Review

Ethological and temporal analyses of anxiety-like behavior:
The elevated plus-maze model 20 years on
A.P. Carobrez
a,
*
, L.J. Bertoglio
b
a
Departamento de Farmacologia, CCB, Universidade Federal de Santa Catarina, Floriano´polis, SC, 88049-900, Brazil.
b
Departamento de Farmacologia, Faculdade de Medicina de Ribeira˜o Preto-USP, Ribeira˜o Preto, SP, 14040-900, Brazil
Abstract
As well as being considered a reliable measurement instrument of animal anxiety-like behavior, the elevated plus-maze (EPM) is also used
as a post-hoc test to evaluate emotionality in genetically modified rodents. The present review considers factors which may further improve
the validity (predictive/face/construct) of the EPM model: (1) the importance of measuring defensive patterns of response such as risk
assessment in addition to traditional measures such as open arm time; (2) other methodological refinements such as min-by-min scoring and
use of a test/retest protocol; and (3) the identification and control of major sources of variability in this test. To estimate whether current use
of the EPM by researchers takes the above factor into account, a survey of the recent literature was conducted. Results showed that the
majority of studies have not yet assimilated these important considerations into their use of the EPM. For example, although risk assessment
measures may be more sensitive to anxiety modulating drugs than traditional measures, only a quarter of studies have adopted them. It is
hoped that this review can provide insights into the optimal use of the EPM, a simple task that can be very complex in terms of behavioral
analysis.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Anxiety; Animal models; Risk assessment; Min-by-min scoring; Test/retest protocol
Contents
1. The elevated plus-maze and ethology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1194
2. Can we study defensive behavior through the aversion elicited by the EPM? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1194
3. Refining the information gathered in the EPM test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1195
4. What is the Trial 1/Trial 2 protocol and why should we use it? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1197
5. Panorama of current EPM use: differences from the ideal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201
The elevated plus-maze (EPM) stands as one of the most
popular in vivo animal tests currently in use. Although it has
been frequently used as a tool to screen anxioselective
effects of drugs (Handley and Mithani, 1984; Pellow et al.,
1985; Lister, 1990), nowadays its usefulness has spread
towards the understanding of the biological basis of
emotionality related to learning and memory, pain,
hormones, addiction and withdrawal, as well as of the
various sub-types of anxiety disorders, such as generalized
anxiety, phobia and post-traumatic stress (Adamec et al.,
1998; File et al., 1998; Lamprea et al., 2000; Carobrez et al.,
2001; Rasmussen et al., 2001; Bannerman et al., 2004). In
addition, the EPM test has been widely used in a post-hoc
fashion as a ‘smoking gun’ piece of evidence of altered
emotionality in animals submitted to biochemical or gene
targeting manipulations (Weiss et al., 2000; Belzung and
Griebel, 2001; Holmes, 2001; Clement et al., 2002). The
EPM has also been successfully used to define brain areas
Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205
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* Corresponding author. Fax: C55 48 337 5479.
E-mail address: adepadua@farmaco.ufsc.br (A.P. Carobrez).
related to fear/anxiety (Jinks and McGregor, 1997; Treit and
Menard, 1997; File et al., 1998; Andrade et al., 1999;
Lacroix et al., 2000; Adamec et al., 2001; Carobrez et al.,
2001; Jardim and Guimaraes, 2001; Adamec et al., 2003).
It is likely that the popularity of the EPM test, with
around 2500 published papers so far (Web of Science,
2005), is due to its obvious and numerous advantages,
namely: economy, rapidity, simplicity of design and
bidirectional drug sensitivity, coupled with the fact that it
does not require lengthy training procedures or the use of
food/water deprivation or electric shock (Pellow et al.,
1985; Rodgers et al., 1997). Despite or possibly by virtue of
these advantages, there are probably as many EPM
paradigms as there are laboratories using the model in
preclinical anxiety research (Hogg, 1996; Rodgers et al.,
1997). This implies that the popularity of the EPM owes
more to practical than theoretical considerations. In view of
this fact, the present review considers important conceptual
and methodological issues which may further improve
the validity, and thus provide a more rational use, of the
EPM test as an experimental tool.
1. The elevated plus-maze and ethology
There are at least two general assumptions among groups
working with the EPM paradigm. First, that it is an
‘ethological’ animal model of anxiety, with the idea that
the aversion elicited during the task has ecological meaning
to the animal (Rodgers et al., 1997). Second, it is also
generally classified as an unconditioned type of model,
implying that the task is aversive per se, and therefore its
usefulness is in modeling unconditioned aversion, predict-
ing its application only in those anxiety states induced by
unconditioned stimuli. However, when trying to understand
the aforementioned concepts more deeply, beyond the
semantic puzzle, we found little evidence to either confirm
or deny these assumptions.
