M 5 Muscarinic Receptor Gene Deletion Accelerates Extinction , But Inhibits Reinstatement of Morphine-Conditioned Reward in Mice

Mesolimbic dopamine neurons in the ventral tegmental area (VTA) play important roles in mediating the rewarding effects of opiates in addicted rats and in drug relapse. M5 muscarinic receptors (M5R) are needed for prolonged dopamine release in the nucleus accumbens induced by morphine. However, little is known about the role of M5Rs in extinction and relapse. Here, we found that the rewarding effects of morphine are similar in wild-type and M5R knockout mice (C57BL/6). Thus, we further investigated the extinction and reinstatement of morphine-conditioned reward. Within group analyses showed that morphine-induced conditioned place preference (CPP) extinguished much faster in M5R KOs (on day 5) than wild-type mice (on 10 day). And cross group and genotype analyses further confirmed this finding. More interestingly, after extinction, either morphine (1, 6 and 10 mg/kg, i.p.) or amphetamine (1 and 3 mg/kg, i. p.) priming reinstated morphineinduced CPP only in wild-type, but not in M5R KOs. Regardless of the genotype, amphetamine but not morphine priming changed the context preference of mice to the black chamber, implying a drug-specific, but M5R-independent increase of anxiety in the mice. These results allow us to conclude that M5Rs play an important role in the extinction and reinstatement of morphine-conditioned reward.

Conditioned responses, produced through Pavlovian associations of drug effects with environmental cues, represent a form of long-term memory [15,16].The conditioned place preference (CPP) paradigm is a useful method for measuring reward learning and plasticity in the presence or absence of drug or environmental cues [17,18].Furthermore, conditioned responses to drug-associated stimuli may be extinguished after repeated exposure to these stimuli in a drug-free state [2,5,7].Throughout extinction, however, previously established responses are not eliminated but rather suppressed by newly formed associations [16,19].Once extinguished, drug-seeking behaviour can be reinstated by a lower dose of noncontingent injection of the drug, called "priming" [20][21][22].
The mesolimbic dopamine pathway, originating from neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc) is one system contributing to the motivational properties of opiates [23,24].M5R mRNA is the only muscarinic receptor mRNA found in dopamine-containing neurons of the substantia nigra and the VTA [25].M5Rs play an important role in activating mesolimbic or nigrostriatal dopamine neurons [26,27].For example, sustained NAc dopamine release induced by mesopontine stimulation was blocked by scopolamine injection (i.p.) in rats, or by M5R knockout (KO) in mice [26,28].The M5R is also needed for social communications in mice during sexual behaviour [29].
Activation of D2-like dopamine receptors in the NAc was found crucial for relapse [4,30].Lesions of the basolateral amygdala impaired cue-and cocaine-induced reinstatement in rats [31], and muscarinic receptor antagonism in the basolateral amygdala, blocked acquisition of cocaine-stimulus association [32].Given the importance of M5Rs in sustained dopamine release and reward expression, we tested whether the M5R gene is needed in extinction and reinstatement.We found that M5R gene deletion accelerates extinction, and suppresses reinstatement of morphineconditioned reward in mice.To our knowledge, this is the first report demonstrating a crucial role of acetylcholine receptors in the extinction and reinstatement of opioid reward.

Subjects
All mice used in these experiments were young adult males (25-35 g, 10-16 weeks).M5 KO mouse breeders were obtained by heterozygotic backcross breeding of homozygous M5 KO mice (129 × 1/SvJ) with C57BL/6 mice for six generations.C57BL/6 and 129 × 1/SvJ mice came from Charles River (Montreal, Canada) and the Jackson Laboratory (Bar Harbor, USA), respectively.Similarly, wild-type control breeder mice were obtained from littermates at the time of backcrossing.All offspring were bred at the University of Toronto.A total of 88 M5 wild-type and 96 M5 KO mice were used, respectively.Subjects were housed in groups of four, in plastic mouse cages in a sound-attenuated room at a temperature of 22°C with lights on from 0700-1900 h.Access to food and water was ad libitum throughout the experiments.Each experimental group consisted of eight mice per treatment.All experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals, and this study was approved by the University of Toronto Animal Care.

