Bo ZHAN, Hong-Yuan MA, Jian-Li WANG, Chao-Bao LIU

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Sex differences in morphine-induced behavioral sensitization and social behaviors in ICR mice

Bo ZHAN, Hong-Yuan MA, Jian-Li WANG*, Chao-Bao LIU

College of Biological Sciences and Engineering,Beifang University of Nationalities, Yinchuan 750021, China

Gender and genetic strain are two prominent variants that influence drug abuse. Although certain sex-related behavioral responses have been previously characterized in ICR mice, little is known about the effects of sex on morphine-induced behavioral responses in this outbred strain. Therefore, in this study, we investigated the sex differences of morphine-induced locomotion, anxiety-like and social behaviors in ICR mice. After morphine or saline exposure for four consecutive days (twice daily), increased locomotion, more time spent in the central area, as well as attenuated rearing and self-grooming behaviors were found in morphine-treated females in an open field; no differences were found in locomotion and the time spent in the central area between male and female controls.When interacting with the same-sex individuals, female controls were engaged in more social investigation, following, body contacting and self-grooming behaviors than controls; morphine exposure reduced contacting and self-grooming behaviors in females; incontrast, these effects were not found in males. These results indicate that female ICR mice are more prosocial and are more susceptible to morphine exposure than males.

Morphine; ICR mice; Locomotion; Social behavior

INTRODUCTION

Gender and genetic strain are two prominent variants that influence drug abuse (Belzung & Barreau, 2000; Orsini et al, 2005; Phillips et al, 2008). It has been reported that females are more sensitive to the addictive properties of drugs compared with males (Anker, et al, 2011; Becker & Hu, 2008; Carroll & Anker, 2010). Besides, differences in behavioral responses to abused drugs may reflect differences in genetic vulnerability to drug abuse (Grabus et al, 2004). Due to the high genetic homogeneity, many inbred strains have been used in drug-related behavioral tests (Eisener-Dorman et al, 2011; Kennedy et al, 2012). For example, C57BL/6 and BALB/c mice, the two of the most commonly used inbred mouse strains (or sometimes their sublines) have been widely implicated in drug abuse research. In fact, outbred mice with genetically variable compositions, vigorous physiques and economical prices have also been considered for neuroethological studies (Dell'omo et al, 1993; Ge et al, 2013). For example, in addition to applications in immunology, pathology and pharmacology, the ICR mouse is also employed in behavioral neuroscience (Ge et al, 2013; Li et al, 2014; Liang et al, 2014) including drug-related studies (Kita-naka et al, 2014; Mao et al, 2011). Recent research has revealed that the ICR males display distinct paternity, including father-pup social interaction, and shed light on parental behaviors (Liang et al, 2014). They spent less time to get used to a new environment and have better environment memory capacity as compared to inbred C57BL/6 and BALB/c mice (Shi et al, 2008). The female ICR mice showed superior learning and memory abilities than males, but there were no sex differences in locomotion (Ge et al, 2013). Although some sex-related responses have been previously characterized in this strain (Aoki et al, 2010; Ge et al, 2013; Yamaura et al, 2013), to our knowledge, little is known about the sex differences of ICR mice in drug-related behaviors.

Behavioral sensitization is a phenomenon in which neural changes are elicited by exposure to repeated intermittent administration of psychostimulants or opio-ids (Stewart & Badiani, 1993; Wang et al, 2010). These neuroadaptations are responsible for drug-induced increases in locomotor activity, reinforcing and rewa-rding effects (Francès et al, 2000; Wang et al, 2010). The endogenous opioid systems are related to changes in social emotional responsivity and social behavior (Kennedy et al, 2011; Nocjar & Panksepp, 2007). The effects of morphine on rewarding effects, locomotion or social play behaviors have been explored in some other mice strains (Cunningham et al, 1992; Campbell et al, 2000; Cole et al, 2013; Vanderschuren et al, 1995). Although morphine-induced conditioned place preference (CPP) has been conducted in male ICR mice (Mao et al, 2011), some studies have proposed the possibility of a disassociation between rewarding effects and behavioral sensitization (Miner, 1997; Wang et al, 2012; Zhang et al, 2002). In this study, we explore the sex differences in morphine-induced beha-vioral sensitization, anxiety and sociability in ICR mice.

