Zero-Maze test is a pharmacologically validated assay
Vogel conicts test Drug-induced aggression of anxiety in animal models that is based on the natural aversion of
Quintero's punished bar pressing Isolation-induced aggression rodents (rats/mice). The maze comprises a black Perspex straannular
Social interaction test Behavior anxiety platform (10.5 cm in diameter; 10 cm wide) elevated to 65 cm above
Acoustic startle
the ground level, divided equally into four quadrants. The two
178 V. Kumar et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 175183
opposite quadrants are enclosed by a black Perspex wall (27 cm high)
on both the inner and outer edges of the platform, while the remaining
two opposite quadrants are surrounded by Perspex lip (1 cm high)
which serves as a tactile guide to animals on these open areas. Rats
are placed on one of the enclosed quadrants for a 5-minute test
period. The maze is cleaned with 10% ethanol/water solution and
dried between test sessions. Time spent in open quadrant, numbers of
head dips over the edges of platform, and numbers of stretch posture
attained from closed to open arms are recorded.
4.5. Unstable elevated exposed plus maze (Jones, Duxon, & King, 2002)
The unstable elevated exposed plus maze (UEEPM) has been
proposed as a novel model of anxiety which elicits unconditioned
escape-related behavior in rats thought to mimic the persistent
ght/ight state exhibited by patients suffering from extreme
anxiety disorders. The UEEPM consists of a plus-shaped maze ele-
vated 50 cm above the oor with four exposed arms measuring
35 cm 12 cm and a center square measuring 12 cm 12 cm.
The oor of the plus maze is covered with matt black rubber and
each arm has a 0.5 cm ledge to reduce slippage. A motor oscillates
the apparatus in the horizontal plane at 85 rpm with movement
amplitude of 2 cm on each side of the central point. Subjects are placed
on the center square facing one of the maze arms 3 s prior to oscillation
commencing. If an animal falls off the apparatus, it is placed back into
the area from which it had exited; if an animal actively escapes, the
trial is terminated. Testing time between 1030 and 1730 h is most suit-
able for this. The UEEPM should be cleaned (20% ethanol solution)
between trials. A black surround is placed around the apparatus to
minimise external visual cues. All trials can be videotaped and later
analyzed by an observer blind to treatment.
4.6. Y-maze model (Monique et al., 1997)
It is well known that spontaneous alternation is a measure of spa-
tial working memory. The Y-maze can be used as a measure of short
term memory, general locomotor activity and stereotypic behavior
(Kokkinidis, Walsh, Lahue, & Anisman, 1976). It is mostly made by
black painted wood or gray plastic. Thirty minutes post-treatment,
each animal is placed in turn in the center of a Y-shape wooden run-
way, Y-maze (70 15 12 cm) placed at a height of 50 cm, with
one of the runways to the open tails close. The cumulative times
spent by each animal in the open and closed arms of the maze are
recorded for 5 min. After each trial, the Y-maze apparatus is wiped
clean with ethanol (10%) solution. This protocol is a modication of
that of Pellow et al. (1985).
4.7. Open eld test
It is one of the tests used to observe general motor activity, explor-
atory behavior and measures of anxiety (Asano, 1986; Mechan et al.,
2002; Whimbey & Denenberg, 1967). The open eld area is made of
plain wood and consists of a square area (60 60 35 cm). The oor
has a square sheet of wood (60 60 cm) with the surface divided
into sixteen squares (15 15 cm). The apparatus is illuminated by a
60 W bulb placed at a height of 100 cm. Forty ve minutes after drugs
treatment, subjects are placed individually in the center of the open
eld and behavioral activity is recorded for ve minutes. Subsequently,
activity can taped by using a video camera to score the following behav-
ioral parameters for a period of 5 min (Dos et al., 2006; Turner, 1972):
(1) the number of entries and time spent in the center, (2) periphery
and corners of the eld, (3) the number of crossings (number of square
oor units entered) as a measure of distance traveled, (4) rearing (num-
ber of times the animal stood on hind legs) and (5) assisted rearing
(forepaws touching the walls of the apparatus).
