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Impaired social interaction and reduced anxiety-related behavior in vasopressin V1a receptor knockout mice
Behavioural Brain Research 178 (2007) 123–127
Research report
Impaired social interaction and reduced anxiety-related behavior in vasopressin V1a receptor knockout mice Nobuaki Egashira a , Akito Tanoue b , Tomomi Matsuda a , Emi Koushi a , Satoko Harada a , Yukio Takano c , Gozoh Tsujimoto d , Kenichi Mishima a , Katsunori Iwasaki a , Michihiro Fujiwara a,∗ a
Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma Jonan-ku, Fukuoka University, Fukuoka 814-0180, Japan b Department of Molecular, Cell Pharmacology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan c Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan d Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan Received 29 May 2006; received in revised form 8 December 2006; accepted 11 December 2006 Available online 15 December 2006
Abstract The arginine vasopressin (AVP) system plays an important role in social behavior. Autism, with its hallmark disturbances in social behavior, has been associated with the V1a receptor (V1aR) gene. Furthermore, impairments of social function are often observed in symptoms of schizophrenia. Subchronic phencyclidine (PCP) produces behaviors relating to certain aspects of schizophrenic symptoms such as impairing social interaction in animals and it reduces the density of V1aR binding sites in several brain regions. Here, we report that V1aR knockout (KO) mice exhibited impairment of social behavior in a social interaction test, and showed reduced anxiety-related behavior in elevated plus-maze and marble-burying behavior tests. Given the current findings, the V1aR may be involved in the regulation of social interaction, and V1aR KO mice could be used as an animal model of psychiatric disorders associated with social behavior deficits, such as autism and schizophrenia. © 2007 Published by Elsevier B.V. Keywords: Vasopressin; V1a receptor; Knockout; Social interaction; Anxiety; Mice
1. Introduction Arginine vasopressin (AVP) is a neurohypophyseal peptide. AVP receptors have been classified into three subtypes: V1a, V1b and V2 receptors [14,29], based on their intracellular transduction mechanisms. The V1a and V1b receptors are associated with phosphoinositol turnover, while the V2 receptor activates adenylate cyclase [9,23]. The V1a receptor (V1aR) is widely distributed in the central nervous system including the septum, cerebral cortex, hippocampus and hypothalamus [10,27,31]. A recent study showed that V1aR play a critical role in regulating behavior such as learning and memory, social recognition and anxiety-like behavior. We have previously reported that V1aR
∗
Corresponding author. Tel.: +81 92 871 6631x6600; fax: +81 92 863 0389. E-mail address: [email protected] (M. Fujiwara).
0166-4328/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.bbr.2006.12.009
knockout (KO) mice exhibit an impairment of spatial learning in comparison to wild-type (WT) mice in an eight-arm radial maze [7]. Moreover, a selective V1aR antagonist mildly impaired spatial memory in WT mice [7]. V1aR KO mice also exhibit a profound impairment in social recognition and reduction in anxiety-like behavior, and re-expressing V1aR in the lateral septum of V1aR KO mice using a viral vector resulted in a complete rescue of social recognition [3,4]. Furthermore, overexpression of the V1aR in the lateral septum of WT mice resulted in a potentiation of social recognition behavior and a mild increase in anxiety-related behavior [3]. AVP plays an important role in social behavior in comparative neurobiological studies [12]. Most recently, intranasally delivered AVP has been reported to affect social communication processes in humans [30]. One of the core features of autism is impairment in social behavior, such as reciprocal social interaction and communication. Interestingly, three
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independent reports demonstrated that autism has been associated with the V1aR gene [15,33,34]. On the other hand, impairments of social function are often observed in symptoms of schizophrenia. Phencyclidine (PCP) has been known to evoke increased severity of symptoms in schizophrenia and to induce schizophrenia-like symptoms in healthy individuals [20,32]. Moreover, PCP decreases social behavior in the rat social interaction test [26]. Similarly, subchronic treatment with PCP results in impaired social interaction and reduced density of V1aR in several brain regions such as the hypothalamus in rats [28]. There is a possibility that V1aR may play an important role in the regulation of social interaction. This idea is of special interest because impaired social interaction is among the core deficits of autistic disorders. However, whether V1aR is involved in the regulation of social interaction has remained untested. Therefore, we investigated whether mice lacking V1aR would exhibit impairment of social interaction. We also examined whether these V1aR KO mice would exhibit changes in anxiety levels and depression-related emotional responses. 2. Materials and methods 2.1. Animals Fifty-six male WT and 60 male V1aR KO mice (V1aR−/−), which were generated by gene targeting as previously reported [16], were used at ages 7–13 weeks for all of the experiments. All mice analyzed were from F3 to F5, which carried the genetic background of 129Sv and C57Black/6J strains. They were kept under a constant light–dark cycle (light 7:00–19:00) in a temperaturecontrolled (23 ± 2 ◦ C) room. Experiments were conducted during the light phase between 9:00 a.m. and 5:00 p.m. The animals had free access to food (CE-2, Crea Japan, Tokyo, Japan) and water in their home cages. All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University.
