Behavioural Brain Research 193 (2008) 277–288

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Behavioural Brain Research

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Research report

Integrating the open field, elevated plus maze and light/dark box to assess
different types of emotional behaviors in one single trial
André Ramos ∗ , Elayne Pereira, Gisele C. Martins, Thaize D. Wehrmeister, Geison S. Izı́dio
Laboratório de Genética do Comportamento, Departamento de Biologia Celular, Embriologia e Genética,
Universidade Federal de Santa Catarina, 88.040-900 Florianópolis, SC, Brazil

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

Article history: Current anxiety tests do not provide, individually, a pure and complete picture of an animal’s emotional
Received 13 April 2008 profile. Therefore, many authors test their experimental hypotheses using a series of anxiety-related tests,
Received in revised form 13 May 2008 which are thought to reflect different facets of emotionality. The objective of this study was to investigate
Accepted 5 June 2008
the potential usefulness of integrating three widely used behavioral tests, the open field (OF), elevated
Available online 11 June 2008
plus maze (EPM), and light/dark box (LDB), to assess a wider range of emotional and non-emotional
behaviors within one single trial. A protocol was developed where rats could freely explore an OF that
Keywords:
was physically connected to an EPM and a LDB during 15 min. Classical anxiety- and locomotion-related
Anxiety
Elevated plus maze
behaviors from each test were measured. Lewis and spontaneously hypertensive rats (SHR) inbred strains,
Open field known to present genetic differences in each of the individual tests, differed for all anxiety-related behav-
Light/dark box iors from the combined apparatus. Factor analyses revealed that similar anxiety- and locomotion-related
Behavior genetics factors were produced by the three tests applied either separately or in combination. Under both condi-
Inbred strains tions, each test produced its own anxiety-related factor. Two benzodiazepines, chlordiazepoxide (at 5 and
Rats 10 mg/kg) and midazolam (at 0.75 mg/kg), facilitated the approach towards the EPM open arms, whereas
pentylenetetrazole (10 mg/kg) specifically inhibited exploration of the three aversive areas (OF center,
EPM open arms, LDB light compartment). Together, these results suggest that the new integrated appa-
ratus may contribute to the study of anxiety, by providing a rapid, comprehensive and reliable method of
assessing emotionality-related behaviors and its underlying components.
© 2008 Published by Elsevier B.V.

1. Introduction imize the consequences of such limitations, many authors submit

Since the creation of the open field test, conceived in 1934 to
provide objective measures of emotionality in rats [18], dozens of
different tests aiming to assess anxiety-related behaviors in labo-
ratory rodents have appeared in the literature, for reviews see Refs.
[1,13,17,23,27,37]. From the field of experimental psychology, the
interest on such tests spread rapidly among neuroscientists, phar-
macologists and, more recently, molecular geneticists. Today, these
tests are part of the screening routine of many laboratories, not only
of those working on drug development but also the ones dedicated
to the behavioral and neurological study of mutant mice and rats
[11,17,40].
Despite their wide diffusion, most users agree that none of
the existing anxiety tests provides a pure, undisputed measure of
emotional reactivity and that each individual test assesses only a
fraction of an animal’s emotional profile [6,13,27]. In order to min-
∗ Corresponding author. Tel.: +55 48 3721 5153; fax: +55 48 3721 5148.
E-mail address: andre@ccb.ufsc.br (A. Ramos).
their experimental subjects to a battery of different tests which,
together, are expected to provide a more complete and reliable
picture of an animal’s emotional reactivity [11,27]. Nevertheless,
several concerns arise from the use of test batteries, such as the
long-lasting influences of previous test experiences, which were
found to be dependent on the animal’s genotype [41], and the need
for allowing inter-test intervals (of a few days or weeks) in order to
minimize the effects of test history [24]. In addition to that, ideally, a
test battery should not be excessively complex or time-consuming
in order to allow its use in high-throughput behavioral phenotyping
studies.
Another issue to be considered when using different tests is
how to interpret treatment effects that are seen in one test but
not in another. Data from correlational studies, in spite of being
somewhat contradictory [28,29], often indicate that there is little
correlation among anxiety-related behaviors measured in different
tests [16,29,38]. Such a lack of correlation is normally interpreted
as a sign of difference in the psychobiological meanings of the var-
ious tests [16,27,38]. However, because an animal cannot be tested
at the same time in two different tests and as most tests only assess

0166-4328/$ – see front matter © 2008 Published by Elsevier B.V.