Drugs
The experimental drugs used in the procedures were morphine sulphate (University of Toronto Drug Dispensary, Ontario, Canada) and d-amphetamine (Sigma, Ontario, Canada).Morphine and amphetamine were dissolved in a 0.9% saline solution at a concentration of 0.9 and 0.15 mg/ml, respectively.Both experimental groups and saline controls were injected at a volume of 6.7 ml/kg.

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that differed in colour and texture, each measuring 15 × 15 × 15 cm [33].One environment consisted of a black box with a smooth, black Plexiglas floor and the other environment was a white box with a white, wire mesh floor.The two boxes were separated by a removable plastic wall, each side painted with the corresponding colour (shade).The ceiling of the boxes was made of removable, clear Plexiglas.Time spent in each chamber was recorded with a video system (Sony, Japan).

CPP Procedure, Extinction and Reinstatement Test
The order of experiments is shown in details in Figure 1.After a preliminary test day measuring weight and open-field locomotion, each mouse was subjected to unbiased place conditioning procedures using a standard unbiased place conditioning procedure [17,34,35].The procedure was unbiased [36] as the mice expressed no baseline biases in preferences for the two conditioning environments (15 min trial: black environment mean ± SEM = 476.6 ± 43.4; white environment mean ± SEM = 423.3± 43.4; t (7) = 0.983, P > 0.05) and no preferences with the injection procedure itself under saline conditions (15 min trial: salinepaired environment mean ± SEM = 477.6 ± 37.4; morphine-paired environment mean ± SEM = 424.7 ± 37.7; t (7) = 0.462, P > 0.05).
Immediately prior to each conditioning trial (Days 2-9 in Figure 1), mice were given an intra peritoneal (i.p.) injection of either 6 mg/kg morphine (or saline), and exposed to one of the two conditioning environments for a 15 min period.On the alternate day, mice were given an injection of saline (or morphine) and exposed to the alternate environment for 15 min.A 15 min conditioning trial was found to produce optimum morphine place preference in mice (data not shown).Mice received one conditioning trial per day and the conditioning procedure was repeated until each mouse received four pairings in each environment for a total conditioning period of eight days.Both treatment compartment and order of drug presentation were counterbalanced within groups.
On the test day, under drug-free conditions, each mouse was given equal access to both chambers simultaneously by removing the shared wall and placing the mouse in the centre of the test area.Time and activity in each environment were recorded over a 15 min period.

Extinction
After CPP test 1, each mouse was given saline for four days, and placed in one chamber (black or white two days each, counterbalanced) for 15 min each day.On test day 2, each mouse was given no injection as on test day 1, and allowed to explore both chambers for 15 minutes.Then, four more days of saline injections (2 in each chamber) were followed by another post-extinction CPP test (test 3) in which the mice were allowed to explore both chambers after no injection.

Priming
Two days later, another CPP test (test 4) was conducted after a single priming injection of morphine (i.p.) 10 min before the CPP test.This evaluated the effect of morphine priming on the reinstatement of CPP.To remove the effect of morphine priming on CPP, after an eight day series of saline conditioning, CPP test 5 was performed.Then another test of CPP (test 6) occurred after a single priming injection of amphetamine (i.p.) 10 min before the CPP test, to evaluate the reinstatement of morphine CPP by amphetamine.

Open-Field Locomotion after Saline and Morphine
Before open-field locomotion testing, we first tested movement coordination and strength using a gravity test, bar test, and rotarod test on day 44 as described before [37].A final test of spontaneous open-field locomotion after saline on day 45 was followed by a test of morphine-induced locomotion on day 46.

Statistical Analyses
Unless specified, results were expressed as mean ± SEM.

Statistical comparisons were performed with paired 2-tail Student's
Conditioning phase: saline and morphine

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t-test [38].A two-way ANOVA (SPSS version 12) was performed and followed by a post-hoc analysis with Student-Newman-Keuls test when appropriate.For the locomotor activity, the effect of genotype and drug was analyzed using two-way analysis of variance (twoway ANOVA) (genotype × dose) and Tuckey's post hoc test where appropriate [39].In all cases, p < 0.05 was considered to be statistically significant.