4.8. Hole board test
The hole-board test has been widely used to check emotionality
or responses to stress in animals. Several behaviors can be readily
observed and quantied in this model, which makes a comprehensive
report of the animal's behavior. It has been established that head
dipping behavior in mice and rats reects exploration distinct from
locomotor activity. Head-dipping behavior was sensitive to changes
in the emotional state of the animal, and suggested that the expres-
sion of an anxiolytic state in animals might be reected by an increase
in head-dipping behavior (Kumar, Bhat, Kumar, & Shah, 2013).
4.9. Hole cross test (Takagi, Watanabe, & Saito, 1971)
A steel partition is xed in the middle of a cage having a size of
30 20 14 cm. A hole of 3 cm diameter that makes at a height of
7.5 cm in the center of the cage. Thirty minutes after treatment, ani-
mals are placed individually in the center of the hole cross apparatus
for 5 min. Subsequently, activity can taped by using a video camera to
score the numbers of hole crossed and analyzed.
4.10. Digital hole-board apparatus (Pathak et al., 2011; Takeda, Tsuji,
& Matsumiya, 1998)
The automatic hole-board apparatus consisted of a gray vinyl
chloride box (50 50 50 cm) with four equidistant holes 3 cm
in diameter on the oor. The area of the hole-board is divided
with white ink into 24 smaller areas. An infrared beam sensor is
installed on the wall to detect the number and duration of head
dipping behaviors. Other behavioral performance such as locus,
distance and speed of movement of mice in the hole-board is
recorded by the overhead color CCD camera. Data from the CCD
camera is collected through a custom-designed interface as a re-
ection signal. All the data are analyzed and stored in a personal
computer using analytical software. During the 5 min experimenta-
tion the number of head-dips, immobility, SAP (Stretched Attend Pos-
ture) rearing, grooming behavior, rears and also displacements between
the different areas are registered.
4.11. Hole-board apparatus
The apparatus consists of a gray Perspex panel (40 40 cm, 2.2 cm
thick) with 16 equidistant holes (3 cm diameter) on the oor (Costa-
Campos, Dassoler, Rigo, Iwu, & Elisabetsky, 2004; Kalueff & Tuohimaa,
2004). Photocells below the surface of the holes provide the measure
of the number of head dip. The board is positioned 15 cm above the
table and is divided into 9 squares of 10 10 cm with black water-
resistant marker. Each animal is individually placed in the center of
the board (facing away from the observer) and the following parame-
ters are noted during 5 min test period: (a) the latency of rst head
dips, (b) number and duration of head dips, and number of rearings,
and (c) spontaneous movements (number of squares crossed with all
four paws).
4.12. Social interaction test (File and Pellow 1985, Min et al., 2005)
The social interaction model of anxiety was developed to provide
an ethologically based test that was sensitive to both anxiolytic and
anxiogenic effects. In general, an increase in social interaction is in-
dicative of an anxiolytic effect, whereas a specic decrease in social
interaction indicates an anxiogenic effect. This test provided a new
approach to the neurobiological mechanisms underlying anxiety disor-
ders. Ge et al. have found that the aversive test condition of the social
interaction test (HU) increases 5-HT and DA turnover throughout
the rat brain. In short, the social interaction test is an extremely
useful animal model for evaluating anxiolytic compounds, which
 V. Kumar et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 175183
are prescribed for treating social phobia, social failure/impairments
and emotional immaturity (Kumar et al., 2012).
4.13. Apparatus and procedure
The test arena is a black Plexiglas box, 60 60 35 cm, with the
base divided into 9 cm squares by lines of white tape. The light inten-
sity of the arena oor is 380 lx. Two test conditions are performed:
high light, unfamiliar arena (HU) and high light, familiar arena (HF).
On the 1st day of the test, each rat is randomly allocated according
to body weight (b 15 g difference) to an unfamiliar partner in groups
of 12 animals (six pairs) which are subsequently administered the
appropriate drug. These rats are then replaced into their home cage
until testing. Following appropriate pretreatment time, members of
each pair of unfamiliar rats are placed in opposite corners of the
arena and observed for social interaction behaviors and overall loco-
motor activity for 10 min. At the end of this period the rats and any
fecal boluses are removed and the arena wiped with a damp cloth.
