Psychopharmacology (2006) 184: 1–12
DOI 10.1007/s00213-005-0238-6
ORIGINA L IN VESTI GATION
C. L. Patti . S. R. Kameda . R. C. Carvalho .
A. L. Takatsu-Coleman . G. B. Lopez . S. T. Niigaki .
V. C. Abílio . R. Frussa-Filho . R. H. Silva
Effects of morphine on the plus-maze discriminative avoidance
task: role of state-dependent learning
Received: 18 October 2005 / Accepted: 20 October 2005 / Published online: 10 December 2005
# Springer-Verlag 2005
Abstract Rationale: The amnesic effects of morphine
may be related to its action on nociception, anxiety, or
locomotion. This effect is also suggested to be related to
state dependency. Objectives: The aims of this study were
to verify the effects of morphine on mice tested in the plus-
maze discriminative avoidance task (DAT) that uses light
and noise as aversive stimuli and allows the concomitant
evaluation of learning, memory, anxiety, and locomotion
and also to verify the possible role of state-dependent learning
in the effects of morphine. Methods and results: The DAT
was conducted in a modified elevated plus-maze. In the
training, the aversive stimuli were applied when mice entered
in one of the enclosed arms, whereas in the test, no stimuli
were applied. The main results showed that (1) pretraining
morphine (5–20 mg/kg i.p.) induced retrieval deficits
(evaluated by the time spent in the aversive arm in the test)
but not acquisition deficits (evaluated by the decrease in
aversive arm exploration along the training); (2) pretest
morphine (5–10 but not 20 mg/kg) counteracted this deficit;
(3) morphine induced hypolocomotion (decreased number of
entries in the arms), irrespective of memory alterations; and
(4) morphine did not alter anxiety-like behavior (evaluated by
the time spent in the open arms) during the training.
Conclusions: Morphine given before training induces re-
trieval deficits in mice tested in the DAT, and these deficits
could be related to morphine-induced state-dependent learn-
ing. Neither the memory deficit induced by pretraining
morphine nor the reversal of this deficit by pretest morphine
seems to be related to anxiety levels or locomotor alterations.
C. L. Patti . S. R. Kameda . R. C. Carvalho .
A. L. Takatsu-Coleman . G. B. Lopez . S. T. Niigaki .
V. C. Abílio . R. Frussa-Filho . R. H. Silva (*)
Department of Pharmacology,
Universidade Federal de São Paulo,
Rua Botucatu, 862-Ed. Leal Prado,
04023-062 São Paulo, Brazil
e-mail: regina.farm@epm.br
Tel.: +55-11-55494122
Fax: +55-11-55792752
Keywords Avoidance paradigms . Morphine .
State dependence . Memory . Anxiety . Mice
Introduction
Several studies have shown the important role of opioid
transmission in learning and memory (Izquierdo et al. 1980;
McGaugh and Baratti 1985; Ragozzino and Gold 1994;
Vaccarino et al. 1998), and several studies have focused on
the effects of morphine on memory. In most of these studies,
pretraining administration of this opiate induced learning/
memory deficits in rodents tested in avoidance paradigms
that use foot shocks as unconditioned stimuli (Izquierdo
1979; Ragozzino et al. 1992; Ragozzino and Gold 1994,
1995; McNay and Gold 1998; Aguilar et al. 2004). Although
a question should be raised of whether pretraining morphine-
induced analgesia would account for the effects of this drug
on memory performance of tasks that involve painful stimuli,
other studies have shown that the amnesic action of morphine
is also present after posttraining or pretesting administration
(Cestari and Castellano 1997; Saha et al. 2001; Mohammad-
Reza et al. 2002; Costanzi et al. 2004). In this respect, when
the drug is given after the acquisition, the effects observed in
the test session are due to an effect of the drug on con-
solidation/retention processes. However, it is still unclear if
pretraining morphine administration induces learning im-
pairments, i.e., alterations in the acquisition of avoidance
tasks. Aguilar et al. (2004), for example, have shown that
morphine induced a behavioral deficit in the acquisition of a
conditioned avoidance response in mice. This deficit, how-
ever, was suggested to be a consequence of morphine-
induced hyperactivity (Aguilar et al. 2004) because the task
applied was dependent on motor behavior (Vinader-Caerols
et al. 1996; Aguilar et al. 1998, 2000).
Another important feature of pretraining morphine-
induced amnesia in avoidance tasks is that it is abolished
by pretesting administration of this drug (Izquierdo and
Dias 1983; Bruins Slot and Colpaert 1999a; Khavandgar et
al. 2002, 2003; Jafari et al. 2004; Mohammad-Reza and
Rezayof 2004; Vakili et al. 2004). This phenomenon is
2
called state dependence, i.e., the retrieval of learned in-
formation requires that the animal be in a state similar to that
in which the memory for this information was acquired
(Izquierdo et al. 1981; Bruins Slot and Colpaert 1999a), and
has been demonstrated after administration of several
psychoactive drugs (Colpaert 1990; Jackson et al. 1992;
Bruins Slot et al. 1999; Colpaert et al. 2001). In addition, it
has been shown that state dependence may act separately on
acquisition, retention, and retrieval (Colpaert et al. 2001).
Because of the failure to respond in the absence of the drug,
morphine state-dependent learning has been proposed as a