Morphine-Induced CPP in Wild-Type and M5 Muscarinic Receptor KO Mice
Before testing place preference, physiological tests of these mice were performed.M5R KO mice did not differ significantly from their wild-type littermates in body weight, body temperature, spontaneous open field locomotion, or motor coordination as shown before [40].To examine whether the lack of M5Rs was associated with changes in the reinforcing properties of opiates, we used the CPP procedure.In this paradigm, mice learn to associate the administration of a drug with one of two compartments which differed visually and texturally.Before drug treatment, preference for the white or black chamber was roughly equal (so called "unbiased") [17,41].In both M5 wild-type and M5 KO mice, administration of morphine (6 mg/kg i.p.) in one of the two chambers, significantly increased the time the mice spent in the morphine-associated chamber on the test day (Figure 2).As shown in Figure 2B (bottom), morphine induced a similar magnitude of CPP in both wild-type and M5 KO mice (t-test, n = 8, p > 0.05).

M5R Deletion Accelerated Extinction of Morphine-Conditioned Reward
To test the effects of the M5R deletion on the rate of CPP extinction, the morphine-conditioned wild-type and M5R KO mice were then treated only with saline for four days.The CPP tests were performed without injection on the next day (day 15, test 2 in Figure 1).Again the mice were treated only with saline for four more days and on the last day (day 20, test 3 in Figure 1) CPP was tested again without injection.
On day 5 (day 15, test 2 in Figure 1), the time M5 KO mice spent in the morphine-conditioned chamber was not significantly longer than that in the saline-conditioned chamber, indicating extinction.However, in the wild-type mice, the morphine-conditioned CPP was still present (Figure 3A).These results indicate that CPP extinguishes faster in M5 KO mice than in wild-type mice.On day 10 (day 20, test 2 in Figure 1

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no difference was observed between the time spent in saline and morphine-paired chambers in either wild-type and M5 mutant mice (Figure 3B), indicating that extinction does eventually occur in both genotypes (n = 8, p > 0.80).
To further confirm these findings, we performed cross group and genotype analyses by comparing the time spent by wild type mice on the morphine paired side with that in all other groups on either day 5 or day 10.We found that with the exception of the time spent by the wild type on the saline paired side (n = 8, p < 0.05), no significant difference was observed (n = 8, p > 0.05).Interestingly, over the four days' saline treatment, the average spent time change on the morphine paired side in the wild type mice was only 14.75 seconds (shorter than day 11), however there was a 60.125 seconds' change (shorter than day 11) in the mutant mice under the same condition.The average rate of extinction over four days was 3.68 s/ day and 15.03 s/day in wild type and M5 mutant mice, respectively.Here, we failed to obtain a significant difference between wild type and M5 mutant mice (n = 8, p > 0.05).Taken together, these results did show that M5 muscarinic receptor gene deletion accelerated the extinction of morphine-conditioned reward learning in the tested C57BL/B6 mice.And an increased number of tested mice may help strengthen the current results statistically.

Lower Dose of either Amphetamine or Morphine Priming Reinstates Morphine-Induced CPP in Wild-Type Mice, But Not in M5R KO Mice
To test the effects of M5R KO on drug-induced reinstatement, the CPP-extinguished mice were then primed by morphine or amphetamine.Surprisingly, 6 mg/kg morphine priming reinstated.CPP only in the wild-type mice, but not in the M5R KO mice (Figure 4) and so did 1 mg/kg amphetamine priming (Figure 5B).Similar priming effects were observed in 3 mg/kg amphetamine and 1 and 10mg/kg morphine, but not in saline (data not shown).These results showed that M5Rs play an important positive role in drugprimed reinstatement of CPP.
Further investigation of context preference in this study showed a shift in preference after drug priming.In particular, 1 mg/ kg amphetamine shifted the preference of both wild-type and M5R KO mice to the black chamber (Figure 5C), but 6 mg/kg morphine priming did not (Figure 4B).No significant difference was found between the wild-type and M5 KO mice.These results indicate amphetamine-specific induction of anxiety-like behavior in the priming test in both genotypes of mice [42,43].