Social interaction time (in seconds) per pair of rats is measured as
time of snifng and mutual grooming, adjacent lying, climbing over
and crawling under the partner, approximation and following (File,
1980). Aggressive-type behaviors (e.g. kicking, aggressive grooming,
biting, boxing and jumping on; (Guy & Gardner, 1985)) are also esti-
mated. These are treated as separate entities since such behaviors are
modulated by different pharmacological agents than social behaviors
(Miczek & Winslow, 1987). Locomotor activity is measured by counting
the number of squares crossed. Following completion of the rst test,
rats are returned to their home cages. On days 2 and 3, the rats are
placed individually, without drug, in the same box for 10 min per day
to familiarize them with the apparatus. On the fourth day, the same
pairs of rats are once again placed in the test arena for 10 min and the
same test procedure is carried out.
4.14. Acoustic startle response in rats
The acoustic startle response is a simple behavior which naturally
occurs in mammals and is affected by a variety of treatments. Startle
response can be used to determine sites and mechanisms of drug ac-
tion. Acoustic startle response consists of a series of rapid movements
beginning at the head involving contraction and extension of major
muscle groups in response to auditory stimuli with a rapid onset of
time. Responses are graded in amplitude in relation to stimulus inten-
sity and may show habituation and sensitization (Davis, 1982). The
test has been modied in various ways, e.g., inhibition by a prepulse
or fear-induced potentiation. Sipes and Geyer (1995) studied the dis-
ruption of prepulse inhibition of the startle response in the rat by DOI
(2,5-dimethoxy-4-iodophenylisopropylamine) which is mediated by
5-HT2A receptors. The authors suggested that studies of the seroto-
nergic substrates of prepulse inhibition may provide a model of the
possible serotonergic role in the sensorimotor gating abnormalities
in schizophrenia and obsessive compulsive disorder patients. Schulz
et al. (1996) performed acoustic startle experiments in rats with a
potent and selective non-peptide antagonist of the corticotropin re-
leasing factor receptors. Walker and Davis (1997) found that the am-
plitude of acoustic startle response in rats was increased by high
illumination levels. Devices to register the intensity of fear-potentiated
startle response in rats were described by Hijzen, Houtzager, Joordens,
Olivier, and Slangen (1995). The detail of the models with modication
and their use in anxiety are described by Sink, Walker, Yang, and Davis
(2011).
4.15. Procedure
Male Wistar rats weighing about 200 g are used. Acoustic startle
reexes are measured in a specially built apparatus, e.g., Coulborn Instru-
ments Acoustic Response Test System. The animals are individually
179
placed in 8 8 16 cm open air cages that restrict locomotion but do
not immobilize the animal, and are placed on one of four platforms within
a sound-attenuating acoustic chamber. A ventilating fan provides an am-
bient noise level. Acoustic stimuli consist of white noise bursts lasting
20 ms at 98 dB and 124 dB SPL. Simultaneous with the rapid onset of
each stimulus, the animal's physical movement within the cage on the
platform is measured for 200 ms as an electrical voltage change via a
strain gage which is converted to grams of weight change following ana-
log to digital conversion. Data are recorded automatically by an interfaced
microcomputer. Pre-tests are performed with all animals to obtain con-
trol values. The animals are treated 2 h prior to the experiment with
test drugs or vehicle given orally or subcutaneously. The results are
given as percentage of the change, related to the values obtained in the
pre-test and assessed by a one-way ANOVA, followed by Dunnett's test
when appropriate (Vogel, Vogel, & Scholkens, 2002).
4.16. Vogel thirsty rat conict test
To verify the anti-anxiety activity, a simple and reliable conict
procedure is described as in Bhattacharya (1994) and Dutt et al.
(2011). Thirty rats are administered shocks while licking water. Rats
are water deprived for 48 h. Two hours before testing, each rat is
placed in a clear Plexiglas box (38 38 cm) with a black Plexiglas
compartment (10 10.5 cm) attached to one wall and an opening
from the large box to the small compartment. The entire apparatus
has a stainless steel grid oor. A water bottle with a metal drinking
tube is tted to the outside of the small compartment so that the
tube is extended into the box at a height of 3 cm above the grid.
Rats lick in bursts with a relatively constant rate of 7 licks per sec.