The ‘ethological’ nature of the EPM was first suggested
by the late Richard Lister (1990) and has been further
suggested since then (File, 1992; Rodgers and Dalvi, 1997;
Rodgers et al., 1997). It is assumed that this kind of test
involves situations or stimuli that are ethologically relevant
to the animal. The fact that the test involves spontaneous
exploration by rodents of the environment, in the absence of
explicit reward or consummatory behavior, accounts for its
classification as an ethological model. On the other hand, a
truly ethological model might study the complete pattern of
animal behavior across the 5 min EPM test session, an
approach which is very rare. Nevertheless, the efficacy of
the EPM in discriminating anxioselective compounds has
been increased with the adoption of a more ethological
analysis (Cruz et al., 1994; Rodgers and Johnson, 1995;
Weiss et al., 1998; Calatayud et al., 2004).
The unconditioned response characteristics seen in the
EPM task are attributed to the spontaneous fear which
the EPM elicits, given that during the regular 5 min session
there is a clear preference to be in the enclosed arms rather
than the open arms (OA). There is also substantial evidence
showing that drugs which increase OA activity are
anxiolytic compounds in other animal models while drugs
which reduce OA activity are anxiogenic. However, as
discussed further below, when the min-by-min structure of
the behavior in the EPM is taken into account, we cannot
rule out the possibility that some learned behaviors are in
fact occurring on the test, and that these influence
performance. Indeed, when analyzing behavior at the
beginning and the end of a 5 min test, the subjects may
show completely different behavior, implying that learning
may be occuring over the duration of the testing session
(see below). Therefore, the test situation will lead to a
gradually increase in the avoidance behavior and the
decision to stay inside the safer place in the maze—the
enclosed arms. If this type of response is learned through
experience, then the EPM could not be classified as a test of
unconditioned responses.
2. Can we study defensive behavior through the aversion
elicited by the EPM?
It was first argued that the reluctance of animals to
explore the open arms of the EPM was a combination of
rodent aversion to open spaces and also to the maze height
(Lister, 1990). However, other studies showed that height
alone is not a sufficiently intense anxiogenic stimulus, and
that rodent aversion to open spaces could be related to
thigmotaxis, a reaction in which the animal remains close to
vertical surfaces, as part of their natural defensive repertoire
(Falter et al., 1992; Treit et al., 1993). Moreover, recent
findings indicate that information gained from the vibrissae
do not play a key role (Cardenas et al., 2001), but that distal
cues gained through visual perception do (Martı ´nez et al.,
2002). The aversive characteristics of the open arms have
been considered sufficient to produce the typical behavioral
patterns exhibited by rodents in the EPM test (File et al.,
1990). Nevertheless, it is still a matter of debate whether
rodents’ exploratory behavior depends on the aversiveness
of the open arms only or the contrasting characteristics of
the open and the enclosed arms. More work is needed to
clarify the stimuli responsible for rodent avoidance of the
open arms of the EPM. In this context, the existence of at
least two environments with different levels of aversion,
open and enclosed arms, appears to be required for animals
to develop open arm avoidance in the EPM test (Bertoglio
and Carobrez, 2000; Salum et al., 2003).
The link between the EPM and defensive responses has
been addressed by using immunohistochemical approaches
in which markers of synaptic function (e.g. Fos protein;
Morgan and Curran, 1991) or of plastic changes in neural
transmission (e.g. pCREB; Adamec et al., 2003) have
allowed the association of defensive behavior with its neural
A.P. Carobrez, L.J. Bertoglio / Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205 1194
substrates. For instance, the amygdala, hippocampal
formation, medial hypothalamic area and midbrain peri-
aqueductal gray are some of the brain areas related to
defensive behavior which show increased expression of Fos
protein after exposure to the EPM (Silveira et al., 1993), a
predator (Canteras and Goto, 1999), the odor of a predator
(Dielenberg et al., 2001), an aversive ultrasonic sound
(Beckett et al., 1997), during fear potentiated startle
(Campeau et al., 1997) or as a result of systemic injections
of anxiogenic drugs (Singewald and Sharp, 2000).
3. Refining the information gathered in the EPM test
Behavioral responses and pharmacological effects
observed in the EPM are under the influence of several
variables, which may be divided into organismic and
procedural (Table 1). Coincidentally, these variables
correspond exactly the main sources of inter-laboratory
variation in the use of the EPM paradigm (Rodgers and
Cole, 1994; Hogg, 1996; Rodgers et al., 1997). Therefore, it
would be imperative that laboratories using, or planning to
use, the EPM test dedicate time and effort in order to define
the optimal test conditions before starting their respective
studies (Hogg, 1996; Rodgers and Dalvi, 1997; Rodgers
et al., 1997). In addition, this variability has lead to calls for
what is known as the ethological approach to data collection
(Rodgers et al., 1997; Roy and Chapillon, 2004). From such
a perspective, this section focuses on the advantages of
applying both ethological measures (instead of conventional
only) and detailed temporal analysis (min-by-min scoring)
of the rodents’ behavior in the EPM. The next section deals
with the importance of the EPM test/retest protocol.
Defensive behavior in mammals refers to any behavior
which reduces the chances of an animal being harmed
(McFarland, 1987). Defensive strategies encompass a
plethora of behavioral manifestations, including the
reactions to predators, conspecifics, certain situations and
inanimate objects (Rodgers and Dalvi, 1997). As such,
flight, freezing, startle, and defensive threat and attack
usually appear in response to proximal danger whereas
environmental features can evoke a different set of reactions
including risk assessment and avoidance behavior, more
subtle defensive adaptations (Blanchard and Blanchard,
1989). A detailed discussion of the behavioral aspects
underlying the concept of defense with respect to the EPM
can be found in the article by Rodgers and Dalvi (1997).