doi:10.1016/j.bbr.2008.06.007
278 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288
temporary emotional states [23], one cannot be sure of how much
of the observed inter-test inconsistency is due to real construct dif-
ferences among tests and how much of it is caused by temporal
changes in the emotional state of an animal that is submitted to
different tests at different times [19].
In view of all the aforementioned matters, several authors have
urged the behavioral neuroscience community to improve the cur-
rent methods of behavioral testing. Such an improvement could be
attained either by conceiving new tests and paradigms [15,21] or by
refining the behavioral analysis [22,33] and/or the current design
of existing tests [13,14].
In the present study, we hypothesize that a more compre-
hensive, reliable and rapid assessment of the emotional profile
of laboratory rodents could be obtained by physically combining
different existing tests that are based on the free exploration of
aversive environments. To this end, we propose a combination of
the three most widely used tests of anxiety, namely the open field
(OF), the elevated plus maze (EPM) and the light/dark box (LDB).
With this integrated test, the experimenter should be able to assess
different aspects of emotionality simultaneously, thus avoiding
undesirable effects of previous test experiences, inter-test inter-
vals and handling between tests. Two pilot studies were devised to
determine: whether or not laboratory rats would indeed explore
all areas of this integrated test; what would be the best starting
place in the apparatus; and what would be the best duration of a
test session. Once a general testing protocol was defined, another
experiment was performed to verify whether the new test, pro-
visionally called the “triple test”, would be capable of detecting
previously known genetic differences in anxiety-related behaviors.
Subsequently, factor analyses were performed on behavioral data
obtained from the OF, EPM and LDB, used either separately or in
combination, in order to elucidate the different behavioral dimen-
sions assessed under each test condition. Finally, pharmacological
experiments were carried out to investigate the effects of three
anxiety-modulating drugs on the new combined apparatus.
2. Materials and methods
2.1. Animals
A total of 375 animals from four rat lines (Wistar, Lewis, spontaneously hyperten-
sive rats and a local heterogeneous stock) were used in a series of eight experiments.
In two pilot studies (experiments 1 and 2), Wistar rats were obtained from the
colonies of the local animal breeding center. Lewis (LEW) and spontaneously hyper-
tensive rats (SHR), used in experiment 3, are inbred strains and have been kept
in our laboratory under a system of brother-sister mating for more than 20 gen-
erations. In experiment 4, a heterogeneous stock, also born in our laboratory, was
used. It derived from the intercross between three different rat lines, as previously
described [32]. Briefly, male Wistar rats were mated with female Hooded rats and
their female offspring were mated with male Lewis rats. Males and females of this
last generation were intercrossed, thus producing a genetically heterogeneous stock
of animals. For experiments 5 and 6, Wistar rats were purchased from UNIVALI (Ita-
jai, Brazil) and were used after a period of 3 weeks of rest inside our laboratory.
Their Wistar offspring, born in our laboratory, were used in experiments 7 and 8. All
animals were weaned and separated by sex at 3–4 weeks of age and thereafter were
kept in collective plastic cages (5–6 rats per cage) with food and water available ad
libitum, under a 12-h light/dark schedule (lights on at 7:00 a.m.), at 22 ± 2 ◦ C. All
animals, naı̈ve to any experimental procedure, were tested between 9 and 11 weeks
of age with all tests being carried out between 1:00 and 6:00 p.m. All procedures
were carried out according to the guidelines of the local Commitee for Animal Care
in Research (CEUA/UFSC).
2.2. Test apparatus
The new test apparatus (Fig. 1) was an integration of three well-known tests
of anxiety/emotionality, namely the open field (OF), elevated plus maze (EPM) and
light/dark box (LDB).
2.2.1. Single format
In experiment 4, each of these tests was used in its regular, individual format,
as previously described [31]. Briefly, the OF was made of wood covered with white
Fig. 1. Diagram representing a top view of the new integrated test, which is a com-
bination of the OF (left), EPM (middle) and LDB (right) tests. All three apparatuses
are positioned at the same height (52 cm from the floor) and are interconnected
so that animals can freely move in all areas. Thicker lines represent 1.5-cm-thick
walls; intermediate lines represent the edges of the EPM open arms; and thinner
lines represent the lines drawn on the apparatuses’ floors (see Section 2 for more
details).
impermeable formica, had a square floor of 100 cm × 100 cm (divided by black lines
into 25 squares of 20 cm × 20 cm) and white walls, 40-cm high. The EPM was made
of wood covered with black formica and had four elevated arms (52 cm from the
floor) 50-cm long and 10-cm wide. The arms were arranged in a cross-like disposi-
tion, with two opposite arms being enclosed (by 40-cm high walls) and two being
open, having at their intersection a central platform (10 cm × 13.5 cm) which gave
access to any of the four arms. The open arms were surrounded by a raised ledge
(1-mm thick and 5-mm high) to avoid rats falling off the arms. Finally, the LDB was
made of wood covered with formica and presented two compartments. One larger
(27 cm × 27 cm × 27 cm) and white, with the floor divided by black lines in nine
squares (9 cm × 9 cm), being strongly illuminated by a 40-W white bulb. The other
smaller (27 cm × 18 cm × 27 cm high) and black, with the floor divided by white lines
in six squares (9 cm × 9 cm), being illuminated by a 40-W red bulb. Both white and
red bulbs were located 30 cm above the apparatus’ floor, thus providing a 750-lx
intensity inside the light compartment and a 25-lx intensity inside the dark com-
partment. The two compartments, separated by a wall, were connected by a small
square opening of 7 cm × 7 cm.
2.2.2. Integrated format
In all the other experiments (1–3 and 5–8), the tests were used in their new,
integrated format (Fig. 1), where the OF, EPM and LDB apparatuses were physically
connected by placing them side by side at the same height (52 cm from the floor) and
by adding rectangular openings with 10 cm of width at the interface between their
respective less aversive areas (i.e. a 10 cm × 40 cm opening between the periphery
of the OF and one enclosed arm of the EPM and a 10 cm × 7 cm opening between
the other enclosed arm of the EPM and the dark compartment of the LDB). This
arrangement was intended to encourage transitions between tests. As shown in
Fig. 1, the connections between the OF and EPM and between the EPM and LDB
were positioned exactly in the mid portions of the OF and LDB walls, respectively.
A dim light in the test room plus the LDB lights provided 10 lx in the OF, 15 lx in the
EPM and 15 and 700 lx in the dark and light areas of the LDB, respectively.
2.3. Testing protocols
2.3.1. Experiment 1
This was a pilot study designed to verify whether the animals would really
explore all three apparatuses as well as to address two basic aspects of the new
triple test: the starting point and the duration of the testing sessions. To this end,
24 Wistar females were randomly dived in three experimental groups that were
placed in one of three starting points: the center of the OF; the central platform of
the EPM facing an open arm; and the white compartment of the LDB. The following
behaviors were registered through direct visual observation from 0 to 15 and from 15
to 30 min. In the OF: number of peripheral squares (adjacent to the walls) crossed
(outer locomotion) and number of central squares (away from the walls) crossed
(inner locomotion). In the EPM: the number of entries with all four paws inside
each type of arm and the time spent with all four paws inside the open arms. In the
LDB: time spent with all four paws in each compartment, and number of transitions
between compartments (one entry into the light, plus one return to the dark). The
apparatus was cleaned with water using a wet sponge and a paper towel before the
introduction of each animal, a procedure that was kept for all experiments.
2.3.2. Experiment 2
Once defined, based on experiment 1, that a 15-min trial initiated in the OF was
enough to produce exploration of all three apparatuses, a second pilot study was
designed to determine the best starting point inside the OF and to investigate the
effects of even shorter trials. To this end, 20 Wistar males were randomly divided in
two experimental groups that were placed either in the center or the periphery (at
the corner of two walls, away from the EPM opening) of the OF. The same behaviors
listed above were measured from 0 to 5, 5 to 10 and 10 to 15 min.
2.3.3. Experiment 3
Once defined, based on the two pilot studies, that the standard test sessions
would start in the center of the OF and last 15 min, an experiment was performed to
 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288 279