A drinkometer circuit is connected between the drinking tube and
the grid oor of the apparatus, so that the rat completes the circuit
whenever it licks the tube. Shock is administered to the feet of the
animal by switching the connections to the drinking tube and grids
from the drinkometer to a shocker which applies an unscrambled
shock between the drinking tube and grid oor. The rat is placed in
the apparatus and allowed to nd the drinking tube and to complete
20 licks before shock (available at the tube for 2 s) is applied. The rat
controls the shock duration by withdrawing from the tube. A 3 min
timer is automatically started after the termination of the rst shock.
During the 3 min period, shocks are delivered following each twen-
tieth lick. The number of shocks delivered during the 3 min session
is recorded for each animal. The number of shocks received after
treatment is compared with untreated animals.
4.17. Mirrored chamber test (Dutt et al., 2011, Kulkarni S.K. 1996)
It has been observed that animal species exhibit approachavoidance
response upon placing of a mirror within their environment. The param-
eters, i.e., latency to enter, and total time spent in the mirrored chamber
can be used to evaluate anxiolytic drugs. The apparatus consists of a mir-
rored cube (30 cm on a side) open on one side that is placed inside a
square wooden box (40 40 30.5 cm). The mirrored cube consists
of ve pieces of mirrored glass. The mirrors used are mirrored on one
surface only (back surface being painted dark brown). The three mir-
rored side panes, a top pane, and the oor pane face the interior of the
cube. The mirrored cube is placed in the center of the wooden container
to form a 5 cm corridor which completely surrounds the mirror
chamber. A mirror is also placed on the container wall so that it
faces the single open side of the mirrored chamber. The other three
walls of the container are painted dark brown. Thirty minutes after
the administration of the test drug or standard, the animals are
placed individually in the chamber of mirrors at a xed corner.
During a 5-min test period, the following parameters are noted:
(a) latency to enter the chamber, i.e., the time spent, in seconds, for
the rst entry into the chamber of mirrors, (b) number of entries
180 V. Kumar et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 175183
in mirrored chamber, and (c) the time spent with each entry is calcu-
lated by dividing the total time spent with number of entries.
4.18. Lightdark test (Crawley, 1981; Hossain et al., 2010)
The lightdark exploration test (LDT) is typically used to assess
anxiety-related responses more directly. This apparatus is based on
the modied model described by Belzung and Le Pape (1994). It con-
sists of wooden boxes (60 cm long 40 cm wide 35 cm deep),
which are divided into two equal compartments by a wooden board
with a 10 10 cm2 opening located centrally at the oor level,
connecting the compartments. One compartment is painted black
and covered with a wooden lid. The other box (transparent glass
covered) is painted white and lit by a 60 Watts light bulb set 30 cm
above the box. At the beginning of the experiment, rats are placed in-
dividually in the center of the illuminated box, facing the opening
away from the dark compartment. Behaviors of the animals are
videotaped for 5 min. Afterwards, videotapes are scored manually
to determine the following parameters: (1) initial latency to enter
the dark compartment, (2) frequency of entries into both the com-
partments and (3) total time spent by subjects in each compartment.
The time spent in illuminated and dark places, as well as the number
of entries in each space, is recorded for 5 min.
4.19. Suok test and modied lightdark Suok test (Kalueff & Tuohimaa,
2005)
The long elevated horizontal rod (Suok test, ST) and its lightdark
modication (LDST) may be used for behavioral characterization in
rats, including simultaneous assessment of their anxiety, activity, and
neurological phenotypes. For the rat exposure test, rats placed in a
small Plexiglas box (20 20 20 cm) divided into two equal compart-
ments by a wire net (1-cm mesh), allowing visual, olfactory, auditory,
and even tactile communication between the animals (to avoid direct
attacks). Subjects are placed individually in the empty compartment
next to the compartment for 5 min prior to testing in the ST. The ap-
paratus is cleaned thoroughly between subjects (wet and dry cloths).
The lighting in the experimental room is similar to that in the hold-
ing room during these procedures.