mechanism for opiate dependence (Colpaert 1990, 1996;
Spanagel 1995). In addition, it has been shown to be dose-
specific and related to time of administration (Nishimura
et al. 1990; Bruins Slot and Colpaert 1999a; Khavandgar
et al. 2003), suggesting that the same strength of morphine
effects should be present for retrieval to occur. In this re-
spect, one of the effects of this drug that could provide cues
for the conditioned learning could be the analgesic state
(Bruins Slot and Colpaert 1999a). This possibility is sup-
ported by the fact that morphine state-dependent learning
occurs at times and at doses at which morphine also pro-
duces analgesia (Bruins Slot and Colpaert 1999b). This
issue seems to be of relevance regarding studies on mor-
phine-induced state dependence performed in avoidance
paradigms that use painful stimuli as unconditioned stimuli.
Finally, another factor that should be taken into account is
the anxiolytic effect of morphine, usually observed in an-
imal models of anxiety (Motta and Brandão 1993; Anseloni
et al. 1999; Kõks et al. 1999; Sasaki et al. 2002; Shin et
al. 2003). The emotional alterations induced by morphine
seem to be important not only because the anxiety level can
be related to memory deficits found in behavioral animal
models (Silva and Frussa-Filho 2000, 2002; Silva et al. 2002a,
2004a; Calzavara et al. 2004) but also because of a possible
role of anxiety alterations (as relevant features of the general
state induced by morphine) in state-dependent learning.
The aims of the present study are (1) to verify the effects
of morphine on learning and memory performance of mice
tested in the plus-maze discriminative avoidance task
(DAT), (2) to verify the possible role of state-dependent
learning in the effects of morphine on this task, and (3) to
investigate the participation of anxiety-like behavior and
motor alterations on the effects of morphine on learning and
memory. In the plus-maze DAT, light and noise, instead
of electric shocks, are used as unconditioned stimuli. In
addition, this behavioral paradigm allows the concomitant
evaluation of learning, memory, anxiety, and motor param-
eters (Silva et al. 1997, 2002b, 2004a; Silva and Frussa-
Filho 2000).
Methods
Subjects
Three-month-old Swiss EPM-M1 male mice (outbred,
raised, and maintained in the Center for Development
of Experimental Models in Medicine and Biology of
Universidade Federal de São Paulo, Brazil, since 1985;
Festing 1993) were used. Animals weighing 30–35 g
were housed under conditions of controlled temperature
(22–23°C) and lighting (12-h light, 12-h dark; lights on at
7 a.m.). Food and water were available ad libitum through-
out the experiments. Animals used in this study were
maintained in accordance with the guidelines of the Com-
mittee on Care and Use of Laboratory Animal Resources,
National Research Council, USA.
Drug
Morphine sulfate (Dimorf, Innovatec) was diluted in saline
solution that was used as control solution. Both morphine
and saline solutions were given intraperitoneally at a dose
of 10 ml/kg of body weight.
Discriminative avoidance task procedure
and statistical analysis
The apparatus employed is a modified elevated plus-maze,
made of wood, containing two enclosed arms with side-
walls, and no top (28.5×7×14 cm, 03 lx at the floor level),
opposite to two open arms (28.5×7 cm, 09 lx at the floor
level). A 100-W lamp was placed exactly over the middle of
one of the enclosed arms (aversive enclosed arm, 660 lx at
the floor level). In the training session, each mouse was
placed in the center of the apparatus and, over a period of
10 min, every time the animal entered the enclosed arm
containing the lamp, an aversive situation was produced
until the animal left the arm. The aversive stimuli were the
100-W light and an 80-dB noise produced by a small
machine placed under the aversive enclosed arm. In the test
session (performed in the same room with the observer in
the same position), the mice were again placed in the center
of the apparatus and observed for 3 min without receiving
the aversive stimulation. In all experiments, the animals
were observed in a random order and in a blind manner, and
the apparatus was cleaned with a 5% alcohol solution after
each behavioral session. Total number of entries in any of
the arms, number of entries in both enclosed arms, percent
time spent in the aversive enclosed arm (time spent in
aversive enclosed arm/time spent in both enclosed arms),
and percent time spent in open arms (time spent in open
arms/time spent in both open and enclosed arms) were
calculated and compared by the one- or two-way analysis of
variance (ANOVA) followed by Duncan’s test or by
Student’s t test. The decrease in percent time spent in the
aversive arm throughout the training session was used to
evaluate learning of the task (experiment II) and was com-
pared by ANOVA with repeated measures. Memory was
evaluated by the time spent in the aversive vs nonaversive
enclosed arms (compared by two- or three-way ANOVA
followed by Duncan’s test) and by percent time in aversive
enclosed arm in the test session. The animal was considered
to be in a certain arm when the four paws passed over its
entrance. Anxiety-like behavior and locomotor activity