Fig. 2. Behavioral variables representing the general exploration level in the three main areas of the triple test: total locomotion in the OF (A), total number of arm entries in
the EPM (B) and total number of transitions in the LDB (C). Bars and vertical lines (top) represent the means and S.E.M. of the behavioral scores accumulated during 30 min,
for Wistar female rats grouped (n = 8) according to the point where they started the test (OF, EPM or LDB). Symbols and error bars connected by lines (bottom) represent the
means and S.E.M. of the behavioral scores accumulated during each half of the test session. No significant effects of the starting point were found. (***) Represents significant
effects of test duration (0–15 vs. 15–30 min, two-way mixed ANOVA, p < 0.001).

investigate whether rats of different genotypes, known to display consistent genetic 2.3.5. Experiments 6–8
differences in anxiety-related behaviors from each of the individual tests [28,29,31], These three experiments were carried out to assess the effects of three phar-
would show similar differences also in the triple test. To this end, 19 LEW rats (12 macological treatments on the behaviors measured in the new triple test. To this
females and 7 males) and 21 SHR rats (11 females and 10 males) were tested in the end, Wistar male rats were submitted to this test after being injected with either a
integrated apparatus, with those behaviors registered in the previous experiments vehicle or the assigned pharmacological compound. Experiments 6 and 7 included
being measured for the total period of 15 min. Males and females were tested in two benzodiazepine drugs known to have anxiolytic-like effects in the EPM, chlor-
alternate days. diazepoxide and midazolam, respectively [7], whereas experiment 8 assessed the
anxiogenic-like effects of pentylenetetrazole. The same protocol used in exper-
2.3.4. Experiments 4 and 5 iment 5 was used in this series of experiments, excepting that here behavior
In order to get an insight into the different behavioral dimensions assessed in was recorded through the use of two video cameras mounted above the appara-
the triple test, in comparison with the underlying behavioral dimensions of the OF, tus (one for the OF and EPM and one for the LDB) and monitored on a TV set
EPM and LDB applied as separate tests, two experiments were carried out using two by an experimenter located in an adjacent room. Adaptation of this protocol to
independent groups of 60 rats (30/sex). In both cases, behavioral data were submit- automated video-tracking systems needs to be considered in future studies. Test
ted to a principal component analysis, as described below. In experiment 4, each order was totally counterbalanced for treatment condition and the experimenter
animal was submitted to the three separate tests successively, with 1-week interval who scored behavior was not informed about the treatment applied to each ani-
between tests. The apparatuses were set up individually, as already described, each mal.
test session lasted 5 min and all aforementioned variables – plus the number of fecal
boli (defecation) dropped in the OF and the number of squares crossed (locomotion)
in each compartment of the LDB – were registered through direct visual observation. 2.4. Drugs
The same general procedure was used in experiment 5, except for the fact that in the
Chlordiazepoxide (CDZ) (Centaur, India) was suspended in 0.5% methylcellu-
latter each animal was submitted once to a single 15-min session in the triple test,
lose (methocel), as previously described [39], and administered intraperitoneally
starting in the center of the OF. Males and females were tested in alternate days.
(i.p.) (2 ml/kg) 30 min prior to the test, at doses of 0.5, 5 and 10 mg/kg. The control
group received a similar volume of 0.5% methocel. Injection volume and tim-
Table 1 ing were chosen based on two previous rat studies testing the effects of CDZ
Behavioral scores (X ± S.E.M.) of female Wistar rats (n = 24) measured at the three in the EPM and LDB [8,25]. The dose range was designed to provide at least
different areas of the triple test from 0 to 15 and 15 to 30 min of test one dose that was known to be effective in the OF and EPM (5 mg/kg) [8,25,39],
one dose that had shown anxiolytic effects in the LDB (10 mg/kg) [8] plus one
Test area Behavior 0–15 min 15–30 min F(1,21) lower dose (0.5 mg/kg) [10]. Midazolam (MDZ) (Cristalia, Brazil) was dissolved
in saline (NaCl 0.9%) and administered i.p. (1 ml/kg) 30 min prior to the test,
OF Inner locomotion 9.0 ± 2.9 3.6 ± 1.4** 8.88
at doses of 0.1875, 0.375 and 0.750 mg/kg. The control group received a simi-
OF Outer locomotion 91.3 ± 13.5 41.4 ± 8.0*** 30.56
lar volume of saline. Volume, doses and timing of MDZ treatment were based
EPM Closed arm entries 22.0 ± 2.2 13.6 ± 2.0** 13.92
in a previous rat study [2] showing anxiolytic effects of this drug in the EPM,
EPM Open arm entries (%) 6.8 ± 1.1 5.0 ± 1.8 1.47
with the present dose range being slightly narrower than the one used in that
EPM Time in open arms (s) 12.9 ± 3.6 9.6 ± 3.9 1.79
previous report (i.e. 0.187–0.75 instead of 0.125–1.0 mg/kg) due to the smaller num-
LDB Time in light (s) 13.8 ± 3.0 6.0 ± 2.4*** 23.35
ber of treatment groups available in the present study. Pentylenetetrazole (PTZ)
LDB Time in dark (s) 251.4 ± 45.8 289.3 ± 63.4 0.57
(Sigma Aldrich, Germany) was dissolved in saline (NaCl 0.9%) and administered
OF = open field; EPM = elevated plus maze; LDB = light/dark box. (**) and (***) repre- i.p. (2 ml/kg) 5 min before testing, as previously described in three rat studies
sent a significant decrease in behavior from first (0–15 min) to second (15–30 min) [12,25,28], at doses of 10 and 20 mg/kg, with the control group receiving a simi-
half of the test session (p < 0.01 and p < 0.001, respectively; time effect in a two-way lar volume of saline. These doses were shown to be effective in rats tested in the
mixed ANOVA). EPM [12,25].
280 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288

Table 2
Behavioral scores (X ± S.E.M.) of male Wistar rats (n = 20) measured at the three different areas of the triple test from 0 to 5, 5 to 10 and 10 to 15 min of test

Test area Behavior 0–5 min 5–10 min 10–15 min F(2,36)