4.20. Suok test (ST) apparatus
The ST apparatus (Kalueff & Tuohimaa, 2005) is a 2.6-m aluminum
tube, 2 cm in diameter, elevated to a height of 20 cm from the cush-
ioned oor. The rod is separated into 10-cm segments by line draw-
ings and xed to two Plexiglas side walls (50 50 cm; 1 cm thick)
preventing the mice from escaping sideways. The experimental
room is dimly lit during this test. The LDST consists of the same alu-
minum rod, with four 60-W bulbs 40 cm above the rod (NB: directed
light) to illuminate the blight Q part of the test, providing the only
lighting in the experimental room (Kalueff & Tuohimaa, 2005).
4.21. Testing protocol of Suok test (Bourin & Hascoet, 2003; Crawley,
1999; Kalueff & Tuohimaa, 2005)
Transport the subjects from their holding room to the experimental
room and leave undisturbed for 1 h prior to testing. Expose the mice to
different stressors for 5 min and then place the rats individually in the
middle part of the ST (snout facing either end) or LDST (snout facing
the dark end). Support the animals by hand during the initial placement
(up to 5 s), if necessary, to avoid a fall due to incorrect positioning.
All test apparatus is thoroughly cleaned (wet and dry cloth) before
each animal. Observe the animal ST or the LDST behaviors for 5 min.
During observations, the experimenter (inter-rated reliability No. 90)
always sits in the same place, 2 m away from the apparatus. Scores for
animal anxiety-related measures, are summarized in Table 1, using a
specially designed register. Horizontal and directed exploration stops,
and defecation scores are crucial measures in this test. In all experi-
ments, the latency measures are reckoned as total observation time
(300 s) in the mice not showing the respective behaviors. Identify and
register separately animal motor behavioral parameters (Kalueff &
Tuohimaa, 2005). Falls and hind leg slips are critical measures in this
test. In view of the importance of light/dark stimuli for LDST, differ-
entiate behavioral measures as a function of their occurrence in the
light or dark part of the test similar to the standard lightdark para-
digm protocol.
4.22. Isolation-induced aggression test (Abramov et al., 2004)
The animal is isolated and kept individually in home cages of
28 20 16 cm for 6 weeks. The isolated animal is prescreened
for aggressive behavior once a day for one or two days prior to the ex-
periment. An intruder animal is introduced into the isolated animals'
home cage for 3 min, and the isolated animals exhibiting bite marks
are used for the drug test experiments on the following day. The
subjects are injected with drugs or vehicle. Thirty minutes later,
two isolated animals are placed in a neutral cage, which is the
same size as their home cages as previously reported. An assessment
of the aggressive behavior (biting attacks, wrestling, lateral threats
and tail switching) of two isolated subjects is conducted and noted
for 5 min as the attack duration.
4.23. Locomotor activity (Rabbani, Wright, & Little, 1995)
The actions of drugs on spontaneous locomotor activity are mea-
sured automatically by breaking of infrared beams (Rabbani et al.,
1995). The units of the activity counts are arbitrary and based on
the beam breaks by movement of the animals. Each animal is treated
with the drug and then, after a 30-min period, placed in a novel cage
in the infrared apparatus. The locomotor activity is counted at 5-min
interval for the next 15 min. The treatments of animals are random-
ized throughout the day, between 08:00 and 13:00 h, to control for
diurnal variations in activity.
4.24. Forced swimming test (FST)
The FST is a model for assessing antidepressant activity (Porsolt,
Bertin, & Jalfre, 1977). The development of immobility when the mice
are placed in an inescapable cylinder lled with water reects the
cessation of persistent escape-directed behavior (Lucki, 1997). The
apparatus consists of a clear Plexiglas cylinder (20 cm high 12 cm di-
ameter) lled to a 15 cm depth with water (24 1 C). In the pre-test
session, every animal is placed individually into the cylinder for 15 min,
24 h prior to the 5 min swimming test. During the test session a trained
observer records the immobility time, considered to be when the sub-
ject makes no further attempt to escape, apart from the movements
necessary to keep its head above the water. It is suggested (Porsolt et
al., 1977) that the immobility reects a state of lowered mood in
which the animals have given up hope of nding an exit and have
resigned themselves to the experimental situation.