Of relevance to the present discussion are avoidance and
the risk assessment (RA) behaviors. Although this may
sound rather obvious, the ethological relevance of
Table 1
Variables proven to influence the behavioral and/or pharmacological response patterns observed in the EPM test
Variables References
a
Organismic
Species Pellow et al., 1985; Lister, 1987; Rex et al., 1994; Yannielli et al., 1996;
Andersen et al., 2000; Varty et al., 2002
Strain Rodgers and Cole, 1993b; Trullas and Skolnick, 1993; Ramos et al., 1997;
Voikar et al., 2001; Carola et al., 2002
Gender Johnston and File, 1991; Imhof et al., 1993; Rodgers and Cole, 1993b;
Voikar et al., 2001
Estrous cycle/lactation Mora et al., 1996; Marcondes et al., 2001
Age Imhof et al., 1993; Boguszewski and Zagrodzka, 2002; Andrade et al.,
2003
Procedural
Housing condition Rodgers and Cole, 1993b; Da Silva et al., 1996
Circadian rhythm/light cycle Griebel et al., 1993; Jones and King, 2001; Bertoglio and Carobrez, 2002b;
Andrade et al., 2003
Prior handling and injection experience Brett and Pratt, 1990; File et al., 1992; Andrews and File, 1993; Lapin,
1995; Schmitt and Hiemke, 1998
Prior stress Steenbergen et al., 1990; Adamec and Shallow, 1993; Rodgers and Cole,
1993a; Padovan and Guimaraes, 2000; Mechiel Korte and De Boer, 2003
Apparatus construction (e.g. floor surface, walls/arms color, arm width,
open arm ledges)
Morato and Castrechini, 1989; Anseloni et al., 1995; Fernandes and File,
1996; Lamberty and Gower, 1996
Illumination level Morato and Castrechini, 1989; Griebel et al., 1993; Jones and King, 2001;
Bertoglio and Carobrez, 2002b
Prior test experience Lister, 1990; File et al., 1990; Griebel et al., 1993; Rodgers and Shepherd,
1993; Treit et al., 1993; Espejo, 1997; Bertoglio and Carobrez, 2000
Measures scored: conventional, ethological Pellow et al., 1985; Cruz et al., 1994; Rodgers and Cole, 1994; Rodgers
et al., 1997; Bertoglio and Carobrez, 2000
Definition/validation of measures (e.g. arm entry) Pellow et al., 1985; Lister, 1990; Espejo, 1997; Holmes and Rodgers, 1998
Method of scoring (i.e. live, manual/automated, videotape) Pellow et al., 1985; Holmes and Rodgers, 1998; Bertoglio and Carobrez,
2002b; Torres and Escarabajal, 2002
a
The criteria for citing were as follows: (1) first report; (2) the most representative; and (3) studies either from different laboratories or species (essentially
rats or mice).
A.P. Carobrez, L.J. Bertoglio / Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205 1195
behavioral tasks is often not taken into account at all
(Rodgers et al., 1997; Ohl, 2003). Indeed, while the primary
indices of EPM anxiety comprise spatiotemporal measures
of open arm avoidance (% of entries and of time spent in),
RA is a significant behavioral dimension, closely related to
fear/anxiety, which has been almost entirely ignored not
only in the EPM but also in other animal tests (Rodgers
et al., 1997). The biological function of RA acts and
postures is to inform behavioral strategies in potentially
dangerous situations (Blanchard et al., 1991; Blanchard
et al., 1993). In fact, it is interesting to note that rodents
continue to display enhanced RA behaviors even after
ceasing to avoid, for example, an unprotected area,
suggesting that this defensive pattern may even be more
sensitive to anxiety modulating drugs than avoidance-
related measures (Rodgers and Cole, 1994; Griebel et al.,
1997; Rodgers, 1997; Setem et al., 1999). For instance,
while doses of 2.5–5.0 mg/kg of the 5-HT
1A
receptor partial
agonist buspirone induce an anxiolytic-like effect on
spatiotemporal measures in the EPM, coupled with a
profound suppression of most active behaviors, at lower
doses significant anxiolysis was observed on several
ethological measures, including reductions in RA behaviors
(Griebel et al., 1997; Rodgers et al., 1997).
The utility of RA measures in discriminating between
anxiogenesis and sedation has also been demonstrated
(Shepherd et al., 1994). The anxiogenic drug mCPP
reduced the time spent in the open areas but increased
the occurrence of RA behaviors, supporting the con-
clusion that the effects of mCPP are due to anxiogenesis
rather than sedation or locomotor impairment (Shepherd
et al., 1994). In this context, there is evidence of the
segregation of behavioral—as shown by factor analysis
studies (Cruz et al., 1994; Rodgers and Johnson, 1995;
Weiss et al., 1998)—and neural mechanisms controlling
RA and open arm exploration in the EPM test (Adamec
et al., 1999; Adamec et al., 2001).