OF Inner locomotion 7.6 ± 1.1 8.5 ± 1.1 5.8 ± 1.1 2.11

OF Outer locomotion 57.1 ± 4.0 45.7 ± 2.9** 29.3 ± 2.5*** 33.66
EPM Closed arm entries 10.0 ± 0.7 11.4 ± 0.9 9.2 ± 1.0 2.74
EPM Open arm entries (%) 15.6 ± 2.6 6.9 ± 1.6* 6.4 ± 2.3** 5.15
EPM Time in open arms (s) 14.3 ± 1.8 7.6 ± 1.9* 7.0 ± 2.3* 4.50
LDB Time in light (s) 0.1 ± 0.1 0.5 ± 0.4 0.5 ± 0.3 2.20
LDB Time in dark (s) 13.5 ± 3.1 29.0 ± 4.0** 24.1 ± 4.7* 4.90

OF = open field; EPM = elevated plus maze; LDB = light/dark box. (*), (**) and (***) represent a significant change in behavior in comparison with the first 5 min of the test
session (p < 0.05, p < 0.01 and p < 0.001, respectively; time effect in a two-way mixed ANOVA followed by Duncan test).

2.5. Statistics
In experiments 1 and 2, two-way mixed ANOVAs for repeated measures were
used to analyze the effects of the starting point (OF, EPM, LDB in experiment 1; inner
vs. outer area of the OF in experiment 2) and the effects of time (0–15 vs. 15–30 min
in experiment 1; 0–5, 5–10, 10–15 min in experiment 2). Whenever necessary, the
mean values were compared using the Duncan test. Data from experiment 3 were
submitted to a two-way ANOVA for strain (LEW and SHR) and sex effects. Duncan
test was used for post hoc comparisons whenever a significant interaction between
strain and sex was detected. Data from experiments 4 and 5 were analyzed using
Pearson correlation and principal component analysis (PCA), followed by an orthog-
onal Varimax rotation, to investigate the relationship among data obtained from the
three tests used separately or in combination. In order to keep a balanced repre-
sentation of each test, the PCA included four variables from each test. Only factors
with eigenvalues greater than 1 were kept. Finally, a one-way ANOVA for treatment
effect, followed by Duncan test whenever necessary, was used in experiments 6–8.
All analyses were performed using the Statistica® 8.0 software package.
3. Results
3.1. Experiment 1
The results of the first pilot study are presented in Fig. 2 and
Table 1. The two-way mixed ANOVA revealed no significant effects
of the starting point (OF, EPM or LDB) and of interaction between
starting point and testing time (0–15 and 15–30) on any of the
behavioral variables considered. However, animals starting the test
session in the OF tended to explore more all the three apparatuses
than animals starting in the EPM or LDB (see Fig. 2). Actually, 100% of
the animals starting in the OF visited all three apparatuses within
the first 15 min of test. Conversely, some animals starting in the
EPM never visited the OF and took more than 15 min to visit the
LDB and some animals starting in the LDB never visited the OF and
never re-entered the light compartment of the LDB. Together, these
preliminary data suggest that starting the test session inside the OF
area would be the best choice to induce maximum exploration of
the whole integrated apparatus.
The same ANOVA revealed a significant decrease from the first
to the second half of the testing session for total OF locomo-
tion (F(1,21) = 32.98, p < 0.001), total EPM arm entries (F(1,21) = 15.35,
p < 0.001) and total LDB transitions (F(1,21) = 20.75, p < 0.001) (see
Fig. 2). This negative time effect was also found for inner OF loco-
motion (F(1,21) = 8.88, p < 0.01), outer OF locomotion (F(1,21) = 30.56,
p < 0.001), EPM closed-arm entries (F(1,21) = 13.92, p < 0.01) and time
in the light compartment of the LDB (F(1,21) = 23.35, p < 0.001) (see
Table 1). Together, these results suggest that the test session could

Fig. 3. Behavioral variables representing the general exploration level in the three main areas of the triple test: total locomotion in the OF (A), total number of arm entries in
the EPM (B) and total number of transitions in the LDB (C). Bars and vertical lines (top) represent the means and S.E.M. of the behavioral scores accumulated during 15 min,
for Wistar male rats grouped (n = 10) according to the point where they started the test (inner or outer area of the OF). Symbols and error bars connected by lines (bottom)
represent the means and S.E.M. of the behavioral scores accumulated during each 5-min period of the test session. No significant effects of the starting point were found. (*)
and (***) Represent significant effects of test duration (0–5 vs. 5–10 or 10–15 min, two-way mixed ANOVA followed by Duncan test, p < 0.05 and p < 0.001, respectively).
 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288 281

Fig. 4. Behavioral variables representing anxiety- (A-F) and locomotion-related (G-H) behaviors in the three main areas of the triple test: inner locomotion in the OF (A), %
entries (B) and time spent (C) in the open arms of the EPM, time spent in the light compartment (D), number of transitions (E) and time spent in the dark compartment (F)
of the LDB, outer locomotion in the OF (G) and number of closed-arm entries in the EPM (H). Bars and vertical lines represent the means and S.E.M. of the behavioral scores
accumulated during 15 min for animals grouped by strain (LEW and SHR) and sex (F and M) (n = 7–12). (*), (**) and (***) Represent significant differences between strains.
(#), (##) and (###) Represent significant differences between sexes (two-way ANOVA, p < 0.05, p < 0.01 and p < 0.001, respectively, with Duncan post hoc test being used in
G only due to a significant strain × sex interaction).

be limited to the first 15 min if one wants to avoid a drastic drop in
the animals’ general exploration level.
3.2. Experiment 2
The results of this experiment are shown in Fig. 3 and Table 2. The
two-way mixed ANOVA revealed no significant effects of the start-
ing point (inner vs. outer area of the OF) and of interaction between
starting point and testing time (0–5, 5–10 and 10–15) on any of the
behavioral variables considered. Overall, there was a slight trend
towards higher exploration levels of all apparatuses in rats starting
the test session in the inner area of the OF (Fig. 3). As one of the two
OF starting points needed to be chosen, the central square seemed
to be the safest choice.
282 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288

Table 3
Factor analysis (PCA) of 12 behavioral variables measured in 60 rats (30/sex) from a genetically heterogeneous stock tested in the open field (OF), elevated plus maze (EPM)
and light/dark box (LDB) as separate tests

Test/behavior Factor 1 Factor 2 Factor 3 Factor 4 Factor 5

OF
Inner locomotion 0.96
Outer locomotion −0.76
Inner locomotion (%) 0.92
Defecation 0.78

EPM
Time in open arms (s) 0.49 0.72
Open arm entries (%) 0.93
Closed arm entries 0.89
Total arm entries 0.88

LDB
Time in light (s) 0.91
Transitions 0.92
Locomotion in light 0.91
Locomotion in dark 0.71

% of total variance 33.6 15.6 14.4 12.9 8.6

Factor loadings higher than 0.4, produced by a varimax rotation, are shown for each factor.