4.25. Learned helplessness test in rats (Bhattacharya, 1994; Katz, 1981;
Seligman & Beagley, 1975)
Rats are subjected to foot shock (60 scrambled shocks, 15 s duration
0.8 mA, every min) in a two compartment jumping box with the escape
door to the unelectried chamber closed. The animals without drug are
reserved in the chamber without shock. The exercise is continued for
1 h. 48 h later, on day 3 after this inescapable shock treatment, the
rats are subjected to avoidance training, using the same apparatus but
keeping the escape route to the unelectried chamber open. During
this avoidance training, the rat is placed in the electried chamber
 V. Kumar et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 175183
and allowed to acclimatise for 5 min before being subjected to 30 avoid-
ance trials, with an inter-trial interval of 30 s. During the rst 3 s of the
trial, a buzzer stimulus (conditional stimulus) is presented, followed by
electric shock (conditioned stimulus) through the grid oor (0.8 mA)
for the next 3 s. The avoidance response characterized by escape to ad-
joining safe chamber, following the buzzer, within 3060 s during the
test session is noted, and the failure to escape is assessed as escape fail-
ure. Escape failure in rats exposed to inescapable shock is postulated to
represent depressive behavior.
4.26. Muricidal behavior (Bhattacharya, 1994)
Muricidal, or mouse killing behavior, is characterized by compulsive
killing by rats of mice introduced in the former's cage within 30 s,
irrespective of the satiety status of the killer rat. Rats are prescreened
for muricidal activity by introducing a mouse in the rat cage. Approxi-
mately 25 to 30% Charles Foster rats exhibit compulsive mouse killing
behavior, the muricidal interval being reduced following repeated ex-
posure to mice. The percentage of prescreened rats exhibiting muricidal
behavior within 30 s of introduction of a mouse into a rat cage following
vehicle or drug administration is noted.
4.27. Geller type conict test (Dutt et al., 2011; Umezu, 2000)
Animals are subjected to food deprivation in order to induce
hunger. Subjects are trained before starting the experiment under
MULT FR20/FR20-punishment schedule (Dutt et al., 2011) of food
reinforcement, using the apparatus for the Geller type conict test
(GT-8510, GT-8005 and GT-7715). The schedule consists of four
pairs of an alternating safe period; the animals' lever pressing is
reinforced by food pellets at FR20 without electric shock. During the
alarm period, which is indicated by a warning stimulus (tonic signal:
80 Hz, 90 dB), every 20th lever press is punished using an electric
shock (5090 V, ca 0.3 mA, 50 Hz AC, duration 0.3 s). The response
rate of the animal is recorded during the safe as well as alarm period.
4.28. Suppression of feeding by novelty (Bhattacharya, Satyam,
& Ramanathan, 1999; Dutt et al., 2011)
The apparatus consists of a wooden box (60 60 35 cm) with a
solid oor. The oor is covered with a 2 cm wooden chip layer, and 15
laboratory chow pellets are evenly placed on the oor. A similar
arrangement is made in home cage rats. The rats are not given any
feed 24 h prior to testing, but are provided with drinking water. The
rats are placed individually in the test chamber and the latency to
begin feeding is recorded. If the rats do not eat within 5 min, the
test is terminated and a latency score of 5 min is recorded.
4.29. Social separation (Dutt et al., 2011)
Social separation stress resulting in vocalizations and nociceptive
responses in animals, and is sensitive to the anxiolytic drugs.
4.30. Chick social separation-stress test (Dutt et al., 2011; Sufka, Roach,
& Chambliss, 2001)
In this procedure, a chick is placed into the observation chamber
in isolation for a 3 min test session (Sufka et al., 2001). To index
stress-induced analgesia, a 50 l injection of 0.10% formalin is admin-
istered into the plantar region of the chick's foot immediately before
placement into the chamber. The following observations are recorded
as: foot lift frequency and foot lift duration in response to formalin;
to index sedation; ventral recumbent latency that resembles a sleep-
like posture. A composite pain score (CPS) is derived from the following
formula CPS = 4 (z-score foot lift) + [z-score (duration / total number
181
of lifts)]. Number of vocalization to index separation-stress (Sufka et al.,
2001).