were evaluated by the percent of time spent in the open arms
and total number of entries in all the arms or in both
enclosed arms of the apparatus, respectively.
Hot plate procedure and statistical analysis
The antinociceptive effects of the doses of morphine used in
the above experiments were evaluated in the hot plate test.
Mice were placed on a heated zinc plate (55°C), and the
latency to hind paw withdrawal was recorded throughout
60 min at 10-min intervals (Woolfe and MacDonald 1944).
A maximal latency value of 30 s was set. The data were
analyzed by ANOVA with repeated measures followed by
Duncan’s test.
Experimental design
Experiment I Groups of ten mice received saline, 5, 10, or
20 mg/kg morphine. Thirty minutes after the injection, all
the animals were submitted to the plus-maze discrimina-
tive avoidance training session, and a test session was
performed 24 h later, 30 min after the saline injection.
Experiment II Groups of 20 mice received saline or
10 mg/kg morphine. Thirty minutes after the injection, all
the animals were submitted to the plus-maze discrimina-
tive avoidance training session, and the behavioral
parameters of this paradigm were registered minute by
minute.
Experiment III Twenty-four hours after the training session
of experiment II, ten animals treated with saline and ten
animals treated with morphine (before the training session,
experiment II) received saline, and the other 20 animals
pretraining treated with saline (n=10) or morphine (n=10)
received 10 mg/kg morphine. A test session was performed
30 min after this injection.
Experiment IV Groups of mice received saline (n=28), 5
(n=10), or 20 (n=10) mg/kg morphine. Thirty minutes after
the injection, all the animals were submitted to the plus-
maze discriminative avoidance training session. Twenty-
four hours after the training session, the animals treated with
saline (before the training session) received saline (n=8), 5
(n=10), or 20 mg/kg morphine (n=10). The animals treated
pretraining with 5 or 20 mg/kg morphine received the same
dose of morphine before the test session. Thirty minutes
after the injection, the animals were submitted to the plus-
maze discriminative avoidance test session.
Experiment V The subjects were placed on the hot plate to
obtain baseline latencies. Immediately after this measure-
ment, groups of animals (n=5) received saline, 5, 10, or
20 mg/kg morphine (each animal were tested only at a
single dose) and placed again on the hot plate 10, 20, 30, 40,
50, and 60 min after the injections.
3
Results
Experiment I: effects of several doses of pretraining
morphine on memory, anxiety-like behavior,
and locomotor activity of mice tested in the plus-maze
discriminative avoidance task
Two-way ANOVA with treatment as between-subjects fac-
tor and arm type (aversive vs nonaversive) as within-
subject factor revealed significant effects of arm type [F
(1,72)=2241.37, p<0.001] and treatment × arm type in-
teraction [F(3,72)=2.82, p<0.05]. Post hoc analysis by
Duncan’s test revealed that all the groups spent signifi-
cantly less time in the aversive than in the nonaversive
enclosed arm (Fig. 1a). In this session, one-way ANOVA
followed by Duncan’s test showed that all the groups treated
with morphine presented significantly decreased percent
time in the aversive enclosed arm (Fig. 1c) when compared
to saline-treated animals [F(3,36)=3.76, p<0.05].
In the test session, two-way ANOVA revealed only a
significant treatment × arm type interaction effect [F(3,72)=
4.16, p<0.01]. Post hoc analysis by Duncan’s test revealed
that only saline-treated animals presented significantly less
time in the aversive enclosed arm than in the nonaversive
one (Fig. 1b). Accordingly, in this session, one-way
ANOVA followed by Duncan’s test showed that all the
groups treated with morphine presented significantly in-
creased percent time in the aversive enclosed arm (Fig. 1d)
when compared to saline-treated animals [F(3,36)=4.77,
p<0.01].
No differences were found in percent time spent in the
open arms in the training or test sessions (one-way ANOVA
followed by Duncan’s test; Fig. 2a,b).
In the training session, when locomotor-activity-related
parameters were compared, no differences were found be-
tween saline controls and mice treated with 5 mg/kg mor-
phine, whereas groups treated with 10 or 20 mg/kg
morphine presented total number of entries [F(3,36)=5.66,
p<0.005], and number of entries in the enclosed arms [F
(3,36)=4.80, p<0.01] significantly decreased (one-way
ANOVA followed by Duncan’s test; Fig. 2c). In the test
session, no differences were found between mice treated
with 5 or 20 mg/kg morphine and saline controls, whereas
groups treated with 10 mg/kg morphine presented number
of entries in the enclosed arms [F(3,36)=4.02, p<0.05]
significantly increased (one-way ANOVA followed by
Duncan’s test; Fig. 2d).
No significant effect of morphine doses was found in any
of the parameters analyzed.
Experiment II: effects of pretraining morphine
on learning of mice during the plus-maze
discriminative avoidance training
ANOVA for the percent time spent in the aversive arm with
treatment as a between-subjects factor and time (minutes of
observation) as a repeated-measures factor revealed signif-
4
600
A C
500
400
Time (s)
300
200
100
* * * *
0 0
0 5 10 20
180
B D
120
Time (s)
60 * 40
0
0 5 10 20
Morphine dose (mg/kg)
Nav Av
Fig. 1 Effects of pretraining morphine on mice tested in the plus-
maze DAT. Mice were treated with saline (0), 5, 10, or 20 mg/kg
morphine 30 min before a 10-min-long training session and tested
during 3 min, 24 h later, in the absence of the drug. Results are
presented as mean±SE of time (seconds) spent in the nonaversive
(Nav) and in the aversive (Av) enclosed arms (a, b) and percent time
icant effects of treatment [F(1,37)=6.91, p<0.05] and time
[F(9,333)=17.21, p<0.001]. Indeed, although the percent
time spent in the aversive arm was decreased in both groups
from the second minute onward, this decrease had a greater
magnitude in morphine-treated mice (Fig. 3a). Similarly to
experiment 1, the percent time spent in the aversive arm
(t=3.27, p<0.005), the total number of entries (t=4.73,
p<0.001), and the number of entries in both enclosed arms
(t=5.56, p<0.001) were significantly decreased in mor-
phine-treated animals, while percent time in the open arms
was not modified (Student’s test; Table 1).
Experiment III: effects of pretest 10 mg/kg morphine
on memory of pretraining morphine-treated mice
tested in the plus-maze discriminative avoidance task
Three-way ANOVA with pretraining and pretest treatments
as between-subjects factors and arm type (aversive vs
nonaversive) as within-subject factor revealed significant
effects of arm type [F(1,72)=79.38, p<0.001], arm type ×
pretraining treatment interaction [F(1,72)=12.51, p<0.001],
arm type × pretest treatment interaction [F(1,72)=21.09,
p<0.001], and arm type × pretraining treatment × pretest
treatment interaction [F(1,72)=5.03, p<0.05]. Post hoc
50
40
30
% TAv
20
10
O O O
0 5 10 20
Morphine dose (mg/kg)
100
80
O
O
60 O
% TAv
20
0
0 5 10 20
Morphine dose (mg/Kg)
spent in the aversive enclosed arm (c, d) of a plus-maze
discriminative avoidance apparatus in the training and test sessions,
respectively. *p<0.05 compared to the time spent in the nonaversive
enclosed arm (two-way ANOVA and Duncan’s test). °p<0.05
compared to saline-treated group (one-way ANOVA and Duncan’s
test)
analysis by Duncan’s test revealed that both groups treated
with saline before training and mice treated with morphine
before both sessions spent significantly less time in the
aversive than in the nonaversive enclosed arm, whereas
mice treated with morphine before training and with saline
before testing showed similar times spent in aversive and
nonaversive enclosed arms (Fig. 4a).
Two-way (pretraining × pretest treatments) ANOVA for
percent time spent in the aversive arm revealed significant
effects of both pretraining [F(1,36)=16.58, p<0.001] and
pretest [F(1,36)=6.36, p<0.05]. Post hoc analysis by
Duncan’s test revealed that (1) mice treated with saline
before training and with morphine before testing did not
differ significantly from control (saline + saline) group, (2)
mice treated with morphine before training and saline be-
fore testing presented significantly increased percent time
spent in the aversive enclosed arm when compared to
control group, (3) mice treated with morphine before both
sessions were not significantly different from the control
group, and (4) mice treated with morphine before both ses-
sions presented significantly decreased percent time spent
in the aversive arm when compared to mice that received
morphine only before training (Fig. 4b).
Two-way ANOVA for total number of entries and num-
ber of entries in the enclosed arms revealed pretest treatment
 5
50 20
A C