The same ANOVA showed a significant decrease along the 15 min
of test for total OF locomotion (F(2,36) = 27.96, p < 0.001) and total
EPM arm entries (F(2,36) = 3.29, p < 0.05) (results of the correspond-
ing post hoc tests are shown in Fig. 3). This negative time effect was
also found for outer OF locomotion (F(2,36) = 33.66, p < 0.001) and %
entries (F(2,36) = 5.15, p < 0.05) and time spent (F(2,36) = 4.50, p < 0.05)
in the open arms of the EPM (results of the corresponding post hoc
tests are shown in Table 2). On the other hand, several other vari-
ables did not show any clear tendency towards a decrease along the
15 min of test (Fig. 3 and Table 2). Finally, there was an increase in
the time spent in the dark compartment of the LDB (F(2,36) = 4.90,
p < 0.05) throughout the test session. Together, these results sug-
gest that limiting the test session to the first 5 or 10 min may not be
advantageous in relation to the proposed 15-min trial if one wants
to assess a range of behaviors as wide as possible.
3.3. Experiment 3
The results of experiment 3 are shown in Fig. 4. There was a sig-
nificant strain × sex interaction (F(1,37) = 11.74, p < 0.01) for the outer
locomotion in the OF, with post hoc tests showing that LEW males
had lower scores when compared with both SHR males (p < 0.01)
and LEW females (p < 0.001). For all the other variables, the two-
way ANOVA revealed a significant effect of strain, regardless of
sex. SHR rats scored higher than LEW for inner locomotion in the
OF (F(1,37) = 46.42, p < 0.001), % entries (F(1,37) = 5.98, p < 0.05) and
time spent (F(1,37) = 4.91, p < 0.05) in the open arms of the EPM,
and time spent in the light (F(1,37) = 10.07, p < 0.01) and number of
transitions (F(1,37) = 20.05, p < 0.001) in the LDB. LEW rats scored
higher than SHRs for the time spent in the dark compartment of
the LDB (F(1,37) = 24.42, p < 0.001) and number of closed arm entries
in the EPM (F(1,37) = 14.24, p < 0.001). Overall sex effects were found
for inner locomotion (F(1,37) = 8.52, p < 0.01), % entries in the open
arms (F(1,37) = 5.86, p < 0.05), time spent in the dark (F(1,37) = 5.11,
p < 0.05) and number of transitions (F(1,37) = 4.79, p < 0.05). In all
cases, females approached more the aversive environments than
males (Fig. 4).
3.4. Experiments 4 and 5
As shown in Tables 3 and 4, the two PCA performed on a set of
12 behavioral variables (four from each test), representing different

Table 4
Factor analysis (PCA) of 12 behavioral variables measured in 60 rats (30/sex) from a Wistar stock tested in the open field (OF), elevated plus maze (EPM) and light/dark box
(LDB) integrated as parts of the triple test

Test/behavior Factor 1 Factor 2 Factor 3 Factor 4 Factor 5

OF
Inner locomotion 0.93
Outer locomotion 0.95
Inner locomotion (%) 0.96
Defecation

EPM
Time in open arms (s) 0.95
Open arm entries (%) 0.97
Closed arm entries 0.97
Total arm entries 0.87

LDB
Time in light (s) 0.95
Transitions 0.91
Locomotion in light 0.94
Locomotion in dark 0.54 0.49 −0.40

% of total variance 36.3 16.2 14.9 12.5 9.8

Factor loadings higher than 0.4, produced by a varimax rotation, are shown for each factor.
 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288 283

Fig. 5. Correlations among six selected variables from the OF, EPM and LDB tests applied either separately (single) or in combination (triple). Two variables potentially
related with anxiety/emotionality were selected from each test: inner locomotion and defecation in the OF; time spent and % entries in the open arms of the EPM; number
of transitions and locomotion in the light compartment of the LDB. Bars represent the correlation coefficients (Pearson r) for each pair of variables measured under the two
different testing conditions. (*) Represent a significant (p < 0.05) correlation.

facets of the behavioral reactivity of rats submitted to the OF, EPM
and LDB applied individually (Table 3) or in combination (Table 4),
produced very similar factor structures. In both situations, five
factors emerged, representing 85.1 and 89.7% of the total variabil-
ity, under the single- and triple-test conditions, respectively. The
factor loadings for each behavioral measure, which represent the
correlation between the measure and a specific factor, are shown.
To facilitate the interpretation, only the more significant loadings
(higher than 0.4) are presented. In both conditions, factor 1 received
the most significant contributions from anxiety-related measures
of the LDB; factor 2 was essentially related to the approach towards
the central area of the OF; and factor 4 received high loadings from
the two main indices of anxiety of the EPM. Also in both cases, fac-
tor 3 correlated with locomotion-related variables from the EPM
and LDB, being slightly contaminated by the time spent in the open
arms of the EPM under the single-test condition. Factor 5, which
represented less than 10% of the total variability, related to outer
locomotion in the OF and either defecation (single) or locomotion in
the dark (triple condition). This last variable, while relating mostly
with the locomotion-related factor (factor 3) in the individual tests,
loaded reasonably evenly over factors 1, 3 and 5 in the triple test.
Fig. 5 illustrates a comparison between the new triple test and
the original single tests in terms of the correlation coefficients of a
matrix made of six selected variables (two from each test) which
284 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288