2.2. Open-field test The ambulation and the number of rearings in the open-field test were measured for 3 min as described previously [1]. Mice (n = 12 WT mice, n = 16 V1aR KO mice, aged 10 weeks) were placed in the center of the open field and the number of ambulation and rearing was measured by an apparatus consisting of a circular floor (diameter = 60 cm) divided by thin black lines into 19 equal blocks. The floor was enclosed by a parapet (height = 50 cm) with an upper opening (diameter = 90 cm). The apparatus was illuminated by a 100 W bulb placed 80 cm above the center of the floor of the apparatus. The activity was measured in a sound-proof dark room under the above-described standard housing conditions.
2.3. Traction-meter test For the traction-meter test, the subjects (n = 12 WT mice, n = 16 V1aR KO mice, aged 11 weeks) had previously been used in the open-field test. In the traction-meter test for muscle tone, we used a tractionmeter (Neuroscience Inc., Tokyo, Japan) that consisted of a muscle tone detector (48.5 cm × 26 cm × 10 cm) with stainless grids (2 mm in diameter, 29 cm × 16 cm) connected to a spring and a printer as described previously [13]. The stainless grids can freely rotate by themselves. The interval of the grids is 2 cm. Each mouse was placed on the stainless grid of the apparatus and the tail was slowly pulled in parallel at a constant speed backwards by an experimenter. The resistance of the forepaw was measured as muscle tone by the detector.
2.4. Rota-rod test After measuring the traction-meter test, the rota-rod test for motor coordination was performed. Mice (n = 12 WT mice, n = 16 V1aR KO mice, aged 12 weeks) were placed on a rotating rod (3 cm diameter; Neuroscience Inc., Tokyo, Japan) with a non-skid surface and the latency to falling was measured for up to 2 min. The test was performed three times a day for three consecutive days. The rotating speed was accelerated in the order of 5, 10 and 15 rpm each day.
2.5. Elevated plus-maze test The procedure is based on that described by Pellow et al. [24]. It was elevated to a height of 50 cm with two open (25 cm × 8 cm) and two enclosed arms (25 cm × 8 cm) arranged so that the arms of the same type were opposite each other. Mice (n = 9 WT mice, n = 11 V1aR KO mice, aged 10 weeks) were placed in the middle of an elevated plus-maze. The time spent in the open arms was used for analysis. The test duration was 5 min.
2.6. Forced swimming test The immobility time in the forced swimming test was measured as described previously [6], which was a modification of that described by Porsolt et al. [25]. Animals (n = 10 WT mice, n = 8 V1aR KO mice, aged 10 weeks) were placed in individual plastic cylinders (diameter 11 cm; height 18 cm) filled with 10 cm of water (23 ± 1 ◦ C) for 15 min, and then removed and dried. A magnet was attached to one of their forelimbs. The immobility time was recorded using the MicroAct Scratching Test (Version 1.03; Neuroscience Inc.) during a 15 min observation period for 2 days in a row.
2.7. Marble-burying behavior test The marble-burying behavior test was measured as described previously [19]. The mice (n = 9 of each genotype, aged 13 weeks) were placed individually in clear plastic boxes (30 cm × 30 cm × 28 cm), containing 25 glass marbles (1.5 cm in diameter) evenly spaced on sawdust 5 cm deep, without food and water. At the same time, the locomotor activity of mice was measured using an automated activity counter (NS-AS01, Neuroscience Inc., Tokyo, Japan) placed 15 cm above the same plastic boxes. The activity was measured with the illumination of a 100 W bulb. The results of marble-burying behavior were expressed as the number of marbles buried at least two-thirds deep in this paradigm within 30 min.
2.8. Social interaction test The social interaction test was performed in a neutral cage (clear plastic box: 30 cm × 25 cm × 17.7 cm) in the animal room. On days 1 and 2, each mouse received one session every day for 10 min in the neutral cage to habituate themselves to the apparatus. On day 3 (test day), pairs of unfamiliar mice (n = 8 pairs of each genotype, aged 7 weeks) with a similar genetic background were placed into the neutral cage for 10 min. Behavior was video-recorded by a camera placed 70 cm above the neutral cage. The time spent in social interaction (active contact such as sniffing, following and grooming the partner) was measured for 10 min.
2.9. Statistical analyses The results are expressed as the means ± S.E.M. The data were analyzed by unpaired t-test. A p-value of less than 0.05 was considered to be statistically significant.