Materials and methods

Animal subjects

Male and female ICR mice (18-22 g) were purch-ased from Ningxia Medical University Laboratory Ani-mal Center (Yinchuan, China). The animals were housed in groups of four in standard transparent Makr-olon cages (32 cm′21.5 cm′17 cm). The colony room was illumi-nated on a 12:12 light-dark cycle (lights on 2000h) and the temperature was maintained at 23±2C. Food and water were available. Mice were allowed to adapt to housing conditions for one week and handled daily by the same experimenter for three days prior to testing. All experiment procedures were performed strictly in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.

Drug administration

Morphine-hydrochloride (Northwest Pharmaceu-tic-al Co., Ltd. Sinopharm, Xi’an, China) was diluted in saline. Mice received subcutaneously a daily binge injection of morphine or saline for four consecutive days. The daily binge pattern was consisted of two injections of morphine (10 mg/kg) or euqal vulume of saline at 0800h and 1400h, respectively.The dose of morphine applied in this study was according to the previous studies onbehavioral sensitization,social motivated behaviors and CPP performance in mice (Mao et al, 2011;Nocjar & Panksepp, 2007; Xu et al, 2007).

Open-field test

Animal subjects were randomly assigned to one of the four treatment groups: male mice receiving physiol-ogical saline (MS,=8) and morphine administration (MC,=8), and female mice receiving physiological saline (FS,=8) and morphine administration (FC,=8). Twenty minutes following the last injection, motor activity and anxiety-like behavior were assessed in an open-field chamber. The chamber is a brightly and evenly illuminated square arena (50 cm×50 cm×25 cm) made of white glacial polyvinyl chloride and illuminated with four 60 W lamps mounted 1.5 m above the arena. The area was divided into 16 quadrants (four central and 12 peripheral) (Fiore & Ratti, 2007). Mice were placed individually in the center of the open-field and left to explore for 5 min and videotaped under white illumin-ation. After the test trial was completed, the open-field was thoroughly cleaned with 70% ethanol solution. To assess anxiety-like behavior, the time spent in the center of the open-field was measured. The number of crossings between quadrants was used to assess locomotion. Additionally, rearing (raising on the hind legs and sniffing into the air or the wall of the box) and self grooming (licking own fur, sometimes using forepaws, passing them over the nose with a series of brief, horiz-ontal movements) were recorded. All focal animals were videotaped during experiments using a Sony camera. The frequency and total duration of these behaviors were later scored by a researcher blind to experimental treat-ment using Jwatcher 1.0.

Same-sex social interaction test

After the open-field test, social interaction test was conducted. The interaction test was performed using only same-sex dyads to eliminate the possibility that even a low frequency of sexually motivated behavior might confound the results of tests. Testing was conducted in a neutral plastic cage (46 cm×31.5 cm×20 cm), with approximately 2 cm of wood shavings covering the floor and a removable opaque divider in the middle. One mouse was placed on each side of the arena with the divider still in place and given 5 min to adapt to the arena. Once the divider was removed a video-recorder mounted 70 cm above the apparatus monitored the arena for 15 min.

The behavior of the individual was classified as social investigation (sniffing the face, body or anogenital area of an individual), following (moving in the direction of or pursuing the test partner who moves away), contact behavior (contact with another individual including staying together or amicable grooming), aggressive beha-vior (pouncing (jumps or lunges), fighting (tumbling and biting) and chasing), self-grooming (cephalocaudal progression that begins with rhythmic movements of the paws around the mouth and face, ears, descending to the ventrum, flank, anogenital area and tail). Since the aggressive behaviors were rarely observed during social interaction, the related data were not shown.