Fig. 6. Anxiety-related behaviors of Wistar male rats treated with either vehicle or different doses of chlordiazepoxide (CDZ at 0.5, 5 or 10 mg/kg, top), midazolam (MDZ
at 0.1875, 0.375 or 0.750 mg/kg, middle) or pentylenetetrazole (PTZ at 10 or 20 mg/kg, bottom). Behavioral variables were measured in a 15-min session in the triple test:
inner locomotion in the OF, % entries and % time spent in the open arms of the EPM, locomotion in the light compartment and number of transitions in the LDB. Bars and
vertical lines represent the means and S.E.M. of animals grouped by pharmacological treatment (n = 13–16). (*), (**) and (***) Represent significant differences between a
given treatment and its respective control group (0 mg/kg) (ANOVA followed by Duncan post hoc test, p < 0.05, p < 0.01 and p < 0.001, respectively).

are potentially related with anxiety/emotionality. Major increases
in inter-test correlations, due to the tests being applied combined
rather than individually, were only observed between defecation
in the OF and each of the other variables (negative correlations)
and between the % entries in the open arms of the EPM and
each of the two variables from the LDB (positive correlations). In
all these cases, significance levels were only attained when the
tests were applied in their integrated format. Correlations between
inner OF locomotion and anxiety-related measures from the EPM
were very low in both testing conditions, whereas some improve-
ment (although not reaching significance) could be observed
between the former variable and anxiety-related measures from
the LDB in the triple test. Finally, the time spent in the open
arms of the EPM was significantly and positively related with both
anxiety-related measures from the LDB, regardless of the testing
condition.
3.5. Experiments 6–8
Results of experiments 6, 7 and 8 are combined in Fig. 6
(anxiety-related measures) and 7 (locomotion-related measures).
The one-way ANOVAs for the behavioral effects of CDZ (experi-
ment 6) revealed no significant effects for the OF variables but a
significant drug effect on the % entries (F(3,60) = 5.98, p < 0.01) and
% time spent (F(3,60) = 3.03, p < 0.05) in the open arms (Fig. 6) and
on the number of entries in the closed arms (Fig. 7) (F(3,60) = 5.13,
p < 0.01) of the EPM. A significant drug effect was also found for the
locomotion in the dark part of the LDB (F(3,60) = 3.16, p < 0.05). Post
hoc tests comparing treatments with the control group indicated
that the intermediate dose of 5 mg/kg increased (p < 0.05) the first
two variables and had no effects on the third variable. The highest
dose of 10 mg/kg, however, had significant (p < 0.05) effects on all
three EPM variables, increasing preference for the open arms and
decreasing closed-arm entries. This same dose also decreased the
locomotion in the dark compartment of the LDB (p < 0.05) (Fig. 7).
Results from the MDZ experiment (experiment 7) showed
basically the same profile as the previous experiment, with no
significant treatment effects on the OF variables and a significant
drug effect on the % entries (F(3,62) = 4.34, p < 0.01) and % time spent
(F(3,62) = 3.73, p < 0.05) in the open arms (Fig. 6) and on the num-
ber of entries in the closed arms (Fig. 7) (F(3,62) = 4.46, p < 0.01)
of the EPM. Post hoc tests showed that only the highest dose of
0.75 mg/kg affected those behaviors, increasing (p < 0.01) prefer-
ence for the open arms and decreasing (p < 0.05) closed-arm entries.
No significant treatment effects were found for the LDB variables.
Finally, the analysis of the PTZ data (experiment 8) showed
significant effects of this compound on all behaviors, except
for locomotion in the dark compartment of the LDB. The one-
way ANOVA effects were the following: OF inner locomotion
(F(2,35) = 5.11, p < 0.05);% entries (F(2,35) = 9.89, p < 0.001) and % time
spent (F(2,35) = 7.82, p < 0.01) in the open arms of the EPM; locomo-
tion in the light compartment (F(2,35) = 4.02, p < 0.05) and transitions
(F(2,35) = 3.76, p < 0.05) in the LDB (Fig. 6); OF outer locomotion
(F(2,35) = 20.83, p < 0.001); and EPM closed-arm entries (F(2,35) = 6.91,
p < 0.01) (Fig. 7). Post hoc tests revealed that 10 mg/kg of PTZ caused
significant reductions only on the inner locomotion in the OF
(p < 0.05), % entries (p < 0.01) and % time spent (p < 0.01) in the open
arms of the EPM and locomotion in the light compartment of the
LDB (p < 0.05) (see Fig. 6). On the other hand, the highest dose of
20 mg/kg had diminishing effects on all variables (p < 0.05), except
for the locomotion in the dark part of the LDB (Figs. 6 and 7).
4. Discussion
The results of the present study reinforce the assumption that
the physical integration of three well-known tests of anxiety – the
OF, EPM and LDB – into one combined apparatus can be useful for
the experimental study of anxiety. Rats of both sexes and from dif-
ferent genetic backgrounds were shown to freely explore all areas
of this combined test within a timeframe that is compatible with
 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288 285

Fig. 7. Locomotion-related behaviors of Wistar male rats treated with either vehicle or different doses of chlordiazepoxide (CDZ at 0.5, 5 or 10 mg/kg, top), midazolam (MDZ
at 0.1875, 0.375 or 0.750 mg/kg, middle) or pentylenetetrazole (PTZ at 10 or 20 mg/kg, bottom). Behavioral variables were measured in a 15-min session in the triple test:
outer locomotion in the OF, number of entries in the closed arms of the EPM, locomotion in the dark compartment of the LDB. Bars and vertical lines represent the means and
S.E.M. of animals grouped by pharmacological treatment (n = 13–16). (*), (**) and (***) Represent significant differences between a given treatment and its respective control
group (0 mg/kg) (ANOVA followed by Duncan post hoc test, p < 0.05, p < 0.01 and p < 0.001, respectively).