3. Results 3.1. Open-field, traction-meter, rota-rod and forced swimming tests No significant differences were observed between WT mice and V1aR KO mice at 10–12 weeks of age in detailed analyses of
N. Egashira et al. / Behavioural Brain Research 178 (2007) 123–127
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Table 1 Performance of WT and V1aR KO mice in open-field, rota-rod, traction meter and forced swimming tests Measurement Open-field test Number of ambulation Number of rearing Traction meter test (kgf) Rota-rod test, latency to fall (s) 5 rpm 10 rpm 15 rpm
WT (n = 12)
V1aR KO (n = 16)
124.4 ± 7.4 18.9 ± 1.9 0.167 ± 0.007
139.4 ± 7.4 18.6 ± 1.8 0.177 ± 0.004
119.1 ± 0.9 119.9 ± 0.1 107.5 ± 3.4
120.0 ± 0.0 116.1 ± 2.9 114.8 ± 4.6
Measurement
WT (n = 10)
V1aR KO (n = 8)
Forced swimming test Immobility time (s), day 1 Immobility time (s), day 2
803.3 ± 23.7 854.0 ± 12.6
774.8 ± 35.4 801.9 ± 35.1
Values represent means ± S.E.M.
their motor function with open-field testing, the traction-meter test for muscle tone and the rota-rod test for motor coordination (Table 1). Moreover, there was no significant difference between WT mice and V1aR KO mice for the immobility time in the forced swimming test for measuring depression. In addition, there was no difference between WT mice and V1aR KO mice for their body weight (WT mice: 23.2 ± 0.5 g; V1aR KO mice: 23.8 ± 0.4 g). 3.2. Elevated plus-maze test The V1aR KO mice spent more time in the open arms of the elevated plus-maze than the WT mice did (p < 0.01 by the unpaired t-test, Fig. 1). 3.3. Marble-burying behavior test The V1aR KO mice buried significantly fewer glass marbles than the WT mice did (p < 0.01 by the unpaired t-test, Fig. 2A). There were no genotypic differences in locomotor activity during the marble-burying behavior (Fig. 2B).
Fig. 1. Elevated plus-maze test performance of WT and V1aR KO mice. The data are expressed as the means ± S.E.M. ** p < 0.01 vs. WT mice. The number of mice is shown at the bottom of each column.
Fig. 2. Number of buried marbles (A) and locomotor activity (B) in the marble burying behavior test in WT and V1aR KO mice. The data are expressed as the means ± S.E.M. ** p < 0.01 vs. WT mice. The number of mice is shown at the bottom of each column.
3.4. Social interaction test The V1aR KO mice exhibited a significant decrease in the social interaction time compared to that of the WT mice (p < 0.001 by the unpaired t-test, Fig. 3). In addition, no incidence of aggressive behavior such as attack biting and wrestling was observed at this time.
Fig. 3. Social interaction time in the social interaction test in WT and V1aR KO mice. The data are expressed as the means ± S.E.M. *** p < 0.001 vs. WT mice. The number of pairs of each genotype is shown at the bottom of each column.
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4. Discussion The major finding in the present study is that V1aR KO mice exhibited an impairment of social interaction compared to the WT mice in the social interaction test. The results of the study also showed that the number of ambulation and rearing in the V1aR KO mice was normal in the open-field test. Moreover, we did not observe differences between V1aR KO mice and WT mice in the traction-meter and rota-rod tests. Thus, motor function in V1aR KO mice was normal. Moreover, there was no significant difference between WT mice and V1aR KO mice for the immobility time in the forced swimming test for measuring depression. On the other hand, we observed that V1aR KO mice showed reduced anxiety-related behavior in the elevated plus-maze test. Therefore, it is unlikely that social interaction is impaired in V1aR KO mice because of a deficit in motor function, depression and high anxiety. These findings suggest that V1aR controls social interaction in particular. This idea is supported by the findings that subchronic treatment with PCP, an animal model of schizophrenia, results in impaired social interaction and reduced density of V1aR in several brain regions [28]. Moreover, the V1aR gene has been linked to autism, providing evidence that the V1aR gene may play a role in social communication and behavior in humans [15,33,34]. The second finding of this study is that the V1aR KO mice showed reduced anxiety-related behavior in the elevated plusmaze test whereas these mice performed normally on the forced swimming test. The present findings are consistent with previous findings [4]. The increased AVP expression in the hypothalamic paraventricular nucleus is considered to underlie anxiety-related behavior [17]. Also, the microinjection of V1 receptor antagonist into the septal nucleus of rats has been reported to reduce the