Morphine-Administration Reduced the Locomotor Activity of the Morphine-Conditioned CPP-Extinguished Mice, But Not Naïve Mice
Before open-field locomotion testing, we first tested movement coordination and strength using a gravity test, bar test, and rotarod test as described before [37].We found that the M5R KO mice had normal movement ability (data not shown).The mean weights of wild-type and M5 KO mice were 28.66 ± 0.92 g and 26.65 ± 0.49 g, respectively.The mean body temperatures of wild-type and M5 KO mice were 36.83 ± 0.10°C and 36.47 ± 0.18°C, respectively.No significant differences were detected (paired t-test, n = 8, p > 0.05).
To test the long-term effects of morphine exposure in CPP conditioning on spontaneous locomotion, the mice were given an open-field locomotion test after 10 days of CPP testing and 10 days of extinction, and compared with naïve mice.As shown in Figure 6, naïve M5R KO mice showed slightly less spontaneous open-field locomotion than wild-type mice, consistent with our group's previous report [40].By contrast, after repeated morphine exposure (6 mg/kg, i. p.) in CPP and extinction testing, locomotor activity in the M5R KO mice was significantly reduced as compared with the wild-type mice (paired t-test, p < 0.05).This shows that the morphine-conditioned M5R KO mice moved less than either the naive mice, or the wild-type mice that were naïve or morphineconditioned.This is consistent with the notion that CPP morphine experience (during both CPP acquisition and reinstatement processes) sustains conditioned spontaneous locomotion more in wild-type than in M5R KO mice.

Discussion
We found that, by using the unbiased procedure, morphineinduced CPP was similar in wild-type and M5R KO mice, but that morphine-induced CPP extinguished faster in M5R KO mice than in wild-type mice.The rate of extinction in wild type mice is about 3 times higher than that of M5KO mice (between day 1 and 5).Subsequently, in the end of extinction (day 11), priming with either morphine or amphetamine reinstated morphine-induced CPP strongly in wild type, but not in M5R KO mice.Morphine experience also reduced the spontaneous locomotor activity of morphine- ISSN: 2474-5049 [33,47], we found less morphine-induced CPP in M5R KO mice than wild-type mice on a CD1x129SvJ background (Steidl et al., 2006 SFN meeting abstract).Knockout mice have most often been produced using the embryonic stem cells from 129 mouse strains [48], but 129 strains perform poorly in CPP tasks, and show more anxiety than C57BL/6 mice.
Another issue concerned with this study is which dose of morphine should be used and why it was used for CPP induction.In this study, it appeared that we only used a single dose of 6 mg/ kg morphine throughout the whole CPP reinforcement.As a matter of fact, our group have tried a range of doses of morphine (1-20 mg/kg, see Steidl et al., and 2006 SFN meeting abstract) and run a series of experiments to determine the proper dose of morphine for induction of CPP.We also referred to several previous publications in CPP induction using morphine [44].The sole reason that led us to choose 6 mg/kg morphine was that 6 mg/kg was shown as the most effective lowest dose in CPP induction in C57BL/B6 background mice.This dose not only allowed us to save time and cost, but also to get stable reliable results, showing our choice was correct.

Faster Extinction and Reduced Reinstatement in M5 KO Mice
The rate of extinction in M5 KO mice (approximately 15.03 s/ day on average from day 1 to 4, see Results) was quicker than that in wild-type mice (approximately 3.68 s/day on average from day 1 to 4, see Results), as indicated by the loss of CPP in M5R KO mice after only four days saline treatment as well as the calculation of preference time difference spent by wild type and M5KO mice on the morphine side change between Day 0 and 5 (see Figure 2, day 10 and 15, respectively).However, as shown in Figure 3B, there was total loss of CPP in both wild type and M5KO mice after eight days of saline exposure, M5Rs did not prevent extinction from exposed M5R KOs, but not that of wild-type mice, compared with naïve mice of both genotypes.These results show that M5Rs play an important role in the maintenance and reinstatement of the drug experience during extinction and after reinstatement.To our knowledge, this is the first report in this field.