40
15

Number of Entries
30
% TO

10
*
20 * *
*
5
10

0 0
0 5 10 20 0 5 10 20

50 20
B D

Numbers of Entries
40 15

30 10
% TO

*
20
5

10
0
0 5 10 20
0 Morphine doses (mg/kg)
0 5 10 20
Total Enclosed Arms
Morphine doses (mg/kg)

Fig. 2 Effects of pretraining morphine on mice tested in the plus-
maze DAT (see legend for Fig. 1). Results are presented as mean±SE
of percent time spent in the open arms (% TO) (a, b) and total
number of entries and number of entries in both enclosed arms (c, d)
of a plus-maze discriminative avoidance apparatus in the training
and test sessions, respectively, presented by mice treated with saline
(0), 5, 10, or 20 mg/kg morphine 30 min before the training session.
*p<0.05 compared to saline-treated group (one-way ANOVA and
Duncan’s test)

[F(1,36)=11.69 and 7.21, respectively, p<0.05] and pre-
training × pretest treatment interaction [F(1,36)=6.43 and
4.77, respectively, p<0.05] effects. Post hoc analysis by
Duncan’s test revealed that only mice that received
40
30
% TAv
morphine before training and saline before testing presented
increased locomotor activity parameters when compared to
animals that were treated with saline before both sessions
(Fig. 4c).
Two-way ANOVA for percent time spent in the open
arms revealed only a significant effect of pretest treatment
[F(1,36)=10.03, p<0.005]. Indeed, post hoc analysis by
Duncan’s test revealed that both groups treated with mor-
phine before testing presented decreased percent time in
the open arms when compared to the respective controls
(Fig. 4d).