Statistical analysis

Statistical analyses were carried out using SPSS 10.0 (SPSS Inc., Chicago, Illinois, USA). Data were che-cked for normality using the one-sample Kolmog-orov–Smirnov test. Data from the open-field test and social interaction test were compared using two-way ANOVA with sex and morphine treatment as factors. Group differences were compared using post-hoc tests. All data are presented here as mean±. Statistical significance was taken at≤0.05.

Results

Behaviors in open-field

Two-way ANOVA revealed the effect of sex on transition ((3,28)=4.479,=0.043) and rearing (duration:(3,28)=5.272,=0.029; frequency:(3,28)=9.810,=0.004). Significant effects of morphine were found on the time spent in the central area((3,28)=6.039,=0.020), rea-ring (duration:(3,28)=5.307,=0.029; frequency:(3,28)=0.016,=0.692) and self-grooming (duration:(3,28)=4.748,=0.038; frequency:(3,28)=7.136,=0.012). The effect of interactions between sex and morp-hine was found in self-grooming frequency ((3,28)=9.017,=0.006).

Specifically, no differences were found in total tran-sition (12.71,=0.195) or time spent in the central area1.75,=0.667) betw-een female and male controls. Morphine-treated females showed more transitions (19.663,=0.049) and spent more time in the central area than the saline-treated females9.789,=0.021) (Figure 1 A, B).

Figure 1 Behaviours of ICR mice during an open-field test

A: Total transitions; B: Total time spent in the central area; C: Duration of rearing and self-grooming behaviors; D: Frequency of rearing and self-grooming behaviors; *:≤0.05.

Compared with male controls, female controls have higher levels of rearing (duration:10.633,=0.012; frequency:=) and self-grooming behaviors (duration:=–0.655,=0.901; frequency=1.875,=0.032)). Moreover, morphine-treatedfemales showed less rearing (duration:–12.743,=0.003; frequency:2.375,=0.361) and self-grooming behaviors (duration:=–7.84,=0.146; frequency:=–3.500,<0.001) than the saline-treated females (Figure1 C, D). No morphine-induced differences were found in the transition numbers, the time spent in the central area, the rearing and self-grooming behaviors in males (0.05, data not shown)(Figure 1).

Social interaction

The effects of sex and morphine on the frequency of social investigation (sex:(3,28)=22.846,<0.001; morphine:F(3,28)=4.892,=0.035) and self-grooming (sex: F(3,28)=23.086,<0.001; morphine:F(3,28)=10.024,=0.004) were revealed by two-way ANOVA test. The significant effects of sex on both the frequency of following (F(3,28)=4.904,=0.035) and con-tact behavior (F(3,28)=5.931,=0.022) as well the self-grooming duration (F(3,28)=4.753,=0.038) were also found. The frequency of contact behavior ((3,28)=7.026,=0.013) and duration of self-grooming behavior (F(3,28)=4.281,=0.048) were significantly affected by the interactions between sex and morphine.

Specifically, in saline groups, compared with male controls,female controls showed higher frequencies of social investigation (6.375,=0.004), follo-wing (3.125,=0.041), contact beha-viors (3.625,=0.004) and self-groom-ing behaviors (duration:52.94,=0.005; frequency:5.500,<0.001) (Figure 2). Compared with saline controls, morphine-treated female mice exhibited lower frequencies of contact behaviors (–3.750,=0.003) and self-grooming behaviors (duration:48.61,=0.009; frequency:–4.750,=0.001) (Figure 2C, D). However, no influences of morphine were found in the social investigation, following, contact and self-grooming behaviors in male mice (0.05, data not shown) (Figure 2).

DISCUSSION

Morphine-induced locomotion and anxiety-like beha-viors

In the present study, no sex differences were observed in locomotion or anxiety levels under saline treatment, which are consistent with previous findings in ICR mice (Ge et al, 2013). According to An et al (2011), similar phenomena in C57BL/6J and BALB/cJ mice were found in the open field and elevated plus maze tests as well. Here, female ICR mice exhibitedmore rearing and self-grooming behaviors than males. Since rearing behaviors of mice, to a certain degree, reflected its exploratory and cognitive ability (Edsbagge et al, 2004; Shi et al, 2008), our results indicate that the female ICR mice have higher levels of exploratory abilities than males. In addition to the time spent in the central zone, the changes in rearing and self-grooming are generally considered as indices of emotional behavioral processes (Carey et al, 2005; To & Bagdy, 1999; To & Bagdy, 1999). Thus, the differences between males and females in rearing and self-grooming behaviors suggest their different emotional changes in an open-field.

