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Psychopharmacology (2006) 184: 1\u201312
DOI 10.1007/s00213-005-0238-6
ORIGINAL INVESTIGATION

C. L. Patti. S. R. Kameda. R. C. Carvalho.
A. L. Takatsu-Coleman. G. B. Lopez. S. T. Niigaki.
V. C. Ab\u00edlio. 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
AbstractRationale: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\u201320 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\u201310 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.

KeywordsAvoidance 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 Baratti1985; Ragozzino and Gold1994; 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 Castellano1997; 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 Dias1983; Bruins Slot and Colpaert1999a; Khavandgar et al.2002,2003; Jafari et al.2004; Mohammad-Reza and Rezayof2004; Vakili et al.2004). This phenomenon is

C. L. Patti. S. R. Kameda. R. C. Carvalho.
A. L. Takatsu-Coleman. G. B. Lopez. S. T. Niigaki.
V. C. Ab\u00edlio. R. Frussa-Filho. R. H. Silva (*)

Department of Pharmacology,
Universidade Federal de S\u00e3o Paulo,
Rua Botucatu, 862-Ed. Leal Prado,
04023-062 S\u00e3o Paulo, Brazil
e-mail: regina.farm@epm.br
Tel.: +55-11-55494122
Fax: +55-11-55792752

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 Colpaert1999a), and has been demonstrated after administration of several psychoactive drugs (Colpaert1990; 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 (Colpaert1990,1996; Spanagel1995). In addition, it has been shown to be dose- specific and related to time of administration (Nishimura et al.1990; Bruins Slot and Colpaert1999a; 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 Colpaert1999a). 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 Colpaert1999b). 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 Brand\u00e3o1993; Anseloni et al.1999; K\u00f5ks 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-Filho2000,2002; 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- Filho2000).

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 S\u00e3o Paulo, Brazil, since 1985; Festing1993) were used. Animals weighing 30\u201335 g were housed under conditions of controlled temperature (22\u201323\u00b0C) 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\u00d77\u00d714 cm, 03 lx at the floor level), opposite to two open arms (28.5\u00d77 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\u2019s test or by Student\u2019st 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\u2019s 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

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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\u00b0C), and the latency to hind paw withdrawal was recorded throughout 60 min at 10-min intervals (Woolfe and MacDonald1944). A maximal latency value of 30 s was set. The data were analyzed by ANOVA with repeated measures followed by Duncan\u2019s test.

Experimental design
Experiment IGroups 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 IIGroups 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 IIITwenty-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 IVGroups 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 VThe 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.

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 \u00d7 arm type in- teraction [F(3,72)=2.82,p<0.05]. Post hoc analysis by Duncan\u2019s 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\u2019s 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 \u00d7 arm type interaction effect [F(3,72)= 4.16,p<0.01]. Post hoc analysis by Duncan\u2019s 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\u2019s 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\u2019s 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\u2019s 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\u2019s 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-

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