20
Table 1 % TAv, % TO, TE, and EE of a plus-maze discriminative
10 avoidance apparatus (mean±SE) in the entire 10 min of the training
session presented by mice treated with saline or 10 mg/kg morphine
30 min before the session (see legend for Fig. 3)
0 Saline Morphine (10 mg/kg)
1 2 3 4 5 6 7 8 9 10
Time (min)
% TAv 08.41±8.71 1.98±1.22*
Sal M10 % TO 07.51±9.30 3.04±6.46
Fig. 3 Effects of morphine on training performance of mice in the TE 20.60±9.40 9.15±5.30*
plus-maze DAT. Mice were treated with saline (Sal) or 10 mg/kg EE 17.40±6.74 7.75±9.85*
morphine (M10) 30 min before a 10-min training session. Data are
presented as mean±SE of percent time spent in the aversive enclosed % TAv Percent time spent in the aversive enclosed arm, % TO
arm (% TAv) of a plus-maze discriminative avoidance apparatus in percent time spent in the open arms, TE total number of entries, EE
each of the 10 min of the training session. ANOVA with repeated number of entries in both enclosed arms
measures revealed treatment and time significant effects *p<0.05 compared to saline-treated animals (Student’s t test)
6
NAv Av Total Enclosed Arms

180 A
20
C
150

Number of Entries
120
Time (s)

O
90 10
O
60 *
*
30 *

0 0
S-S S-M10 M10-S M10-M10 S-S S-M10 M10-S M10-M10

100 50
B D

80 40
O

60 30
% TAv

% TO
40 20

20 10

O O
0 0
S-S S-M10 M10-S M10-M10 S-S S-M10 M10-S M10-M10

Fig. 4 Effects of morphine on testing performance of pretraining-
treated mice in the plus-maze DAT. Mice were treated with saline
(S-) or 10 mg/kg morphine (M10-) 30 min before a 10-min training
session and tested during 3 min, 24 h later, 30 min after an injection
of saline (-S) or 10 mg/kg morphine (-M10). Results are presented as
mean±SE of time (seconds) spent in the nonaversive (NAv) and in
the aversive (Av) enclosed arms (a), percent time in the aversive arm
(% TAv) (b), percent time in the open arms (% TO) (c), total number
of entries and number of entries in the enclosed arms (d) of a plus-
maze discriminative avoidance apparatus in the test session. *p<0.05
compared to the time spent in the nonaversive enclosed arm (three-
way ANOVA and Duncan’s test). °p<0.05 compared to S–S group
(two-way ANOVA and Duncan’s test)

600 180
A B

500 150

400 120
Time (s)
Time (s)

300 90
*
200 60
*
100 30

* * * * *
0 0
Sal M5 M20 Sal-Sal Sal-M5 Sal-M20 M5-M5 M20-M20

Nav Av Nav Av

Fig. 5 Effects of morphine on testing performance of pretraining-
treated mice in the plus-maze DAT. Mice were treated with saline
(Sal), 5 (M5), or 20 (M20) mg/kg morphine 30 min before a 10-min
training session and tested during 3 min, 24 h later, 30 min after an
injection of saline (-Sal), 5 (-M5), or 20 (-M20) mg/kg morphine.
Results are presented as mean±SE of time (seconds) spent in the
aversive enclosed arm (Av) and in the nonaversive enclosed arm
(Nav) of a plus-maze discriminative avoidance apparatus in the
training (a) and test (b) sessions. *p<0.05 compared to the time
spent in the nonaversive enclosed arm
 7
Table 2 % TAv, % TO, TE, and EE of a plus-maze discriminative avoidance apparatus (mean±SE) presented by mice treated with saline, 5,
or 20 mg/kg morphine 30 min before training or test sessions (see legend for Fig. 5)
Training Test
Sal M5 M20 Sal–Sal Sal–M5 Sal–M20 M5–M5 M20–M20

% TAv 4.24±0.60 7.47±3.85 3.42±4.74 39.16±11.15 0.68±0.68a,b 0.34±0.34a,b 33.66±12.47 56.09±14.72

% TO 15.93±3.69 13.92±7.94 6.00±1.39 17.66±4.56 0.74±0.74a 1.70±1.70a 2.42±1.40a 2.92±2.44a
TE 17.64±1.60 14.20±2.79 14.10±2.34 7.63±1.35 1.20±0.25a 2.00±1.12a 3.60±1.17a 2.90±1.38a
EE 14.07±1.36 11.2±2.03 11.2±1.91 5.00±0.82 1.10±0.18a 1.60±0.72a 3.00±0.93 2.10±0.81a
% TAv Percent time spent in the aversive enclosed arm, % TO percent time spent in the open arms, TE total number of entries, EE number of
entries in both enclosed arms
a
p<0.05 compared to Sal–Sal group in the test session
b
p<0.05 compared to M5–M5 and M20–M20 groups in the test session (ANOVA followed by Duncan’s test)