anxiety in the elevated plus-maze test [18]. Furthermore, overexpression of the V1aR in the lateral septum of WT mice resulted an increase in anxiety-related behavior [3]. Therefore, V1aR may be involved in the anxiety-related behavior. The third finding of the present study is that the V1aR KO mice buried significantly fewer glass marbles than the WT mice did in the marble-burying behavior test. This phenomenon does not seem to contribute to a change in locomotor activity, since there were no genotypic differences in locomotor activity during the marble-burying behavior. It has been suggested that the burying behavior would be rewarding or compulsive [5]. In general, the marble-burying behavior test has been considered as an animal model of obsessive-compulsive disorder (OCD) [11,19,21]. In fact, serotonin (5-hydroxytryptamine, 5-HT)reuptake inhibitor (SRI) and selective 5-HT-reuptake inhibitor (SSRI), which have been used to treat human OCD symptoms, inhibit marble-burying behavior without affecting locomotor activity [11,21,22]. Therefore, the reduced number of marbles buried by V1aR KO mice might reflect an anti-OCD effect of the mutation. Alternatively, the interpretation that mice have less anxiety when they are burying foreign objects is based on the assumption that the animals would consider marbles startling by virtue of their novelty. Therefore, the reduced number of marbles buried by V1aR KO mice might reflect an anxiolytic effect of the mutation. This idea is supported by the present finding that
the V1aR KO mice showed reduced anxiety-related behavior in the elevated plus-maze test. Anxiolytic drugs have been proposed to function in the social interaction test, a rat model for detecting compounds with anxiolytic properties, by increasing the social interaction time [8]. In the present study, we found that V1aR KO mice exhibited the impairment of social interaction in spite of reduced anxietyrelated behavior. In addition, we found that V1aR KO female mice also exhibited the impairment of social behavior in the social interaction test (N. Egashira et al., unpublished data). Thus, there are no gender differences in the social interaction test. Our findings support the possibility that V1aR regulates social interaction. Finally, the results of the present experiments demonstrate that mice lacking the V1aR exhibit impaired social interaction. Autistic patients express social interaction deficits [2], and autism has been associated with the V1aR gene [15,33,34]. On the other hand, impairments of social function are often observed in symptoms of schizophrenia. Hence, V1aR may be involved in the regulation of social interaction, and V1aR KO mice could be used as an animal model for investigating social behavior deficits that co-occur with such illness as autism and schizophrenia. Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 18591318). References [1] Al-Khatib MH, D¨okmeci I, Fujiwara M. Differential role of nucleus accumbens and caudate-putamen in mediating the effect of nomifensine and methamphetamine on ambulation and rearing of rats in the open-field test. Jpn J Pharmacol 1995;67:69–77. [2] American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed. Washington, DC: American Psychiatric Association; 1994. pp. 66–71. [3] Bielsky IF, Hu S-B, Ren X, Terwilliger EF, Young LJ. The V1a vasopressin receptor is necessary and sufficient for normal social recognition: a gene replacement study. Neuron 2005;47:503–13. [4] Bielsky IF, Hu S-B, Szegda KL, Westphal H, Young LJ. Profound impairment in social recognition and reduction in anxiety-like behavior in vasopressin V1a receptor knockout mice. Neuropsychopharmacology 2004;29:483–93. [5] Broekkamp CL, Rijk HW, Joly-Gelouin D, Lloyd KL. Major tranquillizers can be distinguished from minor tranquillizers on the basis of effects on marble burying and swim-induced grooming in mice. Eur J Pharmacol 1986;126:223–9. [6] Egashira N, Iwasaki K, Takashima A, Watanabe T, Kawabe H, Matsuda T, et al. Altered depression-related behavior and neurochemical changes in serotonergic neurons in mutant R406W human tau transgenic mice. Brain Res 2005;1059:7–12. [7] Egashira N, Tanoue A, Higashihara F, Mishima K, Fukue Y, Takano Y, et al. V1a receptor knockout mice exhibit impairment of spatial memory in an eight-arm radial maze. Neurosci Lett 2004;356:195–8. [8] File SE. Animal models for predicting clinical efficacy of anxiolytic drugs: social behaviour. Neuropsychobiology 1985;13:55–62. [9] Gouzenes L, Sabatier N, Richard P, Moos FC, Dayanithi G. V1a- and V2type vasopressin receptors mediate vasopressin-induced Ca2+ responses in isolated rat supraoptic neurons. J Physiol 1999;517:771–9.