A number of studies have employed a supplementary
test, such as the holeboard apparatus, immediately
preceding or following EPM testing to assess locomotor
activity (Pellow et al., 1985; Lister, 1987). Although
exploration in the holeboard apparatus does not correlate
with indices of anxiety in the EPM (Weiss et al., 1998), it
can still be argued that any test of exploration of an
unfamiliar environment, such as the holeboard apparatus,
will contain elements of novelty-induced behavioral
inhibition, and therefore be influenced to some degree by
changes in fear/anxiety. In view of these facts, current
opinion has strengthened the idea of the RA behavioral
analysis as a valuable tool to measure more precisely
emotional reactivity in rodents (Roy and Chapillon, 2004).
Therefore, these behaviors should be systematically studied
to minimize false-negative results, for example when a
pharmacological treatment does not provide significant
results with classically recorded parameters (Rodgers et al.,
1997; Roy and Chapillon, 2004).
During recent years, a tendency towards automation of
the EPM test has occurred in order to supposedly
standardize measurements and to avoid subjective
interpretation of the animals’ behavior (e.g. Torres and
Escarabajal, 2002). Although automated scoring programs
permit analysis of patterns of spatial locomotor explora-
tion, only a few behavioral patterns can be recorded.
Therefore, the sensitivity of the EPM test may be
compromised since subtle behavioral alterations resulting
from drug effects, such as changes in RA behaviors,
remain unrecognized (Ohl, 2003). Moreover, as yet
unknown behavioral characteristics occurring in geneti-
cally modified animals might be missed by automatic
recordings, and changes in exploratory strategies are
likely to be misinterpreted when analyzed according to
predefined parameters. Thus, more complex observation-
based analyses should be performed, with appreciation of
the rich behavioral repertoire displayed by rodents
(Rodgers and Dalvi, 1997; Rodgers et al., 1997; Belzung,
1999). Based on this discussion, other tests of anxiety
may well benefit from greater attention to behavioral
detail (Rodgers et al., 1997).
A fascinating feature of the EPM concerns the effect
of prior test experience on subsequent behavioral and
pharmacological responses. The next section will discuss
this in detail with reference to the changes in
pharmacological responsiveness observed in EPM-experi-
enced rodents. With regard to the behavioral changes,
although early results showed that repeated testing did
not modify baseline open arm exploration (Pellow et al.,
1985; Lister, 1987; File et al., 1990), nowadays there is
growing evidence that it increases open arm avoidance
during Trial 2 (Lee and Rodgers, 1990; Almeida et al.,
1993; Griebel et al., 1993; Treit et al., 1993; Rodgers
and Cole, 1994; Fernandes and File, 1996; Bertoglio and
Carobrez, 2000; 2002b; 2002c). This pattern of response
appears to be acquired early on Trial 1, as revealed by
min-by-min scoring (Fig. 1).
The main results of the present analysis, which in general
harmonize with those obtained using mice (e.g. Rodgers
et al., 1996; Holmes and Rodgers, 1998), are as follows:
1. Up until the second min of Trial 1, roughly equal open
and enclosed arm exploration were observed, suggesting
novelty/curiosity as the main motivational stimulus
during this phase. As the session continued, however,
rats showed a clear enclosed arm preference. In other
words, open arm avoidance—representing possibly a
behavioral response to cope with the aversiveness of the
situation—is clear as early as the third min, further
increasing throughout the session, and also being
maintained on Trial 2. Interestingly, one can prevent
this increase in open arm avoidance by retesting with
intervals of 3-weeks using the same maze placed in a
different room (Adamec and Shallow, 2000; Adamec
and Young, 2000);
A.P. Carobrez, L.J. Bertoglio / Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205 1196
2. Both RA behaviors and general exploratory activity,
represented by stretched attend postures and enclosed
arms entries, respectively, remained stable throughout
Trial 1, suggesting that the pattern of change in open arm
exploration cannot be merely attributed to a general
behavioral suppression (Fernandes and File, 1996;
Rodgers et al., 1996). This proposition is supported by
the fact that the plasma corticosterone response to EPM
exposure does not appear to habituate with retesting
(File et al., 1994; Rodgers et al., 1999). Moreover,
although after the third min subjects remain inside the
enclosed arms, a continuous tendency to explore the
maze, as evidenced by RA behaviors, reveals an
approach/avoidance conflict sensitive to anxiolytic-like
drugs. With regard to Trial 2, the first minutes were
characterized by higher levels of these RA and general
exploratory activity measures relative to the end of Trial
1 (Fig. 1C–D). This profile may reflect an initial and
rapid re-familiarization with the EPM apparatus before
resorting to the typical open arm avoidance (Holmes and
Rodgers, 1998).
4. What is the Trial 1/Trial 2 protocol and why
should we use it?