Experiment IV: effects of pretest 5 and 20 mg/kg
morphine on memory of pretraining morphine-treated
mice tested in the plus-maze discriminative
avoidance task
Two-way ANOVA with treatment as between-subjects
factor and arm type (aversive vs nonaversive) as within-
subject factor revealed significant effects of arm type [F
(1,90)=470.67, p<0.001]. Post hoc analysis by Duncan’s
test revealed that all the groups spent significantly less time
in the aversive than in the nonaversive enclosed arm
(Fig. 5a). In this session (training), one-way ANOVA fol-
lowed by Duncan’s test revealed that there was no signifi-
cant difference in any of the groups for percent time in the
aversive enclosed arm (Table 2).
In the test session, two-way ANOVA revealed only a
significant arm-type effect [F(1,90)=23.195, p<0.001]. Post
hoc analysis by Duncan’s test revealed that all the groups
except M20–M20 (animals that received 20 mg/kg mor-
phine before both sessions) presented significantly less time
in the aversive enclosed arm than in the nonaversive one
(Fig. 5b). In this session, one-way ANOVA followed by
60
Duncan’s test showed that the groups treated with saline
before training and 5 or 20 mg/kg morphine before test
presented significantly decreased percent time in the aver-
sive enclosed arm (Table 2) when compared to all the other
groups [F(4,43)=5.8421, p<0.001].
No differences were found in percent time spent in the
open arms in the training session (one-way ANOVA fol-
lowed by Duncan’s test; Table 2). In the test session, all
the animals that received morphine before testing pre-
sented a decrease in percent time spent in the open arms
[F(4,43)=4.0917, p<0.01].
In the training session, no differences were found when
mice treated with 5 or 20 mg/kg morphine were compared to
saline controls for total number of entries [F(2,45)=1.0154,
p>0.05] or number of entries in the enclosed arms [F
(2,45)=1.0291, p>0.05] (one-way ANOVA followed by
Duncan’s test; Table 2). In the test session, one-way
ANOVA followed by Duncan’s test showed a decrease in
the total number of entries [F(4,43)=2.9584, p<0.05] and
number of entries in the enclosed arms [F(4,43)=2.9443,
p<0.05] presented by all the animals treated with morphine
before the test session, except by the animals treated with
5 mg/kg morphine before both sessions (Table 2).