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[23] Omura T, Nabekura J, Akaike N. Intracellular pathways of V1 and V2 receptors activated by arginine vasopressin in rat hippocampal neurons. J Biol Chem 1999;274:32762–70. [24] Pellow S, Chopin P, File SE, Briley M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14:149–67. [25] Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977;229:327–36. [26] Sams-Dodd F. Phencyclidine in the social interaction test: an animal model of schizophrenia with face and predictive validity. Rev Neurosci 1999;10:59–90. [27] Szot P, Bale TL, Dorsa DM. Distribution of messenger RNA for the vasopressin V1a receptor in the CNS of male and female rats. Brain Res Mol Brain Res 1994;24:1–10. [28] Tanaka K, Suzuki M, Sumiyoshi T, Murata M, Tsunoda M, Kurachi M. Subchronic phencyclidine administration alters central vasopressin receptor binding and social interaction in the rat. Brain Res 2003;992:239–45. [29] Thibonnier M, Coles P, Thibonnier A, Shoham M. Molecular pharmacology and modeling of vasopressin receptors. Prog Brain Res 2002;139:179–96. [30] Thompson RR, George K, Walton JC, Orr SP, Benson J. Sex-specific influences of vasopressin on human social communication. Proc Natl Acad Sci USA 2006;103:7889–94. [31] Tribollet E, Raufaste D, Maffrand JP, Serradeli-Le Gal C. Binding of the non-peptide vasopressin V1a receptor antagonist SR-49059 in the rat brain: an in vitro and in vivo autoradiographic study. Neuroendocrinology 1999;69:113–20. [32] Tsai G, Coyle JT. Glutamatergic mechanisms in schizophrenia. Annu Rev Pharmacol Toxicol 2002;42:165–79. [33] Wassink TH, Piven J, Vieland VJ, Pietila J, Goedken RJ, Folstein SE, et al. Examination of AVPR1a as an autism susceptibility gene. Mol Psychiatr 2004;9:968–72. [34] Yirmiya N, Rosenberg C, Levi S, Salomon S, Shulman C, Nemanov L, et al. Association between the arginine vasopressin 1a receptor (AVPR1a) gene and autism in a family-based study: mediation by socialization skills. Mol Psychiatr 2006;11:488–94.
Research report
Impaired social interaction and reduced anxiety-related behavior in vasopressin V1a receptor knockout mice Nobuaki Egashira a , Akito Tanoue b , Tomomi Matsuda a , Emi Koushi a , Satoko Harada a , Yukio Takano c , Gozoh Tsujimoto d , Kenichi Mishima a , Katsunori Iwasaki a , Michihiro Fujiwara a,∗ a
Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma Jonan-ku, Fukuoka University, Fukuoka 814-0180, Japan b Department of Molecular, Cell Pharmacology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan c Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan d Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan Received 29 May 2006; received in revised form 8 December 2006; accepted 11 December 2006 Available online 15 December 2006
Abstract The arginine vasopressin (AVP) system plays an important role in social behavior. Autism, with its hallmark disturbances in social behavior, has been associated with the V1a receptor (V1aR) gene. Furthermore, impairments of social function are often observed in symptoms of schizophrenia. Subchronic phencyclidine (PCP) produces behaviors relating to certain aspects of schizophrenic symptoms such as impairing social interaction in animals and it reduces the density of V1aR binding sites in several brain regions. Here, we report that V1aR knockout (KO) mice exhibited impairment of social behavior in a social interaction test, and showed reduced anxiety-related behavior in elevated plus-maze and marble-burying behavior tests. Given the current findings, the V1aR may be involved in the regulation of social interaction, and V1aR KO mice could be used as an animal model of psychiatric disorders associated with social behavior deficits, such as autism and schizophrenia. © 2007 Published by Elsevier B.V. Keywords: Vasopressin; V1a receptor; Knockout; Social interaction; Anxiety; Mice
1. Introduction Arginine vasopressin (AVP) is a neurohypophyseal peptide. AVP receptors have been classified into three subtypes: V1a, V1b and V2 receptors [14,29], based on their intracellular transduction mechanisms. The V1a and V1b receptors are associated with phosphoinositol turnover, while the V2 receptor activates adenylate cyclase [9,23]. The V1a receptor (V1aR) is widely distributed in the central nervous system including the septum, cerebral cortex, hippocampus and hypothalamus [10,27,31]. A recent study showed that V1aR play a critical role in regulating behavior such as learning and memory, social recognition and anxiety-like behavior. We have previously reported that V1aR
∗
Corresponding author. Tel.: +81 92 871 6631x6600; fax: +81 92 863 0389. E-mail address: [email protected] (M. Fujiwara).