Besides RA analysis and min-by-min scoring of the
rodents’ behavior in the EPM task, several laboratories have
also employed a test/retest protocol (e.g. File et al., 1990;
Lister, 1990; Griebel et al., 1993; Rodgers and Shepherd,
1993; Treit et al., 1993; Espejo, 1997; Bertoglio and
Carobrez, 2000). As already mentioned, this approach has
proven that prior test experience produces enduring changes
in behavioral responses. That is, after the initial overall
apparatus exploration it seems that such rodents acquire,
consolidate and retrieve some kind of memory related to
exploration of potentially dangerous areas of the maze—the
open arms. As discussed below, this learning appears to
influence future drug responsiveness in the test (File, 1993;
File et al., 1993; Bertoglio and Carobrez, 2002a, 2002c;
Cruz-Morales et al., 2002).
Naive rodents tested in the EPM display a characteristic
increase in open arm exploration and reduced RA
behaviors after the administration of anxiolytic-like drugs.
Fig. 1. Min-by-min scoring of the rats’ behavior in the EPM Trial 1 and Trial 2. Data are presented as mean GSEM (nZ60). Statistical
analysis comprised two-way repeated measures ANOVA followed by post-hoc Tukey-HSD test (p!0.05). For further details of procedures see
Bertoglio and Carobrez, 2002b. * Significantly different from the respective first min of Trial 1;
#
significantly different relative to the respective
fifth min of Trial 1.
A.P. Carobrez, L.J. Bertoglio / Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205 1197
However, if the subjects have already performed a 5 min
EPM session (Trial 1), they no longer respond to these drugs
on Trial 2. This phenomenon was initially observed for the
benzodiazepine chlordiazepoxide (Lister, 1987) and
referred to as ‘one-trial tolerance’ (OTT; File et al., 1990).
Afterwards, the loss of the anxiolytic-like effect of several
drugs, which act on different molecular targets to produce
their effects, was systematically observed during the retest
of rodents in the EPM (Table 2). Interestingly, it was shown
that this phenomenon actually is not exclusive to the EPM
model (Hascoet et al., 1997; McGregor and Dielenberg,
1999; Holmes et al., 2001).
Considerable attention has been directed at attempts to
either explain or prevent this pharmacoresistance to
anxiolytics with EPM retesting. Regarding the former
issue, several hypotheses have been proposed, including a
locomotor habituation (Dawson et al., 1994), an altered state
of the binding-sites and/or the receptor complexes involved
(Gonzalez and File, 1997; Bertoglio and Carobrez, 2002a,
2002b; 2003), an experimentally induced sensitization of
fear/anxiety (Treit et al., 1993; Bertoglio and Carobrez,
2000), and/or a qualitative shift in the nature of the aversive
response elicited between trials, from an unconditioned
fear response to one of learned avoidance (Holmes and
Rodgers, 1998; Dal-Col et al., 2003; Bertoglio and
Carobrez, 2004), against which the anxiolytic-like drugs
tested were ineffective (File and Zangrossi, 1993; Bertoglio
and Carobrez, 2003). This latter proposal is supported by
studies showing that the spatiotemporal behavioral
measures obtained from Trial 1 and Trial 2 load on separate
factors when they are submitted to factor analysis (File and
Zangrossi, 1993; Fernandes and File, 1996; Holmes and
Rodgers, 1998).
In relation to prevention of the OTT phenomenon, it was
demonstrated that rodents continue to display an anxiolytic-
like profile in response to drugs on the EPM Trial 2 when: 1)
a new motivational conflict situation was introduced in the
task (Pereira et al., 1999; Andreatini et al., 2003), 2) the time
length on Trial 1 was limited to 1 min (Dal-Col et al., 2003),
and 3) learning-disruptive doses of chlordiazepoxide (File
et al., 1990) or scopolamine (Bertoglio and Carobrez, 2004)
were administered prior to Trial 1.
In addition, it has been shown that brain areas related to
fear/defense systems may modulate this loss of a drug’s
anxiolytic-like effect on Trial 2. For example, it was
reported that a reversible deactivation of the basolateral
nucleus of the amygdala with lidocaine immediately after
Trial 1 restores the anxiolytic-like effect of a benzo-
diazepine on Trial 2 (File et al., 1998). A similar pattern of
results was observed after the deactivation of both the
dorsomedial hypothalamus (File et al., 1999) and the
dorsolateral periaqueductal gray matter just prior to Trial
2 (Bertoglio et al., 2005). This suggests that the
phenomenon may be related to different sets of brain
structures being preferentially involved in the expression of
fear/defensive behaviors during Trial 1 and Trial 2 (see also
Staples et al., this issue).
An approach/avoidance conflict is produced when the
animal is exposed to a novel situation that supposedly
creates a conflict between the motivation to explore it and an
unconditioned fear of novelty (Ohl, 2003). This may be the
underlying mechanism rendering the EPM test sensitive to
anxiolytic-like drugs (Handley and McBlane, 1993).