Figure 2 Behaviors of ICR mice in the same-sex social interaction test

A: Social investigation; B: Following behavior; C: Contact behavior; D: Self-grooming; *:≤0.05.

Several studies have shown that mice exhibit a height-ened sensitivity to the effects of morphine (Niu et al, 2013; Xu et al, 2009). In this study, morphine increased the locomotion whereas attenuated the levels of anxiety, rearing and self-grooming behaviors in females; the time spent in the central area of an open field implies the morphine-induced anxiolytic effects in females.Similarly, cocaine treatments also decrease levels of rearing and grooming behaviors (Carey et al, 2005). These findings illustrate the importance of rearing and self-grooming phenotypes in drug-related behavioral researches. Additionally, our results showed that the locomotion, the time spent in the central zone or the rearing and self-grooming behaviorsin males were not altered by morphine, indicating that female and male ICR mice respond differently to repeated morphine exposure.

Morphine-induced social behaviors

When interacting with the same-sex individuals, we found that under saline treatment, females were engaged in more social investigation, following, body contact and self-grooming behaviors than males. And these differences between males and females were inconsistent with their patterns in locomotion and anxiety-like behaviors. Interestingly, similar phenomena of high levels of sociability in females were also found in C57BL/6J and BALB/cJ mice (An et al, 2011; Wang et al, 2014). In addition, the decreased contact and self-grooming behaviors in morphine-treated females showed that morphine changed the social behaviors in females, whereas, morphine did not alter any social behaviors in males, combining our findings that morphine increased locomotor activity in females, indicating that female ICR mice are more susceptible to morphine than males.

Addition to ovarian hormones, sensitivity to the effects of drugs has been proposed to be associated with the sociability, emotion and memory (Curtis & Wang, 2007; Niigaki et al, 2010; Perrine et al, 2008). Compare with males, female ICR mice exhibit more sociabilityand better memory ability (Ge et al, 2013). These traits may influence behavioral responses to morphine in females. Besides, it has been reported that female ICR mice exhibit higher responses to stress and higher levels of serum corticosterone under both basal and stressed conditions as compared with males (Aoki et al, 2010; Yamaura et al, 2013). Sex differences in stress may contribute to sexually dimorphic patterns in morphine-induced behavioral sensitization (Holly et al, 2012; Wang et al, 2010).

In conclusion, this study compared the morp-hine-induced behavioral sensitization and social behaviors in female and male ICR mice. Their different behavioral responses to repeated morphine exposure indicate that female ICR mice are more susceptible to morphinethan males.

REFERENCES

An XL, Zou JX, Wu RY, Yang Y, Tai FD, Zeng SY, Jia R, Zhang X, Liu EQ, Broders H. 2011. Strain and sex differences in anxiety-like and social behaviors in C57BL/6J and BALB/cJ mice.60(2): 111-123.

Anker JJ, Carroll ME. 2011. Females are more vulnerable to drug abuse than males: evidence from preclinical studies and the role of ovarian hormones.: Neill J C, Kulkarni J. Biological Basis of Sex Differences in Psychopharmacology., 8: 73-96.

Aoki M, Shimozuru M, Kikusui T, Takeuchi Y, Mori Y. 2010. Sex differences in behavioral and corticosterone responses to mild stressors in ICR mice are altered by ovariectomy in peripubertal period.27(10): 783-789.

Becker JB, Hu M. 2008. Sex differences in drug abuse.29(1): 36-47.

Belzung C, Barreau S. 2000. Differences in drug-induced place conditioning between BALB/c and C57Bl/6 mice.and65(3): 419-423.