0166-4328/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.bbr.2006.12.009
knockout (KO) mice exhibit an impairment of spatial learning in comparison to wild-type (WT) mice in an eight-arm radial maze [7]. Moreover, a selective V1aR antagonist mildly impaired spatial memory in WT mice [7]. V1aR KO mice also exhibit a profound impairment in social recognition and reduction in anxiety-like behavior, and re-expressing V1aR in the lateral septum of V1aR KO mice using a viral vector resulted in a complete rescue of social recognition [3,4]. Furthermore, overexpression of the V1aR in the lateral septum of WT mice resulted in a potentiation of social recognition behavior and a mild increase in anxiety-related behavior [3]. AVP plays an important role in social behavior in comparative neurobiological studies [12]. Most recently, intranasally delivered AVP has been reported to affect social communication processes in humans [30]. One of the core features of autism is impairment in social behavior, such as reciprocal social interaction and communication. Interestingly, three
124
N. Egashira et al. / Behavioural Brain Research 178 (2007) 123–127
independent reports demonstrated that autism has been associated with the V1aR gene [15,33,34]. On the other hand, impairments of social function are often observed in symptoms of schizophrenia. Phencyclidine (PCP) has been known to evoke increased severity of symptoms in schizophrenia and to induce schizophrenia-like symptoms in healthy individuals [20,32]. Moreover, PCP decreases social behavior in the rat social interaction test [26]. Similarly, subchronic treatment with PCP results in impaired social interaction and reduced density of V1aR in several brain regions such as the hypothalamus in rats [28]. There is a possibility that V1aR may play an important role in the regulation of social interaction. This idea is of special interest because impaired social interaction is among the core deficits of autistic disorders. However, whether V1aR is involved in the regulation of social interaction has remained untested. Therefore, we investigated whether mice lacking V1aR would exhibit impairment of social interaction. We also examined whether these V1aR KO mice would exhibit changes in anxiety levels and depression-related emotional responses. 2. Materials and methods 2.1. Animals Fifty-six male WT and 60 male V1aR KO mice (V1aR−/−), which were generated by gene targeting as previously reported [16], were used at ages 7–13 weeks for all of the experiments. All mice analyzed were from F3 to F5, which carried the genetic background of 129Sv and C57Black/6J strains. They were kept under a constant light–dark cycle (light 7:00–19:00) in a temperaturecontrolled (23 ± 2 ◦ C) room. Experiments were conducted during the light phase between 9:00 a.m. and 5:00 p.m. The animals had free access to food (CE-2, Crea Japan, Tokyo, Japan) and water in their home cages. All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University.
2.2. Open-field test The ambulation and the number of rearings in the open-field test were measured for 3 min as described previously [1]. Mice (n = 12 WT mice, n = 16 V1aR KO mice, aged 10 weeks) were placed in the center of the open field and the number of ambulation and rearing was measured by an apparatus consisting of a circular floor (diameter = 60 cm) divided by thin black lines into 19 equal blocks. The floor was enclosed by a parapet (height = 50 cm) with an upper opening (diameter = 90 cm). The apparatus was illuminated by a 100 W bulb placed 80 cm above the center of the floor of the apparatus. The activity was measured in a sound-proof dark room under the above-described standard housing conditions.
2.3. Traction-meter test For the traction-meter test, the subjects (n = 12 WT mice, n = 16 V1aR KO mice, aged 11 weeks) had previously been used in the open-field test. In the traction-meter test for muscle tone, we used a tractionmeter (Neuroscience Inc., Tokyo, Japan) that consisted of a muscle tone detector (48.5 cm × 26 cm × 10 cm) with stainless grids (2 mm in diameter, 29 cm × 16 cm) connected to a spring and a printer as described previously [13]. The stainless grids can freely rotate by themselves. The interval of the grids is 2 cm. Each mouse was placed on the stainless grid of the apparatus and the tail was slowly pulled in parallel at a constant speed backwards by an experimenter. The resistance of the forepaw was measured as muscle tone by the detector.
2.4. Rota-rod test After measuring the traction-meter test, the rota-rod test for motor coordination was performed. Mice (n = 12 WT mice, n = 16 V1aR KO mice, aged 12 weeks) were placed on a rotating rod (3 cm diameter; Neuroscience Inc., Tokyo, Japan) with a non-skid surface and the latency to falling was measured for up to 2 min. The test was performed three times a day for three consecutive days. The rotating speed was accelerated in the order of 5, 10 and 15 rpm each day.
2.5. Elevated plus-maze test The procedure is based on that described by Pellow et al. [24]. It was elevated to a height of 50 cm with two open (25 cm × 8 cm) and two enclosed arms (25 cm × 8 cm) arranged so that the arms of the same type were opposite each other. Mice (n = 9 WT mice, n = 11 V1aR KO mice, aged 10 weeks) were placed in the middle of an elevated plus-maze. The time spent in the open arms was used for analysis. The test duration was 5 min.
2.6. Forced swimming test The immobility time in the forced swimming test was measured as described previously [6], which was a modification of that described by Porsolt et al. [25]. Animals (n = 10 WT mice, n = 8 V1aR KO mice, aged 10 weeks) were placed in individual plastic cylinders (diameter 11 cm; height 18 cm) filled with 10 cm of water (23 ± 1 ◦ C) for 15 min, and then removed and dried. A magnet was attached to one of their forelimbs. The immobility time was recorded using the MicroAct Scratching Test (Version 1.03; Neuroscience Inc.) during a 15 min observation period for 2 days in a row.
2.7. Marble-burying behavior test The marble-burying behavior test was measured as described previously [19]. The mice (n = 9 of each genotype, aged 13 weeks) were placed individually in clear plastic boxes (30 cm × 30 cm × 28 cm), containing 25 glass marbles (1.5 cm in diameter) evenly spaced on sawdust 5 cm deep, without food and water. At the same time, the locomotor activity of mice was measured using an automated activity counter (NS-AS01, Neuroscience Inc., Tokyo, Japan) placed 15 cm above the same plastic boxes. The activity was measured with the illumination of a 100 W bulb. The results of marble-burying behavior were expressed as the number of marbles buried at least two-thirds deep in this paradigm within 30 min.