4.31. Cat odor exposure (Dutt et al., 2011; Johnston & File, 1988)
This test is based on defensive behavior displayed by rodents when
confronted by a predator or by its odor. Mice/rats are divided into three
groups, i.e., control (no odor), neutral door (modeling clay) and preda-
tor odor (cat feces). Cat feces are obtained from at least 2 year old male
domestic cat, collected outdoors shortly after defecation. The neutral
odor is provided by a 2 cm diameter ball of blue modeling clay dough
of the same volume as cat feces. The mice/rats are individually brought
to the testing room dimly lit with a 40-W red bulb (15-lx on the testing
location). After the removal of the grid, and depending on the group, an
odorant stimulus is placed on the saw dust, at the opposite location of
the food compartment. The same procedure is followed for the control
group except that no stimulus is given. In order to avoid any disruption
between odors, the mice/rats from the control group are always tested
rst followed by those from the neutral odor group and the predator
odor group. The grid is then replaced and the behavior of each mouse/
rat is recorded from the side during 5 min (Dutt et al., 2011; Johnston
& File, 1988; Roy, Belzung, Delarue, & Chapillon, 2001). The parameters
of recording are as follows (a) the time spent at the opposite location
of the odorant stimulus, (b) the number of entries into the tow parts of
the cage (i.e. under the food compartment or stimulus location), (c) the
number of contacts with the odorant stimulus, (d) the number of
stretch attended posture; and (e) the number of burrows in odorant
stimulus. The cat odor can also be obtained by rubbing a damp cloth
(20 20 cm) against the fur of male laboratory housed domestic cats
for 5 min (Onusic et al., 2003). Clothes with cat odor are reserved in
sealed plastic bags. Each cloth is used for four exposures only.
4.32. Staircase test (Vogel & Vogel, 1997)
The staircase test is used for evaluating anxiolytic activity by
purporting step-climbing to reect exploratory or locomotor activ-
ity, while rearing behavior is an index of anxiety state. For experi-
ments with mice, the staircase is composed of ve identical steps
2.5 cm 10 cm 7.5 cm. The internal height of the wall is constant
along the whole length of the staircase. In this test, each animal is
used only once. The drug is administered 1 h or 30 min prior to the
test. The animal is placed on the oor of the box with its back to
the staircase. The number of step climbings and the number of
rearings are counted for a 3 minute period. A step is considered to
be climbed only if the subject has placed all four paws on the step.
After each test, the box is cleaned (10% ethanol) in order to eliminate
any olfactory cues which might modify the behavior of the next ani-
mal. In this experimental model, the average number of steps and
rearing of control group is taken as 100%, and the values of treated
animals are expressed as percentage of the control.
4.33. Foot shock induced aggression test (Tedeschi et al., 1959)
A pair of male mice/rats is placed in a box with a grid oor consisting
of steel rods with a distance of 6 mm. A constant AC current of 0.8 mA is
supplied to the grid oor. A constant shocker delivers a 60 Hz current
for 5 s, follow by 5 s intermission. Forty-ve minutes after the treat-
ment, the total number of ghts is recorded for 3 min. The ghting
behavior consists of vocalization, leaping, running, rearing and facing
each other with some attempts to attack by biting.
4.34. Foot shock-induced freezing behavior in rats (Dutt et al., 2011;
Vogel & Vogel, 1997)
In this test, anxiolytic activity is determined by freezing behavior.
The drug is administered 30 min prior placing the animal in a
182 V. Kumar et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 175183
standard conditioning chamber (e.g. Coulborn Instrument) for a 6.5 min
session. Two minutes after the start of the session, a scrambled foot
shock (0.5 mA, 0.5 s) is delivered through the grid oor of the chamber.
Using an assembly of push buttons interfaced with a computer, an ob-
server monitors the amount of time each animal spends in the following
mutually exclusive types of behaviors like freezing: immobility with rigid
body posture; sedated posture: sitting or sleeping; small exploratory
movements: movements involving the torso or front paw only, vertical
movements of the head or snifng; and locomotion: activity involves
hind jaws, grooming or rearing. Frequency of rearings is also counted.
All types of behaviors are monitored for the entire 6.5 min session. Eval-
uation of the anti-anxiety effect can be done on the basis of duration of
foot-shock induced freezing after administration of test compound and
control.