Morphine Induced Similar CPP in both M5 KO and Wild-Type C57BL/6 Mice
In the present study, we found similarly reliable morphine conditioned place preference (rewarding effects) in wild-type and M5 KO mice, as measured by the unbiased CPP procedure, in which mice showed equal spent time exploring both chambers in the initial test.This is consistent with several previous studies of unbiased morphine-induced CPP showing that dopamine receptor blockers do not block morphine CPP in naïve rats (Nader and van der Kooy, 1996; Laviolette and van der Kooy, 2004).This contrasts, however, with a previous study that reported reduced CPP in M5R KO mice [44], using a "biased procedure" (i.e., a strong initial preference for the black chamber).A strong preference in baseline testing can reflect a strong aversion to one chamber [45,46], and therefore, a removal of that bias by morphine can be due to anxiolytic effects rather than rewarding effects of morphine is important [17,33,47].In this study, we adopted an "unbiased" approach since no rewarding effects were observed.This greatly helped avoid aversive effects of saline to CPP learning.However, due to the limitation of experimental design, it is impossible for us to remove the brain structure that lead to aversive effects, the interpretation of our results generated by our current CPP model should be with careful caution.
In addition, we used C57BL/6 strain mice in the present study, in contrast with several 129 background crossed strains reported previously [44].Consistent with this idea and the previous work

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happening completely after more days' saline treatment.Therefore, it appears that the M5 receptor may help maintain responding to the morphine paired chamber during the early phase of extinction.To our knowledge, this is the first time to demonstrate the role of M5Rs in maintaining morphine reward learning and memory.
After eight days' continuous saline treatment, both morphine (6 mg/kg) and amphetamine (1 mg/kg) priming reinstated CPP reward learning in wild-type mice, but not M5 KO mice, suggesting that intact dopamine function is important in reinstatement of a previously learned morphine CPP.These results support a role for M5Rs in maintaining and restoring previously learned CPP, possibly through the sustained activation of dopamine neurons by M5Rs [26,[49][50][51].Previous authors have argued that dopamine neural activity is important in the learning of extinction and spontaneous recovery in different paradigms [52,53].Considering the role of M5Rs in sustaining dopamine release of midbrain dopaminergic neurons [26,44,50] in response to opiates [44], the faster extinction and reduced reinstatement in M5R KO mice may result from a lack of sustained dopamine release after M5R knockout.However, we cannot rule out the possibility that M5Rs may also play its roles via basolateral amygdala or other sites in the rest of the brain [54].
Given that the different types and doses of drugs (1, 6 and 10 mg/kg) morphine.Here, the data obtained with 1 and 10 mg were not shown.For 1 and 3 mg/kg amphetamine, the data obtained with 3 mg/kg were not shown) used in drug priming tests, our current studies may have a comprehensive pharmacology meaning.Nevertheless, a more systemic study using a wide range of morphine may further strengthen the results.
One minor concern is about the best timing of drug priming in wild type and M5KO mice.There are two options: 1. Separately priming.This is to prime the group of mice as soon as this group of mice (here is M5KO mice) demonstrated extinguished CPP, and meanwhile leave another group of mice to go through the process of extinction, and then to prime them using same drugs as soon as it was found that CPP extinguished.2. Simultaneously priming.For this option, experimenters should wait for the final extinction in both compared groups.Since the aim of our current research is to prove the role of M5R in extinction and restatement in C57BL/ B6 mice, we want to see whether the extinction will be faster or not in M5KO mice and whether it is it more difficult or not for drug priming in M5KO mice after same procedure of treatments.Therefore, we chose the latter one.
It is worth mentioning that knockdown of M5R expression by antisense oligos in ventral tegument area (VTA) decreased the sensitivity of rats to long-term rewarding hypothalamic stimulation, in which each trial begins with priming stimulation [27].This is similar to our current drug priming results showing that morphine-induced CPP could be reinstated in wild type, but not in M5R KO, mice.If we had VTA region or dopamine neuron specific M5 knockout mice to be tested our current CPP models, we could further clarify the findings obtained in our current constitutively M5R deleted mice.This remains to be done in the future.Considering the size and the number of mice as well as the complexity of the experiments, it is hard for us to run pump delivering oligo-antisense experiments in the current model.