high-throughput behavioral testing. Once the basic protocol was
defined, 97.5% of all the animals submitted to the triple test visited
all three apparatuses during the regular 15-min trial. Two inbred rat
strains, LEW and SHR, known to display high and low anxious-like
profiles, respectively, in all three individual tests, also differed in a
consistent manner for all anxiety-related measures from the triple
test. Multivariate analyses suggested that the three tests used in
combination assess the same basic behavioral dimensions as those
assessed by the individual tests. Finally, pharmacological data pro-
vided further support to the use of the triple test in the preclinical
investigation of anxiety.
The first experiment showed that rats explore the three major
areas of the triple test regardless of where they were placed in the
beginning of the test session. However, a non-significant but con-
sistent trend towards higher general exploration levels was found
in animals initially placed inside the OF. Such a tendency indicated
that the OF could be considered as a good starting point for this test.
As far as the test duration is concerned, most standard protocols
of the OF, EPM and LDB use sessions of 5 min [9,25,26]. Thus, one
could suppose that a 15-min session should be adequate to evaluate
animals in an integrated set including the three tests. It was shown
here, indeed, that a 15-min period was enough for all animals start-
ing in the OF to visit all three apparatuses. Increasing the time from
15 to 30 min, however, produced a drop in most behavioral scores,
which is suggestive of habituation [1]. The further refinement of the
new protocol, which was the object of a second study, showed that
starting the test session in either the center or the periphery of the
OF did not cause any changes in behavior, although a slight trend
towards higher general exploration was seen in animals starting in
the inner OF area. Reducing even further the test sessions to either
10 or 5 min did not bring a real benefit to the test. Whereas some
behaviors dropped with time, probably as a result of familiarization,
others remained stable or even tended to increase throughout the
15 min of test. In summary, the present results, together with pre-
liminary pharmacological data (not shown), suggested that 15-min
sessions would be adequate for allowing a satisfactory expression
of all behaviors. Thus, from the two pilot studies on, a protocol
of 15 min starting in the center of the OF was chosen for the new
integrated test.
The results obtained with the inbred strains LEW and SHR
(experiment 3) corroborated the hypothesis that this new paradigm
is capable of detecting genetic differences in anxiety-related behav-
iors. LEW and SHR rats have been used as a genetic model of anxiety
for more than 10 years based on the fact that they respond dif-
ferently to several anxiety- and stress-related tasks, including the
OF, EPM and LDB [3,4,28,29,31]. LEW rats, when compared with
SHRs, have repeatedly shown higher avoidance of the aversive
compartments of each individual test [28,29,31]. Pharmacological
experiments indicated that such differences were anxiety-related
[28,39], whereas genetic analyses showed them to result from the
direct effects of genes [29,30]. The present results are in total agree-
ment with the aforementioned previous studies, as LEW rats of both
sexes, when tested in the triple test, were shown to ambulate less
in the inner OF area, to do less entries and spend less time in the
EPM open arms, to spend less time in the light and more time in
the dark compartments of the LDB and to perform less transitions
in the LDB.
As far as locomotion measures are concerned, the present results
are not in total agreement with previous studies, which have shown
LEW and SHR rats not to differ in general locomotion in the OF
[28,29,31,39]. Herein, SHR males displayed more peripheral loco-
motion in the OF than LEW males. In the EPM, on the other hand,
286 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288
LEW rats of both sexes did more closed-arm entries than SHRs.
This is in agreement with some previous studies [20,39] but not
with others [28,29,31], which showed no strain differences for this
measure. It is important to recognize that the new test is not only
a sum of the individual tests, because in each separate appara-
tus an animal would not have the choice to exit the test area,
whereas here it can choose to be anywhere at any point in time.
This fact, added to other unavoidable design and protocol differ-
ences, might account for any inconsistency between the triple test
and the regular single tests. However, the present strain results
strongly suggest that many similarities exist between these two
testing conditions.
In order to further investigate the degree of similarity between
the triple and single test conditions, principal component analy-
ses (PCA) were performed for each situation. The results showed a
striking similarity between the five-factor solutions found in both
experimental conditions. The LDB, OF and EPM, applied either sepa-
rately or in combination, produced three independent, test-specific
anxiety factors (factors 1, 2 and 4, respectively), if one considers
that the variables loading on these factors have been validated as
indices of anxiety [9,10,25,26]. Factor 3, on the other hand, could be
seen as a locomotion-related factor, for it related with measures of
locomotion in two protected areas, the closed arms of the EPM [12]
and the dark part of the LDB. Outer OF locomotion loaded heavily
on Factor 5, together with either defecation or locomotion in the
dark, suggesting that this factor is at least partially related with
locomotion. These results are in agreement with previous factor
studies that showed that different anxiety tests seem to reflect dif-
ferent facets of emotionality [16,29,38]. The present PCA analyses
also suggest that the triple test preserves important features of its
individual components and therefore it can be used to assess differ-
ent types of emotional states as well as locomotor activity in novel
but not highly aversive areas. The main difference between the fac-
tors obtained under the two testing conditions was related to the
locomotion in the dark part of the LDB, which loaded only on factor
3 in the individual tests, but on factors 1, 3 and 5 in the triple test.
Factor 1 reflected mostly the approach towards the LDB light com-
partment. Thus, the relatively high loading (0.54) of locomotion in
the dark compartment on this factor means that animals exploring
more the light also explored more the dark LDB areas. This rela-
tionship makes sense in the triple test only, where there is a choice
between the LDB and the other test areas and a higher preference
for the LDB, as a whole, might cause an increase in all LDB variables.
The correlation analyses further clarified the relationships
among variables from the three different tests applied either
individually or in combination. Fig. 5 shows the correlation coef-
ficients among six measures (two from each test) selected for
being anxiety- or emotionality-related [9,10,18,25,26]. Because in
the triple apparatus an animal can express its emotional state in all
tests within the timeframe of a single trial, one could expect the
correlations to increase in comparison with the single-test condi-
tion. Such an increase, however, was only observed in some pairs
of variables, such as OF defecation in relation to all the others vari-
ables and % of entries in the open arms of the EPM in relation to
the two variables from the LDB. Defecation was the first measure
of emotionality proposed by Hall [18], but it is rarely considered