4.35. Exploratory rearings (Hiller & Zetler, 1996; Oliva, Gonzalez-Trujano,
Arrieta, Enciso-Rodrguez, & Navarrete, 2004; Ugalde et al., 2005)
Each mouse is individually placed on a lter paper-covered oor of a
glass cylinder (16 cm in height and 11 cm in diameter). The number of
spontaneous rearings on its posterior limbs is counted during the rst
5 min. Reduced exploratory rearing is shown by naive mice after place-
ment in an unfamiliar environment revealing an anxiolytic-like effect.
4.36. Four plate test in mice
The four plate test is used for evaluating anxiolytic activity by deliv-
ering the shocks to reect locomotor activity (Vogel & Vogel, 1997).
The shape of the testing apparatus is rectangular (25 10 16 cm).
The oor is covered with four identical rectangular metal plates
(8 11 cm) separated from each other by a gap of 4 mm. The plates
are connected to a source of continuous current which are applied to
two adjacent plates, with a mild electrical shock of 0.35 mA for 0.5 s.
Thirty minutes before the test; the animals are administered the drug.
At the beginning of the test, the subject is gently dropped onto a plate
and is allowed to explore the enclosure for 15 s. After this, every time
the animal crosses from one plate to another the experimenter elec-
tries the whole oor for 0.5 s which evokes a clear ight reaction in
the animal. The number of times the apparatus is electried is counted
each minute for 10 min. The delivery of shock decreases dramatically
the motor activity. The number of shocks received during the rst
minute is taken as the parameter for evaluating anxiolytic activity.
5. Anxiogenic agent-using model
5.1. Antagonism of discrimination stimuli produced by anxiogenic drugs
Mostly anxiolytic drugs block the discriminative effect of
pentylenetetrazole (PTZ), whereas anticonvulsants without anxiolytic
effects do not (Dutt et al., 2011; Tecott & Nestler, 2004). In this model,
a sub-convulsive dose of PTZ is used as an anxiogenic stimulus in rats.
The basic method involves training rats to press an appropriate lever
for food in the presence of an injection of PTZ. Responses to a second
lever are also recorded. A rat is considered trained, when it can reliably
press the lever appropriate to a PTZ versus saline injection. Treatment
with anxiolytic drug blocks this discriminative effect of PTZ.
6. -Carboline-induced behavioral syndrome in monkeys
The ethyl ester of -carboline-3-carboxylate (-CCE) possesses a
high afnity for BZD receptor binding site and therefore, it causes a
behavioral syndrome, which is blocked by diazepam (Barnett, 1986;
Dutt et al., 2011). The procedure involves chaired monkeys with
in-dwelling intravenous catheters. -CCE is administered i.v., and
type of behavior including head and body turning, and distress vocal-
ization is recorded. Plasma samples are collected for estimation of
difference in cortisol, epinephrine, and norepinephrine during the
experiment. Drugs with anti-anxiety activity are considered to block
these behavioral changes.
7. Limitation of animal models
The main concern about animal models is the lack of standardiza-
tion between the different laboratories. Some are square in shape and
others circular; some are clear and others opaque; some are bright
and others totally dark; some have tops and others are open. The
presence of objects within the arena, placement of the animal (center
or close to the walls), recording period (220 min, usually 5 min) and
items recorded are the main variations observed across the literature.
The -carboline-induced behavioral syndrome in monkey model is
time consuming. Moreover, rhesus monkeys are expensive and dif-
cult to obtain. These disadvantages associated with this model limit
its use for preliminary screening of anxiolytic drugs. Therefore, this
procedure ts best as a tertiary evaluation, after there is sufcient
evidence of anxiolytic potential from other preliminary measures.
8. Conclusion
The present comprehensive review article enlightens the various
aspects of animal model of anxiety disorder, which may be used by
young researchers for evaluation of anxiolytic agents in this eld.
Acknowledgment
Vijender Kumar, ZA Bhat, and Dinesh Kumar would like to thank the
Central Scientic Industrial Research (CSIR), New Delhi for providing
nancial assistance and the Department of Pharmaceutical Sciences,
University of Kashmir, Srinagar (J & K), India for providing necessary
facility for the same work.
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