Campbell JO, Wood RD, Spear LP. 2000. Cocaine and morphine-induced place conditioning in adolescent and adult rats., 68(4): 487-493.

Carey RJ, DePalma G, Damianopoulos E. 2005. Evidence for Pavlovian conditioning of cocaine-induced responses linked to emotional behavioral effects.and80(1): 123-134.

Carroll ME, Anker JJ. 2010. Sex differences and ovarian hormones in animal models of drug dependence.58(1): 44-56.

Cole SL, Hofford RS, Evert DJ, Wellman PJ, Eitan S. 2013.Social influences on morphine conditioned place preference in adolescent mice.,18(2): 274-285.

Cunningham CL, Niehus DR, Malott DH, Prather LK. 1992. Genetic differences in the rewarding and activating effects of morphine and ethanol.107(2-3): 385-393.

Curtis JT, Wang Z. 2007. Amphetamine effects in microtine rodents: a comparative study using monogamous and promiscuous vole species.148(4): 857-866.

Dell'omo G, Laviola G, Chiarotti F, Alleva E, Bignami G. 1993. Prenatal oxazepam effects on cocaine conditioned place preference in developing mice.15(3): 207-210.

Edsbagge J, Zhu SW, Xiao MY, Wigstr?m H, Mohammed AH, Semb H. 2004. Expression of dominant negative cadherin in the adult mouse brain modifies rearing behavior.25(3): 524-535.

Eisener-Dorman AF, Grabowski-Boase L, Tarantino LM. 2011. Cocaine locomotor activation, sensitization and place preference in six inbred strains of mice.and7(1): 29.

Fiore L, Ratti G. 2007. Remote laboratory and animal behaviour: an interactive open field system.49(4): 1299-1307.

Francès H1, Graulet A, Debray M, Coudereau JP, Guéris J, Bourre JM. 2000. Morphine-induced sensitization of locomotor activity in mice: effect of social isolation on plasma corticosterone levels.860(1-2): 136-140.

Ge JF, Qi CC, Qiao JP, Wang CW, Zhou JN. 2013. Sex differences in ICR mice in the Morris water maze task.62(1): 107-117.

Grabus SD, Glowa JR, Riley AL. 2004. Morphine- and cocaine-induced c-Fos levels in Lewis and Fischer rat strains.998(1): 20-28.

Holly EN, Shimamoto A, Debold JF, Miczek KA. 2012. Sex differences in behavioral and neural cross-sensitization and escalated cocaine taking as a result of episodic social defeat stress in rats.224(1): 179-188.

Kennedy BC, Panksepp JB, Runckel PA, Lahvis GP. 2012. Social influences on morphine-conditioned place preference in adolescent BALB/cJ and C57BL/6J mice.219(3): 923-932.

Kennedy BC, Panksepp JB, Wong JC, Krause EJ, Lahvis GP. 2011. Age-dependent and strain-dependent influences of morphine on mouse social investigation behavior.22(2): 147-159.

Kitanaka N, Kitanaka J, Hall FS, Uhl GR, Watabe K, Kubo H, Takahashi H, Tanaka K, Nishiyama N, Takemura M. 2014. Agmatine attenuates methamphetamine-induced hyperlocomotion and stereotyped behavior in mice.25(2): 158-165.

Li M, Fu Q, Li Y, Li SS, Xue JS, Ma SP. 2014. Emodin opposes chronic unpredictable mild stress induced depressive-like behavior in mice by upregulating the levels of hippocampal glucocorticoid receptor and brain-derived neurotrophic factor.98:1-10.

Liang MK, Zhong J, Liu HX, Lopatina O, Nakada R, Yamauchi AM, Higashida H. 2014. Pairmate-dependent pup retrieval as parental behavior in male mice.8:186.

Mao Yu, Yang SC, Liu C, Ma YY, Hu XT. 2011. Effects of propranolol on acquisition and retrieval of morphine- induced conditioned place preference memories in ICR mice.32(6): 670-674.