as an index of anxiety in pharmacological studies [26,27]. Yet, the
present results suggested a mild but significant negative correla-
tion between OF defecation and classical anxiety measures from
the EPM and LDB. These, in turn, seem to be more strongly related to
each other, which is compatible with their extensive use as anxiety
models [13,17,27], than with the most popular measure of anxiety
from the OF. Actually, the central OF locomotion was not signif-
icantly correlated with any of the other variables, regardless of
the experimental condition. Overall, the present data suggest that
the correlation between emotionality measures from different tests
varies from low (r < 0.25) to moderate (r < 0.5) and that testing ani-
mals in an integrated apparatus may improve such a correlation in
some cases.
Finally, a preliminary pharmacological investigation was carried
out using three different drugs with known effects on anxiety-
related behaviors. The benzodiazepine chlordiazepoxide (CDZ), at
5 mg/kg, produced anxiolytic-like effects on two anxiety measures
from the EPM (% entries and % time spent in the open arms), thus
confirming previous studies [8,25,39], without showing any influ-
ences on other variables, either locomotion- or anxiety-related. At
10 mg/kg, CDZ produced the same anxiolytic-like effects but also
seemed to provoke sedation, as shown by the reduced number of
closed-arm entries in the EPM and of squares crossed in the dark
compartment of the LDB. The effects of midazolam (MDZ) were very
similar to those of CDZ, except that only the highest dose of MDZ
(0.75 mg/kg) had significant anxiolytic-like effects and these were
not devoid of sedative influences in the EPM. Such anxiolytic and
sedative effects agree with a previous study using similar doses
of MDZ in the regular EPM [2]. However, based on that previous
study, an anxiolytic-like effect was expected to be seen also with the
present intermediate dose (0.375 mg/kg), but this was not observed
herein. In summary, the present results are consistent with the
known anxiolytic effects of CDZ and MDZ and the possible sedative
effects of CDZ in the common EPM [7,12,25,39]. Nevertheless, these
anti-anxiety agents did not have the expected effects on behav-
iors measured in the OF and LDB areas. Two alternative hypotheses
could be put forward to explain this lack of effect: (1) in rats, the
OF and LDB, even individually, are not consistently sensitive to ben-
zodiazepines; (2) these tests, individually, respond consistently to
benzodiazepines, but when physically combined they change their
nature and loose such sensitivity.
In the single OF test, anxiolytic effects of acute CDZ and MDZ
treatments were found in about only half of the rat studies reviewed
by Prut and Belzung [26], which brings some support to the first
aforementioned hypothesis. Such an inconsistency, however, might
be partially explained by the huge inter-laboratory variation found
in OF protocols [36]. Yet, data from our own laboratory [39] have
shown that CDZ at 5 mg/kg can have anxiolytic-like effects in rats
tested in the single OF test, depending on the genotype and gender
of the animals. As far as the LDB is concerned, CDZ and other benzo-
diazepines have been shown to increase exploration of mice in the
aversive light compartment [9,10], an effect also produced by sero-
tonin reuptake inhibitors [35]. In rats, however, CDZ at 5 mg/kg,
a treatment that reduced anxiety in the EPM, did not change the
approach towards the white area of the regular LDB test [8], which
is in total agreement with the present results of the triple test.
Likewise, at 10 mg/kg, CDZ was shown to reduce the latency to
enter the light area of the LDB but it did not change other clas-
sical indices of anxiety from this test [8], which also agrees with
the present results. Citalopram, which was shown to be anxiolytic
in mice [35], had anxiogenic-like effects in the rat LDB [34] and,
in another comprehensive study, rats receiving a wide range of
anxiolytic drugs (including benzodiazepine and serotonin-related
compounds) showed no signs of anxiolysis in a LDB [5]. Together,
these results support the aforementioned hypothesis 1 and suggest
that the LDB may not be as efficient for anxiolytic drug screening
in rats as it is in mice.
The aforementioned issues need to be addressed in future
studies, by testing rats of the same strain and under the same phar-
macological treatments used herein, in each of the individual tests,
specially the OF and LDB. If hypothesis 1 is confirmed, this may
bring some concerns about the meaning of the regular individual
tests for rats. On the other hand, if hypothesis 2 is corroborated,
future studies will have to investigate the potential usefulness of
 A. Ramos et al. / Behavioural Brain Research 193 (2008) 277–288
the triple test by verifying, for example, if its OF and LDB areas
are sensitive to other classes of anxiolytic drugs, such as buspirone
and selective serotonin reuptake inhibitors, which tend to produce
inconsistent results when tested in each of the individual compo-
nents of the triple test [5,7,26]. Moreover, the effects of different
treatment schedules (i.e. acute vs. chronic) will have to be consid-
ered.
The effects of pentylenetetrazole (PTZ), on the other hand, were
clear-cut and test-independent. At 10 mg/kg, PTZ had anxiogenic-
like effects in all aversive areas of the triple test, without showing
any influences on measures of protected locomotion. It signifi-
cantly reduced inner locomotion in the OF, % entries and % time
spent in the open arms of the EPM and locomotion in the light
compartment of the LDB, but it did not affect outer locomotion,
closed-arm entries and locomotion in the dark compartment of the
three respective tests. At 20 mg/kg, however, PTZ reduced all vari-
ables (except for locomotion in the dark), which is in agreement
with its known anxiogenic and hypolocomotor effects [12,25,28].
The present pharmacological results demonstrate that the triple
test provides anxiety-related indices, which respond to anxliolytic
and/or anxiogenic treatments (e.g. CDZ 5 mg/kg and PTZ 10 mg/kg),
as well as locomotion-related indices, which are not influenced by
these treatments. However, these data should be considered with
caution because they are quite preliminary and narrow in terms of
tested compounds.
In summary, the present study demonstrates that the inte-
gration of three widely used tests of anxiety, the OF, EPM and
LDB, into one single experimental set can be useful for measur-
ing, in a relatively simple way, a wide range of unconditioned
exploratory behaviors in just one trial, minimizing common con-
cerns about previous test experiences, inter-test intervals and
handling between tests. The behaviors measured were shown to
reflect distinct emotional and motor dimensions, in line with the
recently proposed notion of “hybrid models”, which are expected
to assess different behavioral domains simultaneously [21]. The
present results also point to this new integrated paradigm as a
promising tool for genetic and pharmacological screening, as it
seems to provide a more informative snapshot of an animal’s emo-
tional status in comparison with each of the original tests. Further
studies involving behavioral, pharmacological and genetic exper-
iments will be necessary before the usefulness of this model can
be confirmed and generalized to other species, in particular to
mice.
Acknowledgements
This work was supported by a grant (Ed 612005) from
CNPq/Brazil. A. Ramos was recipient of a fellowship from CNPq and
G.S. Izidio and E. Pereira had scholarships from CNPq. The authors
wish to thank Ligia F.G. de Oliveira for her technical assistance.
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