50 Experiment V: effects of morphine in nociception

40 H evaluated by the hot plate procedure
Latency (s)

O O O
O * * *
30 * * ANOVA for latency to hind paw withdrawal with treatment
* *
*
* as between-subjects factor and time (minutes of observa-
20
tion) as a repeated measure revealed significant effects of
10 treatment [F(3,16)=8.67, p<0.01], time [F(6,96)=5.39,
p<0.001], and treatment × time interaction [F(18,96)=
0 2.84, p>0.01] (Fig. 6). All the animals presented a similar
0 10 20 30 40 50 60
baseline latency. After treatment, the dose of 5 mg/kg
Time (min)
morphine did not modify latency to hind paw withdrawal.
Sal M5 M 10 M 20 The dose of 10 mg/kg morphine induced an analgesic effect
Fig. 6 Effects of morphine on hind paw withdrawal from a hot from 30 min onward (increased latency when compared to
plate. Mice were treated with saline (Sal), 5 (M5), 10 (M10), or 20 saline-treated animals). In addition, the dose of 20 mg/kg
(M20) mg/kg morphine. Latency for hind paw withdrawal was morphine was effective in inducing an analgesic effect from
measured upon exposure to a hot plate immediately before (0) and 20 min onward. Finally, at 40 min of observation, the group
10–60 min after treatment. Data are presented as mean±SE latency
to hind paw withdrawal (s).*p<0.05 compared to Sal group. °p<0.05 treated with 20 mg/kg morphine was significantly different
compared to the M5 group. Hp<0.05 compared to M10 group from the group treated with 10 mg/kg morphine, suggesting
(ANOVA with repeated measures followed by Duncan’s test) a dose-dependent antinociceptive effect of morphine.
8
Discussion
In the plus-maze discriminative avoidance paradigm, learn-
ing is indicated by a decrease in the exploration of the
aversive enclosed arm throughout the training session
(Silva et al. 2004a), while retrieval of the task is indicated by
decreased exploration of this arm in the test session when
the aversive stimuli are no longer present. Indeed, amnesic
procedures increase (Claro et al. 1999; Silva et al. 1999,
2002a,b, 2004a; Silva and Frussa-Filho 2000, 2002), while
memory-improving manipulations decrease (Silva et al.
1997, 1999, 2000; Claro et al. 1999) aversive arm explo-
ration in the test session. In this respect, results from
experiment I showed that all doses of morphine adminis-
tered before the training induced retrieval deficits in the test
session. Indeed, besides spending the same amount of time
in the aversive and in the nonaversive enclosed arms (see
Fig. 1b), all the groups treated with morphine presented
significantly increased percent time in the aversive enclosed
arm when compared to saline-treated animals (Fig. 1d).
These data are in agreement with other studies reporting a
memory deficit in avoidance tasks after pretraining mor-
phine administration (Izquierdo 1979; Ragozzino et al.
1992; Ragozzino and Gold 1994, 1995; McNay and Gold
1998; Aguilar et al. 2004).
The possibility that the retrieval deficit shown by mor-
phine-treated animals in experiment I was the result of some
effect of the drug in the training session—which would
modify the learning process and therefore impair retention
and/or retrieval—could be raised. Regarding motor behav-
ior, it was observed that 10 and 20 mg/kg morphine de-
creased total number of entries and entries in the enclosed
arms of the apparatus (Fig. 2c; Table 1). Although morphine
has been mostly reported as a stimulant of motor behavior in
mice (Hynes and Berkowitz 1983; Stevens et al. 1986;
Kuribata 1995; Kuzmin et al. 2000) and rats (Vivian and
Miczek 1999; Kalinichev et al. 2004), there are some studies
showing that morphine, depending on the dose and route of
administration, can decrease locomotion in rats (Havemann
et al. 1982; Boyer et al. 1998; Vivian and Miczek 1998;
Timar et al. 2005). In addition, our data are in accordance
with the fact that systemic administration of morphine,
especially at doses over 10 mg/kg, is associated with a
biphasic effect on locomotor activity. Indeed, when mor-
phine is administered to rats, an initial period of hypo-
locomotion (lasting 30–60 min) and a subsequent period of
hyperactivity are observed (Babbini and Davis 1972; Vasko
and Domino 1978; Roberts et al. 1978; Walter and
Kuschinsky 1989; Johnson and Glick 1993; Kosten and
Bombace 2000). This biphasic pattern of locomotor
alterations has also been demonstrated in hamsters (Schunr
et al. 1983) and mice (Saito 1990). Notwithstanding, the
alterations induced by morphine in the locomotor param-
eters did not seem to be related to the amnesic effect of the
drug. In this respect, 5 mg/kg morphine, despite inducing
retrieval deficits, did not modify locomotor activity in the
training session.
Pretraining morphine administration (especially at the
dose of 10 mg/kg) induced an increase in the parameters
related to locomotor activity in the test session (Figs. 2d
and 4c). This increase does not seem to be related to a
stimulant effect of the previously given (24 h before) mor-
phine treatment, since the stimulant phase of morphine
effect on locomotor activity in mice ends approximately
240 min after the injection (Kosten and Bombace 2000). It
is possible that the animals that received pretraining mor-
phine were not able to remember not only the presentation
of aversive stimuli in a specific arm but also the general
information about the apparatus. In other words, the im-
pairment effect of morphine may not have been specifically
related to the associative learning induced by pairing light
and noise to the aversive arm. Habituation (i.e., the decre-
ment of exploratory activity when a rodent is exposed to a
new environment; Conceição et al. 1994; Silva et al. 1996)
deficit may have also occurred in pretraining-morphine-
treated mice.
Another modification induced by morphine administra-
tion in the training session was the decrease in percent time
spent in the aversive enclosed arm (Fig. 1c; Table 1). Al-
though controversial to a decreased retrieval, this result
could reflect an improvement in learning of the task; that is,
the animals treated with morphine could have spent less
time in the aversive arm because they learned faster to avoid
it. Indeed, the analysis of the aversive enclosed arm explo-
ration throughout the training session does provide an
indication of better learning by morphine-treated animals
(Fig. 3). In fact, although both saline- and morphine-treated
mice presented decreased percent time in the aversive arm
from the second minute onward, this decrease had a greater
magnitude in morphine-treated mice. This increased learn-
ing of the task by morphine-treated mice could be related to
the aversive properties of this drug, which can induce
increased responses to aversive situations. Indeed, Anseloni
et al. (1999) demonstrated that although low doses of sys-
temically administered morphine (0.1–0.3 mg/kg) induced
an anxiolytic-like behavior, a high dose of this drug injected
into the dorsal periaqueductal gray increased anxiety-like
behaviors in an elevated plus-maze.
The analysis of percent time spent in the open arms
(currently used as a measurement of anxiety-like behavior
in rodents; Pellow and File 1986; Lister 1987; Frussa-Filho
et al. 1991, 1999; Goto et al. 1993; Silva et al. 2004b)
showed that morphine had no effect on anxiety-like be-
havior of mice in the training session of the plus-maze
discriminative avoidance apparatus (Fig. 2a; Table 1). This
result is not in accordance with other studies that show an
anxiolytic effect of this drug in the conventional elevated
plus-maze (Motta and Brandão 1993; Anseloni et al. 1999;
Kõks et al. 1999; Sasaki et al. 2002; Shin et al. 2003). These
discrepant results could be due to qualitative differences
between the exploration of the open arms of the conven-
tional elevated plus-maze apparatus and the plus-maze ap-
paratus used here. However, percent time spent in the open
arms of the apparatus employed here has been extensively