Drug-specific induction of anxiety in morphine-conditioned mice
Psychostimulants act on both anxiety and reward circuits [55][56][57].Both 6 mg/kg morphine and 1 mg/kg amphetamine priming reinstated CPP in morphine-conditioned mice, but only amphetamine induced anxiety-like preferences for the dark chamber in morphine-conditioned mice that previously showed no bias.
In mice, blocking or activating postsynaptic M1 receptors in the infralimbic prefrontal cortex modulate anxiety-like behaviour [58].In the shock-probe burying model, M4 but not M2 muscarinic receptor knockout mice showed increased anxiolysis compared with wild-type mice, suggesting that muscarinic M4 receptors are of particular significance in anxiety modulation [59].Most recently, it was shown that both non-selective and selective M1 and M4 mAChR agonists increased the time spent on exploring in the open arms, whereas antagonists decreased exploration [60].In the present study, we found that M5R deletion had little effect in morphine CPP reward learning, and did not affect the amphetamine-induced anxiety response.These results suggest that, unlike M1 and M4 but like M2 receptors, M5Rs do not appear to play a significant role in amphetamine-induced anxiety in C57BL/B6 mice.

Drug Craving and Locomotion
In cocaine-craving rats after forced abstinence, cocaine priming before extinction increased locomotor activity [61].A morphine priming dose (5 mg/kg) similarly stimulated locomotor activity in GDNF (+/-) mice [62].However, in CREB (alpha-delta) KO  ISSN: 2474-5049 mice, similar locomotor activity was observed in KOs and wildtype littermates after similar doses of morphine (5 and 10 mg/ kg).In the present study, morphine experience (6 mg/kg) did not change locomotor activity of either wild-type or M5R KO mice, but significantly reduced spontaneous locomotor activity of M5R KO mice at two weeks after morphine CPP reinforcement treatment.This suggests that M5Rs may play a positive role in morphine craving, as indicated indirectly by conditioned locomotion after morphine exposure and extinction.In particular, loss of M5Rs reduced the ability of previous morphine experience to sustain locomotion in an open field environment.
In conclusion, these results suggest that M5 muscarinic receptors play an important role in the extinction and reinstatement of morphine CPP in previously trained mice, but not in the acquisition of unbiased CPP in naïve C57BL/B6 mice.In particular, M5Rs are needed for maintenance of CPP during extinction after acquisition, and are also crucial for drug-induced priming of CPP after extinction.To our knowledge, this is the first report showing an important role of acetylcholine receptors in the extinction and reinstatement of opioid reward.

Figure 2 :Figure 3 :
Figure 2: Unbiased morphine-induced conditioned place preference in wild type and M5R KO mice.(A) Before conditioning, wild-type and M5 KO mice were tested for the time spent (in seconds) in the different chambers.No preferences for black (B) vs. white (W) chambers were observed in either genotype."S" and "M" represent saline and morphine treatments, respectively.(B) Morphine (6 mg/kg) induced similarly reliable place preference in wild-type and M5 KO mice of C57BL/6 background.

Figure 4 :
Figure 4: Morphine priming reinstated morphine-induced CPP in wild-type mice, but not M5R KOs.CPP-extinguished mice were primed with a single dose (6 mg/kg, i.p.) of morphine.The 4th CPP test was performed in the beginning 10 min after morphine administration (panel A).S1 (saline) and M1 (morphine) and S2 and M2 represent saline and morphine treatments in wild-type and M5 KO mice, respectively.W1 and B1 represent the time spent in white and black boxes, respectively, by wild-type mice.W2 and B2 represent the time in white and black boxes by M5R KO mice.A single dose of morphine (6 mg/kg) priming did not affect preference of mice to different shades of chambers (panel B).

Figure 5 :
Figure5: Amphetamine priming reinstated CPP in wild type mice, but not M5R KO mice.After the 4 th place preference test, the mice were consecutively treated with saline for 8 days, and the 5 th CPP test was performed to check CPP extinction after morphine priming (panel A).The 6th CPP test was performed at 10 min after a single dose of amphetamine (1 mg/kg) (panel B).Amphetamine priming (1 mg/kg) increased the place preference of morphine-conditioned mice for the black box (panel C).PP = Place Preference.

Figure 6 :
Figure 6: Morphine-administration significantly reduced the locomotor activity of morphine-conditioned wild-type and M5 KO mice.After 8-days of consecutive saline treatment of the morphine conditioned mice, CPP extinction test was performed and then the mice were given 6 mg/kg morphine (i.p.) and subjected to two consecutive days' locomotion tests.On Day 1: The locomotion of the mice wastested in an open field for 20 minutes (F20), and then the mice were treated with saline followed by another 20 minute (S20) test (A).On Day 2: The mice were tested with the same procedure except 6 mg/kg morphine was used instead of saline (B).