Interestingly, these drugs shift the balance of this conflict
from avoidance towards approach (Gray and McNaughton,
2000). However, the tendency to approach and the
propensity to avoid such stimuli is a function of the
experience with the stimuli concerned. Based on this fact,
it was recently proposed that the OTT phenomenon could
indicate that the approach/avoidance conflict is no longer
present on the EPM Trial 2 (Bertoglio and Carobrez,
2003). In other words, throughout the initial overall EPM
Table 2
Studies reporting the loss of the drug’s anxiolytic-like effect in EPM-experienced rodents
Drug Species Dose (mg/kg), adminis-
tration route
Reference (s)
Chlordiazepoxide Mouse 5.0–10, i.p. Lister, 1987; Holmes and Rodgers, 1999; Rodgers et al., 2002
Rat 2.5–10, i.p. File, 1990; File, 1993; File and Zangrossi, 1993; File et al., 1990; File et
al., 1992; File et al., 1993; File et al., 1998; File et al., 1999; Pereira et al.,
1999; Frussa-Filho and Ribeiro, 2002; Escarabajal et al., 2003
Diazepam Mouse 2.0–4.0, i.p. Rodgers et al., 1992
Rat 2.0–4.0, i.p. Rodgers and Shepherd, 1993; Treit et al., 1993; Boerngen-Lacerda and
Souza-Formigoni, 2000
Midazolam Rat Rat 0.25–1.5, i.p 0.001, i.c. Bertoglio and Carobrez, 2002c; 2004; Cruz-Morales et al., 2002;
Dal-Col et al., 2003; Gonzalez and File, 1997
Phenobarbital Rat 20–60, i.p. File, 1993; Bertoglio and Carobrez, 2002a
Ethanol Rat 1200–1400, i.p. Bertoglio and Carobrez, 2002a
Memantine Rat 8.0, i.p. Bertoglio and Carobrez, 2003; 2004
MK-801 Rat 0.06, i.p. Bertoglio and Carobrez, 2003
HA-966 Rat 3.0, i.p. Bertoglio and Carobrez, 2003
WAY-100635 Mice 0.003, i.c. Canto-de-Souza et al., 2002; Nunes-de-Souza et al., 2002
8-OH-DPAT Rat 0.08, i.p. Bertoglio et al., 2004
Legend: i.p.Zintraperitoneal; i.c.Zintra-specific brain sites.
A.P. Carobrez, L.J. Bertoglio / Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205 1198
exploration, subjects would develop and adopt non-conflict
(and thus anxiolytic-insensitive) behaviors, such as
enclosed arms preference, in the subsequent EPM
exposure. In view of this, the maintenance of a drug’s
anxiolytic-like activity seen when learning-disruptive drugs
are given pre-Trial 1 (see below) implies a failure to
develop this anxiolytic-insensitive behavioral strategy
(Bertoglio and Carobrez, 2004). Altogether, these findings
suggest that such learning underlies either the acquisition
of open arm avoidance or the qualitative shift in the
defensive responses seen in EPM Trial 2, which render the
subjects unresponsive to drugs.
In consonance with this proposition are the results
showing that only a prior experience in the whole EPM
apparatus, and not a confinement in one of the open or
enclosed arms for 5 min, elicits these behavioral and
pharmacological changes (Bertoglio and Carobrez, 2000;
2002c). Alternatively, drug unresponsiveness on retesting in
the EPM could be explained by the following reasoning. An
anxiolytic-like effect appears as decreased open arm
avoidance because reduced anxiety removes the inter-
ference of fear with exploratory tendencies and hence drives
greater exploration of the open arms. Prior experience in the
EPM reduces motivation to explore the open arms per se
and even if an anxiolytic-like drug were given there would
be no added motivation to explore familiar open arms, and
hence no ‘anxiolytic-like’ effect of drugs on Trial 2.
We have recently examined whether Trial 2 performance
on the EPM is affected by a prior test exposure in which
drugs were given that interfere with open arm avoidance
(Bertoglio and Carobrez, 2003; 2004), Male Wistar rats
were systemically administered with pentylenetetrazole
(30 mg/kg), midazolam (0.25 mg/kg) or scopolamine
(1.5 mg/kg) and 30 min later submitted to the EPM Trial
1. All groups were retested (Trial 2) undrugged in the EPM
48 h later. The results of this experiment are shown in Fig. 2.
Considering the Trial 1 session data (Fig. 2A), one could
argue that midazolam and scopolamine both produced
anxiolytic-like effects since they increased open arm
exploration. Nevertheless, while midazolam undoubtedly
possesses an anxiolytic-like effect (e.g. Bertoglio and
Carobrez, 2003; 2004), a scopolamine-induced learning
acquisition deficit has been systematically reported using
several animal models of learning and memory (e.g. Zanotti
et al., 1986; McNaughton and Morris, 1987; De-Mello and
Carobrez, 2002). Evidence that scopolamine given prior to
Trial 1 disrupted the usual behavioral strategy adopted
throughout Trial 2 performance (open arm avoidance) is
given by the min-by-min analysis. Whereas the group
treated with midazolam prior to Trial 1 displayed a % open
arms time score similar to controls on Trial 2 (Fig. 2B,D),
the group receiving scopolamine prior to Trial 1 performed
differently (Fig. 2E).
Therefore, the usefulness of the test-retest approach rests
on the fact that a non-selective and/or false-positive
anxiolytic-like effect of a given drug can be easily detected
in the EPM task. This attempt to refine the EPM is important
in view of the fact that it is the most popular animal model
employed in screening for novel therapeutic agents.