used for evaluation of anxiety-related behaviors in mice,
presenting similar results to those obtained in the conven-
tional elevated plus-maze (Silva et al. 1997, 2002a, 2004b;
Silva and Frussa-Filho 2000, 2002; Calzavara et al. 2004).
This concern, notwithstanding the above-mentioned aver-
sive properties of morphine, may increase responses to
aversive situations (Anseloni et al. 1999). Accordingly,
morphine administration induced an anxiogenic-like be-
havior in the plus-maze apparatus when administered at
high doses in the dorsal periaqueductal gray (Anseloni et al.
1999). Furthermore, the opioid antagonist naltrexone po-
tentiated the anxiolytic effect of chlordiazepoxide in rats
and mice submitted to this and other animal models of
anxiety (Belzung and Agmo 1997; Frussa-Filho et al.
1999). Interestingly, the facilitation of the anxiogenic com-
ponent of morphine produced by an aversive environment
could also explain the observed decrease in the locomotor
activity parameters. Indeed, open-arm exploration and lo-
comotor activity in the plus-maze apparatus are closely
related (Moser 1989; Silva et al. 2004b).
In experiment III, we attempted to verify whether the
amnesic effect reported in the plus-maze DAT would be
related to state-dependent learning. The results were in ac-
cordance with the first experiment, showing that 10 mg/kg
morphine induced a retention deficit in mice tested in the
plus-maze apparatus (Fig. 4). Corroborating previous stud-
ies of morphine effects in avoidance paradigms (Izquierdo
1979; Ragozzino et al. 1992; Ragozzino and Gold 1994,
1995; McNay and Gold 1998; Aguilar et al. 2004), the
pretest administration of 5 or 10 mg/kg morphine com-
pletely counteracted the amnesic effect of pretraining ad-
ministration of this drug. In this respect, it has been
suggested that the improvement in performance induced by
pretest morphine would be related to a direct effect of this
drug on memory retrieval mechanisms (Nishimura et
al. 1990; Shiigi and Kaneto 1990). Accordingly, in the
present study, mice that received morphine only before the
test session presented better retrieval when compared to
control mice (decreased percent time in the aversive arm in
the test session; Table 2).
The present results lend support to the state-dependent
learning induced by morphine hypothesis. Indeed, mice that
received morphine (5 or 10 mg/kg) before both sessions
(training and test) were not different from controls, showing
the same amount of retrieval (experiments III and IV). In
contrast, the present results do not corroborate the obser-
vation that state-dependent learning is bidirectional, i.e.,
animals that are trained after a saline injection should pre-
sent retention deficits if morphine is administered before the
test session. In the present study, mice treated with mor-
phine before testing showed similar or even better retention
levels than that presented by the control group (Figs. 4
and 5; Table 2). In this respect, previous data have shown
that drug-to-vehicle state alterations often exert greater
effects than vehicle-to-drug changes do (Colpaert 1990;
Jackson et al. 1992).
The impaired retrieval induced by the dose of 20 mg/kg
was not counteracted by pretest administration of this dose
9
(Fig. 5b). This finding suggests that pretraining adminis-
tration of morphine, depending on the dose, can induce a
memory deficit that is not related to state-dependent learn-
ing. Indeed, opioid transmission was suggested to exert an
inhibitory modulation of memory consolidation, since post-
training administration of opioid agonists and antagonists
impair and improve retention, respectively (see Izquierdo
1982 for a review; Castellano et al. 1994; Cestari and
Castellano 1997).
Although no effect of morphine on anxiety levels in the
training session was detected, this drug was able to induce
an anxiogenic-like effect in the test session. In this respect,
there are several studies showing that the anxiety-like be-
havior related to the open arms of a plus-maze is quali-
tatively different in animals that had been previously
exposed to the maze (Rodgers et al. 1997; Holmes and
Rodgers 1999). Indeed, benzodiazepines—classical anxio-
lytic drugs—do not modify open-arm exploration in plus-
maze-experienced rodents (File 1990; Pereira et al. 1999;
Frussa-Filho and Ribeiro 2002; Calzavara et al. 2005).
Anxiogenic substances, however, usually maintain their
effects (decreased open-arm exploration) upon retesting
(File 1993). In this context, morphine, which has been
shown to induce aversion (Anseloni et al. 1999), could be
potentiating the already increased preference for the en-
closed arms in a second exposition to the plus-maze ap-
paratus. This concern, notwithstanding the anxiety state
generated by morphine, does not seem to be related to the
state-dependent learning induced by this drug, since this
effect was not present in the training session.
As mentioned in the “Introduction,” morphine-induced
analgesia should be considered when evaluating the effects
of this drug on learning and memory. In this respect, in the
present study, we have used nonnociceptive stimuli (light
and noise) to engender avoidance behavior. In addition,
results from experiment V (morphine-induced analgesia)
seem to corroborate the absence of a relationship between
the effects of this opiate on nociception and memory.
Indeed, while the three doses induced retrieval deficits, a
significant antinociceptive effect was found only at 10 and
20 mg/kg. Additionally, while the analgesic effect was
dose-dependent, no dose relation was found in the plus-
maze DAT test session. Finally, the results depicted in Fig. 6
also suggest dissociation between antinociceptive effects
and state-dependent learning, since the range of doses that
induced these effects was not the same.
In conclusion, the results reported here indicate that
morphine given before the training session induces retrieval
deficits in mice tested in the plus-maze DAT, an avoidance
paradigm that does not involve painful stimulation. This
deficit was shown to be related, in part, to a morphine-
induced state-dependent learning, since pretest morphine
administration restored retention performance to control
levels. Neither the memory deficit induced by pretraining
morphine nor the reversion of this deficit by pretest mor-
phine seems to be related to anxiety-related, locomotor, or
antinociceptive effects of this opiate.
10
Acknowledgements This research was supported by grants from
Fundação de Amparo a Pesquisa do Estado de São Paulo (proc.
01/10713-7 and FAPESP/CEPID proc. 98/14303-3), Fundo de
Auxílio ao Docente e Aluno da UNIFESP (FADA), Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and
Associação Fundo de Incentivo à Psicofarmacologia (AFIP).
The authors would like to thank Ms. Teotila R. Amaral, Mr.
Cleomar S. Ferreira, and Mr. Antônio Rodrigues dos Santos for
capable technical assistance. The experiments performed in the
present study comply with the current Brazilian laws and are in
accordance with the Ethics Committee of Universidade Federal de
São Paulo.
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