Miner LL. 1997. Cocaine reward and locomotor activity in C57BL/6J and 129/SvJ inbred mice and their F1 cross.and58(1): 25-30.

Niigaki ST, Silva RH, Patti CL, Cunha JL, Kameda SR, Correia-Pinto JC, Takatsu-Coleman AL, Levin R, Abílio VC, Frussa-Filho R. 2010. Amnestic effect of cocaine after the termination of its stimulant action. Progress in Neuro-Psychopharmacology and34(1): 212-218.

Niu HC, Zheng YW, Huma T, Rizak JD, Li L, Wang GM, Ren H, Xu LQ, Yang JZ, Ma YY, Lei H. 2013. Lesion of olfactory epithelium attenuates expression of morphine-induced behavioral sensitization and reinstatement of drug-primed conditioned place preference in mice.and103(3): 526-534.

Nocjar C, Panksepp J. 2007. Prior morphine experience induces long-term increases in social interest and in appetitive behavior for natural reward.181(2): 191-199.

Orsini C, Bonito-Oliva A, Conversi D, Cabib S. 2005. Susceptibility to conditioned place preference induced by addictive drugs in mice of the C57BL/6 and DBA/2 inbred strains.181(2): 327-336.

Perrine SA, Sheikh IS, Nwaneshiudu CA, Schroeder JA, Unterwald EM. 2008. Withdrawal from chronic administration of cocaine decreases delta opioid receptor signaling and increases anxiety- and depression-like behaviors in the rat.54(2): 355-364.

Phillips TJ, Kamens HM, Wheeler JM. 2008. Behavioral genetic contributions to the study of addiction-related amphetamine effects.32(4): 707-759.

Shi JW, Zou H, Jin ML. 2008. Assessing exploratory behavior and memory in ICR, BALB/c and C57BL/6 mice using habituation.29(1): 49-55.

Stewart J, Badiani A. 1993. Tolerance and sensitization to the behavioral effects of drugs.4(4): 289-312.

To CT, Bagdy G. 1999. Anxiogenic effect of central CCK administration is attenuated by chronic fluoxetine or ipsapirone treatment.38(2): 279-282.

Vanderschuren LJMJ, Spruijt BM, Van Ree JM, Niesink RJM. 1995. Effects of morphine on different aspects of social play in juvenile rats.117(2): 225-231.

Wang JL, Wang B, Chen W. 2014. Differences in cocaine-induced place preference persistence, locomotion and social behaviors between C57BL/6J and BALB/cJ mice.35(5): 426-435.

Wang YC, Wang CC, Lee CC, Huang AC. 2010. Effects of single and group housing conditions and alterations in social and physical contexts on amphetamine-induced behavioral sensitization in rats.486(1): 34-37.

Wang JL, Zhang LX, Zhang P, Tai FD. 2012. Cocaine-induced rewarding properties, behavioural sensitization and alteration in social behaviours in group-housed and postpuberty isolated female mandarin voles.23(7): 693-702.

Xu ZW, Hou B, Gao Y, He FC, Zhang CG. 2007. Effects of enriched environment on morphine-induced reward in mice.204(2): 714-719.

Xu YF, Su RB, Yang RF, Wu N, Lu XQ, Li J. 2009. Effects of Y-IP5 on morphine-induced behavioral sensitization and conditioned place preference in mice.25(12): 1578-1583.

Yamaura K, Bi Y, Ishiwatari M, Oishi N, Fukata H, Ueno K. 2013. Sex differences in stress reactivity of hippocampal BDNF in mice are associated with the female preponderance of decreased locomotor activity in response to restraint stress.30(12): 1019-1024.

Zhang Y, Mantsch JR, Schlussman SD, Ho A, Kreek MJ. 2002. Conditioned place preference after single doses or "binge" cocaine in C57BL/6J and 129/J mice.and73(3): 655-662.

Received: 28 September 2014; Accepted: 12 December 2014

This study was supported by the National Natural Science Foundation of China (31260513); the National Innovation Experiment Program for University Students (XJCX-2014-106)

, E-mail: wang_jianli@163.com