Furthermore, it is interesting to observe the changes
produced by anxioselective drugs in the structure of EPM
behavior. Pentylenetetrazole reduces the open arm
exploration at the beginning of Trial 1 while midazolam
fails to further increase it beyond the control values in the
first two min of testing. As the session continues, while
the controls express increased open arm avoidance around
the third min of Trial 1, the midazolam-treated group
maintains a relatively stable level of high open arm
activity (Fig. 2B,D). No difference could be detected
when comparing controls and the group treated with
pentylenetetrazole (Fig. 2B,C) in the last 3 min of testing.
This implies that the anxiogenic-like effect is essentially due
to changes appearing during the first 2 min of testing while
Fig. 2. Utility of Trial 1/Trial 2 protocol in overcoming confounding results in the EPM. Rats were administered prior to Trial 1 with pentylenetetrazole (PTZ),
midazolam (MDZ) or scopolamine (SCO), and 48 h later all groups were retested (Trial 2) undrugged. Full data (5 min) of Trial 1 are represented on graph ‘A’
whereas the others show the min-by-min scoring of the Trial 2 rats’ behavior. Data are presented as mean or meanCSEM. (nZ10–12). Statistical analysis
comprised one-way (A) or two-way repeated measures ANOVA (B–E), followed by post-hoc Tukey-HSD test (P!0.05). For further details of procedures see
Bertoglio and Carobrez, 2002b; 2003 or Bertoglio and Carobrez, 2004). * Significantly different from saline-treated rats (Trial 1);
#
significantly different
relative to the respective minute of controls.
A.P. Carobrez, L.J. Bertoglio / Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205 1199
the anxiolytic drug effects are due to changes observed in
the remaining 3 min.
5. Panorama of current EPM use: differences
from the ideal
The utility of the EPM test as a measurement instrument
for animal anxiety-like behavior is beyond doubt. However,
its reliability/sensitivity will be progressively refined by our
better understanding of some conceptual issues underlying
the test and the introduction of a more comprehensive
methodological approach to data collection, such as the
analysis of RA behaviors, the use of min-by-min scoring and
of the Trial 1/Trial 2 protocol. A panorama of the recent
literature (Table 3) shows that adoption of these methodo-
logical refinements has been rather slow to occur. For
example, although the recording of RAbehaviors in the EPM
test was introduced more than 10 years ago (e.g. Rodgers
et al., 1992; Cole and Rodgers, 1993; Cruz et al., 1994), even
now only a quarter of recent studies have adopted this
approach. One might explain this by the fact that it demands a
substantially greater investment of time than conventional
scoring. However, this argument has proven to be unfounded
(Shepherd et al., 1994; Dourish et al., 1995; Rodgers et al.,
1997). In fact, it is likely that neither the min-by-min scoring
nor the adoption of a test/retest protocol significantly
increases the time spent on data collection. Whatever
additional time investment were required for these analyses,
it would be compensated for by their numerous advantages,
such as the enhancement of sensitivity (and specificity) to
(anxioselective) drug action and the improved distinction
between general activity and emotional reactivity (Griebel
et al., 1997; Rodgers et al., 1997; Bertoglio and Carobrez,
2003; 2004; Roy and Chapillon, 2004).
Recent advances in neuroscience and understanding in the
etiology of anxiety have led researchers to new potential
treatments that are proving to be at least as effective as
benzodiazepines, the traditional treatment for anxiety for over
40 years (Nutt and Malizia, 2001; Kent et al., 2002; Millan,
2003). The GABA system has long been targeted in anxiety
interventions via benzodiazepines, but better understanding of
its role in anxiety disorders has led to the fine-tuning of drugs
that act onspecific subunits of the GABA-Areceptor (Nutt and
Malizia, 2001; Gorman, 2003). Meanwhile, recent findings
have highlighted the neurotransmitter glutamate as an
important element in anxiety and anxious behaviors (Caro-
brez, 2003; Holden, 203; Bergink et al., 2004; Javitt, 2004).
Moreover, the recognition that chronic treatment with
antidepressants, such as the selective serotonin reuptake
inhibitors (SSRIs), is effective in anxiety disorders (Argyr-
opoulos et al., 2000; Millan, 2003; Vaswani et al., 2003) has
stimulated researchers to develop anxiolytics that influence
the serotonin and/or noradrenergic systems (Kent et al., 2002;
Gorman, 2003; Vaswani et al., 2003). Apart from these
conventional neurotransmitters, many other modulators have
been implicated in anxiety, including: adenosine, cannabi-
noids, numerous neuropeptides, hormones, neurotrophins,
Table 3
Some details of methodological issues in the use of the EPM test according to a survey
a
including 200 studies (25 per year/species) published since 2001
Variable Year Average
2001 2002 2003 2004
Rat Mouse Rat Mouse Rat Mouse Rat Mouse Rat Mouse
Sex used
Male 85 60 84 72 73 73 76 72 80 69
Female 10 05 00 08 03 03 12 04 06 05
Both 05 35 16 20 24 24 12 24 14 26
Test room illumination
Low (!50 lux) 55 64 66 64 81 58 78 62 70 62
Moderate
(50–250 lux)
45 36 28 36 15 38 22 23 28 33
High (O250 lux) 00 00 06 00 04 04 00 15 02 05
EPM construction
Wood 40 00 25 05 40 15 47 5 38 06
Clear Plexiglas 13 43 20 33 10 33 12 37 14 37
Dark Plexiglas 47 57 55 62 50 52 41 58 48 57
Open-arm ledges
(height)
20 20 28 24 20 20 35 25 26 22
Measures scored
Conventional only 85 80 80 68 60 77 76 72 75 74
CEthological
b
15 20 20 32 40 23 24 28 25 26
Data are presented as percentage.