2.8. Social interaction test The social interaction test was performed in a neutral cage (clear plastic box: 30 cm × 25 cm × 17.7 cm) in the animal room. On days 1 and 2, each mouse received one session every day for 10 min in the neutral cage to habituate themselves to the apparatus. On day 3 (test day), pairs of unfamiliar mice (n = 8 pairs of each genotype, aged 7 weeks) with a similar genetic background were placed into the neutral cage for 10 min. Behavior was video-recorded by a camera placed 70 cm above the neutral cage. The time spent in social interaction (active contact such as sniffing, following and grooming the partner) was measured for 10 min.
2.9. Statistical analyses The results are expressed as the means ± S.E.M. The data were analyzed by unpaired t-test. A p-value of less than 0.05 was considered to be statistically significant.
3. Results 3.1. Open-field, traction-meter, rota-rod and forced swimming tests No significant differences were observed between WT mice and V1aR KO mice at 10–12 weeks of age in detailed analyses of
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Table 1 Performance of WT and V1aR KO mice in open-field, rota-rod, traction meter and forced swimming tests Measurement Open-field test Number of ambulation Number of rearing Traction meter test (kgf) Rota-rod test, latency to fall (s) 5 rpm 10 rpm 15 rpm
WT (n = 12)
V1aR KO (n = 16)
124.4 ± 7.4 18.9 ± 1.9 0.167 ± 0.007
139.4 ± 7.4 18.6 ± 1.8 0.177 ± 0.004
119.1 ± 0.9 119.9 ± 0.1 107.5 ± 3.4
120.0 ± 0.0 116.1 ± 2.9 114.8 ± 4.6
Measurement
WT (n = 10)
V1aR KO (n = 8)
Forced swimming test Immobility time (s), day 1 Immobility time (s), day 2
803.3 ± 23.7 854.0 ± 12.6
774.8 ± 35.4 801.9 ± 35.1
Values represent means ± S.E.M.
their motor function with open-field testing, the traction-meter test for muscle tone and the rota-rod test for motor coordination (Table 1). Moreover, there was no significant difference between WT mice and V1aR KO mice for the immobility time in the forced swimming test for measuring depression. In addition, there was no difference between WT mice and V1aR KO mice for their body weight (WT mice: 23.2 ± 0.5 g; V1aR KO mice: 23.8 ± 0.4 g). 3.2. Elevated plus-maze test The V1aR KO mice spent more time in the open arms of the elevated plus-maze than the WT mice did (p < 0.01 by the unpaired t-test, Fig. 1). 3.3. Marble-burying behavior test The V1aR KO mice buried significantly fewer glass marbles than the WT mice did (p < 0.01 by the unpaired t-test, Fig. 2A). There were no genotypic differences in locomotor activity during the marble-burying behavior (Fig. 2B).
Fig. 1. Elevated plus-maze test performance of WT and V1aR KO mice. The data are expressed as the means ± S.E.M. ** p < 0.01 vs. WT mice. The number of mice is shown at the bottom of each column.
Fig. 2. Number of buried marbles (A) and locomotor activity (B) in the marble burying behavior test in WT and V1aR KO mice. The data are expressed as the means ± S.E.M. ** p < 0.01 vs. WT mice. The number of mice is shown at the bottom of each column.
3.4. Social interaction test The V1aR KO mice exhibited a significant decrease in the social interaction time compared to that of the WT mice (p < 0.001 by the unpaired t-test, Fig. 3). In addition, no incidence of aggressive behavior such as attack biting and wrestling was observed at this time.
Fig. 3. Social interaction time in the social interaction test in WT and V1aR KO mice. The data are expressed as the means ± S.E.M. *** p < 0.001 vs. WT mice. The number of pairs of each genotype is shown at the bottom of each column.