a
Performed on the PubMed site (www.pubmed.com) using the entering expression ‘elevated plus-maze’. Within each year/species, the studies were
randomly chosen.
b
Essentially comprising risk assessment behaviors (e.g. stretched attend postures); Legend to figures.
A.P. Carobrez, L.J. Bertoglio / Neuroscience and Biobehavioral Reviews 29 (2005) 1193–1205 1200
cytokines and several cellular mediators (Griebel, 1999; Kent
et al., 2002; Hood et al., 2003; Millan, 2003). In viewof these
considerations, the so-called animal tests of anxiety should be
sensitive to this wide spectrum of drugs in order to have
predictive validity. Regarding the EPM, it provides an
excellent tool for detecting benzodiazepine/GABA- and
glutamate-related compounds (Rodgers et al., 1997; Menard
and Treit, 1999; Carobrez et al., 2001; Bertoglio and Carobrez,
2003; Carobrez, 2003), but inconsistent results have been
reported for those drugs which modulate the serotoninergic
(Menard and Treit, 1999; Borsini et al., 2002) or some of the
neuropeptide systems (Griebel, 1999; Menard and Treit,
1999). This profile has led certain authors to doubt the value of
the EPM model (Moser, 1989; Dawson and Tricklebank,
1995; Griebel, 1999; Borsini et al., 2002). However, it is
worthy of note that most of the animal tests of anxiety,
including the EPM, have been validated pharmacologically by
benzodiazepines (Lister, 1990; Green and Hodges, 1991;
Rodgers, 1997; Rodgers et al., 1997), and therefore, it would
be inappropriate to criticize them for relative insensitivity to
agents operating through entirely different mechanisms
(Rodgers, 1997; Rodgers et al., 1997). Furthermore, incon-
sistent results obtained in this test may indicate that the
associated emotional state/reaction is critically dependent on
stimulus parameters (Hogg, 1996; Rodgers et al., 1997).
Clearly, the behavioral expressions displayed in the EPM
test represent a combination of exploratory and avoidance
behaviors, as well as general activity, all of which are
influenced by both genetic and environmental factors
(Table 1). As a consequence, results on the EPM can be
heavily influenced by testing conditions and the procedure
used (Tables 1 and 3), and it is essential to carefully recognize
and control these variables when using the EPM to infer
anxiety-like behavior in animals. On the basis of these
considerations, it is to be hoped that future studies will further
disclose the precise applicability of the EPMtest in studies of
the pharmacology and neurobiology of fear and anxiety.
6. Concluding remarks
As noted by Rodgers (1997), many of the inconsistencies
in this area might be solved if the emphasis were directed
not only to pharmacological but also to behavioral
validation of animal models. As disorders of anxiety
represent an inappropriate activation or exaggeration of
normally adaptive defensive responses, and their form,
function and mechanisms are highly conserved in evolution,
an improvement in test validity could be achieved when the
full defensive repertoire of animals is considered. An
important caveat is the fact that the defensive repertoire of
animals will seldom match a specific anxiety disorder in
humans. For example, when analyzing the behavioral
symptoms of generalized-anxiety disorder, avoidance and
risk assessment appear to be predominant, but flight/escape
responses may also occur. On the other hand, risk
assessment and avoidance behaviors might arise as a
consequence of panic disorder as well.
With regard to the EPM model, the structure of behaviors
revealed on Trial 1 shows that the novelty and the initial
tendency to approach both arms equally is gradually
replaced by an increased tendency to avoid the open arms.
The predominance of approach during the initial 2 min on
Trial 1 can be blocked by anxiogenic drugs, causing
increased avoidance of the open arms. Furthermore, the
increased tendency to avoid the open arms over the
remaining 3 min can be prevented by anxiolytic drugs
such as benzodiazepines. During Trial 2, the increased open
arm avoidance, acquired during Trial 1 and consolidated
thereafter, is insensitive to anxiolytics (Table 2), suggesting
a different type of defensive response when compared to the
avoidance seen during Trial 1. In addition to the above
profile, the experiments using lidocaine injected into brain
structures lead to the idea that a different set of brain
structures might participate in the behavior exhibited in the
EPM over successive trials, which might also account for
the differences in sensitivity to anxioselective drugs (e.g.
File et al., 1998; File et al., 1999; Bertoglio et al., 2005).
Acknowledgements
We are greatful to Dr. I. McGregor and the anonymous
reviewers for their critical comments and to Gareth Cuttle
for English corrections on the manuscript. This work was
supported by FAPESP (02/13197-2; 03/13032-6), CAPES
and CNPq (521864/96-8).
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