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4. Discussion The major finding in the present study is that V1aR KO mice exhibited an impairment of social interaction compared to the WT mice in the social interaction test. The results of the study also showed that the number of ambulation and rearing in the V1aR KO mice was normal in the open-field test. Moreover, we did not observe differences between V1aR KO mice and WT mice in the traction-meter and rota-rod tests. Thus, motor function in V1aR KO mice was normal. Moreover, there was no significant difference between WT mice and V1aR KO mice for the immobility time in the forced swimming test for measuring depression. On the other hand, we observed that V1aR KO mice showed reduced anxiety-related behavior in the elevated plus-maze test. Therefore, it is unlikely that social interaction is impaired in V1aR KO mice because of a deficit in motor function, depression and high anxiety. These findings suggest that V1aR controls social interaction in particular. This idea is supported by the findings that subchronic treatment with PCP, an animal model of schizophrenia, results in impaired social interaction and reduced density of V1aR in several brain regions [28]. Moreover, the V1aR gene has been linked to autism, providing evidence that the V1aR gene may play a role in social communication and behavior in humans [15,33,34]. The second finding of this study is that the V1aR KO mice showed reduced anxiety-related behavior in the elevated plusmaze test whereas these mice performed normally on the forced swimming test. The present findings are consistent with previous findings [4]. The increased AVP expression in the hypothalamic paraventricular nucleus is considered to underlie anxiety-related behavior [17]. Also, the microinjection of V1 receptor antagonist into the septal nucleus of rats has been reported to reduce the anxiety in the elevated plus-maze test [18]. Furthermore, overexpression of the V1aR in the lateral septum of WT mice resulted an increase in anxiety-related behavior [3]. Therefore, V1aR may be involved in the anxiety-related behavior. The third finding of the present study is that the V1aR KO mice buried significantly fewer glass marbles than the WT mice did in the marble-burying behavior test. This phenomenon does not seem to contribute to a change in locomotor activity, since there were no genotypic differences in locomotor activity during the marble-burying behavior. It has been suggested that the burying behavior would be rewarding or compulsive [5]. In general, the marble-burying behavior test has been considered as an animal model of obsessive-compulsive disorder (OCD) [11,19,21]. In fact, serotonin (5-hydroxytryptamine, 5-HT)reuptake inhibitor (SRI) and selective 5-HT-reuptake inhibitor (SSRI), which have been used to treat human OCD symptoms, inhibit marble-burying behavior without affecting locomotor activity [11,21,22]. Therefore, the reduced number of marbles buried by V1aR KO mice might reflect an anti-OCD effect of the mutation. Alternatively, the interpretation that mice have less anxiety when they are burying foreign objects is based on the assumption that the animals would consider marbles startling by virtue of their novelty. Therefore, the reduced number of marbles buried by V1aR KO mice might reflect an anxiolytic effect of the mutation. This idea is supported by the present finding that
the V1aR KO mice showed reduced anxiety-related behavior in the elevated plus-maze test. Anxiolytic drugs have been proposed to function in the social interaction test, a rat model for detecting compounds with anxiolytic properties, by increasing the social interaction time [8]. In the present study, we found that V1aR KO mice exhibited the impairment of social interaction in spite of reduced anxietyrelated behavior. In addition, we found that V1aR KO female mice also exhibited the impairment of social behavior in the social interaction test (N. Egashira et al., unpublished data). Thus, there are no gender differences in the social interaction test. Our findings support the possibility that V1aR regulates social interaction. Finally, the results of the present experiments demonstrate that mice lacking the V1aR exhibit impaired social interaction. Autistic patients express social interaction deficits [2], and autism has been associated with the V1aR gene [15,33,34]. On the other hand, impairments of social function are often observed in symptoms of schizophrenia. Hence, V1aR may be involved in the regulation of social interaction, and V1aR KO mice could be used as an animal model for investigating social behavior deficits that co-occur with such illness as autism and schizophrenia. Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 18591318). References [1] Al-Khatib MH, D¨okmeci I, Fujiwara M. Differential role of nucleus accumbens and caudate-putamen in mediating the effect of nomifensine and methamphetamine on ambulation and rearing of rats in the open-field test. Jpn J Pharmacol 1995;67:69–77. [2] American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed. Washington, DC: American Psychiatric Association; 1994. pp. 66–71. [3] Bielsky IF, Hu S-B, Ren X, Terwilliger EF, Young LJ. The V1a vasopressin receptor is necessary and sufficient for normal social recognition: a gene replacement study. Neuron 2005;47:503–13. [4] Bielsky IF, Hu S-B, Szegda KL, Westphal H, Young LJ. Profound impairment in social recognition and reduction in anxiety-like behavior in vasopressin V1a receptor knockout mice. Neuropsychopharmacology 2004;29:483–93. [5] Broekkamp CL, Rijk HW, Joly-Gelouin D, Lloyd KL. Major tranquillizers can be distinguished from minor tranquillizers on the basis of effects on marble burying and swim-induced grooming in mice. Eur J Pharmacol 1986;126:223–9. [6] Egashira N, Iwasaki K, Takashima A, Watanabe T, Kawabe H, Matsuda T, et al. Altered depression-related behavior and neurochemical changes in serotonergic neurons in mutant R406W human tau transgenic mice. Brain Res 2005;1059:7–12. [7] Egashira N, Tanoue A, Higashihara F, Mishima K, Fukue Y, Takano Y, et al. V1a receptor knockout mice exhibit impairment of spatial memory in an eight-arm radial maze. Neurosci Lett 2004;356:195–8. [8] File SE. Animal models for predicting clinical efficacy of anxiolytic drugs: social behaviour. Neuropsychobiology 1985;13:55–62. [9] Gouzenes L, Sabatier N, Richard P, Moos FC, Dayanithi G. V1a- and V2type vasopressin receptors mediate vasopressin-induced Ca2+ responses in isolated rat supraoptic neurons. J Physiol 1999;517:771–9.
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