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Locomotor activity, ultrasonic vocalization and oxytocin levels in infant CD38 knockout mice
Neuroscience Letters 448 (2008) 67–70
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Locomotor activity, ultrasonic vocalization and oxytocin levels in infant CD38 knockout mice Hong-Xiang Liu a,b,1 , Olga Lopatina a,b,1 , Chiharu Higashida a , Takahiro Tsuji a , Ichiro Kato c , Shin Takasawa d , Hiroshi Okamoto e , Shigeru Yokoyama a,b , Haruhiro Higashida a,b,∗ a
Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan The Kanazawa University 21st Century COE Program on Innovative Brain Science for Development, Learning and Memory, Kanazawa 920-8640, Japan Department of Biochemistry, Toyama University School of Medicine, Toyama 930-0194, Japan d Department of Biochemistry, Nara Medical University, Nara 634-8521, Japan e Department of Advanced Biological Science for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan b c
a r t i c l e
i n f o
Article history: Received 11 July 2008 Received in revised form 22 September 2008 Accepted 24 September 2008 Keywords: Oxytocin Social behavior Ultrasonic vocalization CD38 ADP-ribosyl cyclase Development
a b s t r a c t Oxytocin (OT), a neurohormone involved in reproduction, plays a critical role in social behavior in a wide range of mammalian species from rodents to humans. The role of CD38 in regulating OT secretion for social behavior has been demonstrated in adult mice, but has not been examined in pups or during development. Separation from the dam induces stress in 7-day-old mouse pups. During such isolation, locomotor activity was higher in CD38 knockout (CD38−/− ) pups than in wild-type (CD38+/+ ) or heterozygous (CD38+/− ) controls. The number of ultrasonic vocalizations was lower in CD38−/− pups than in CD38+/+ pups. However, the difference between the two genotypes was less severe than that in OT knockout or OT receptor knockout mice. To explain this, we measured plasma OT levels. The level was not lower in CD38−/− pups during the period 1–3 weeks after birth, but was significantly reduced after weaning (>3 weeks). ADP-ribosyl cyclase activities in the hypothalamus and pituitary were markedly lower from 1 week after birth in CD38−/− mice and were consistently lower thereafter to the adult stage (2 months old). These results showed that the reduced severity of behavioral abnormalities in CD38−/− pups was due to partial compensation by the high level of plasma OT. © 2008 Elsevier Ireland Ltd. All rights reserved.
Oxytocin (OT), a nonapeptide involved in reproduction, is synthesized in the paraventricular nucleus and supraoptic nucleus of the hypothalamus and travels down neuronal axons to the posterior pituitary [4]. It is secreted into the general circulation from the nerve endings of the neurohypophysis and into the brain from dendrites [4]. It is well known that OT is linked to complex social behavior [9,12,13,21,28]. In humans, intranasal OT may promote trust [16] and infusion with OT can increase generosity [31]. In rodents, OT is highly involved in social interaction, social recognition, pair bonding, and maternal behavior [8,15,17,18,19,22,25,30]. In addition, animal studies have shown that increased levels of OT in the early postnatal period may affect behavior and last into adulthood [3,17], and that subcutaneous administration of low doses of OT facilitates social recognition [23]. Two types of OT and OT recep-
∗ Corresponding author. Department of Biophysical Genetics, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. Tel.: +81 76 265 2455; fax: +81 76 265 2455. E-mail address: [email protected] (H. Higashida). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.09.084
tor gene knockout mice show profound social amnesia that can be fully rescued by injection of OT into the medial amygdala [7,27,29]. In these knockout mice, impairment of social behavior is clearly observed even in pups. These observations suggest that OT plays an important role in social behavior during brain development throughout the juvenile to adult stages. We demonstrated previously that adult mice with a null mutation in CD38, a “niceness” protein with ADP-ribosyl cyclase activity, showed deficiency in social behavior due to abnormalities in central and peripheral OT secretion [14]. We also showed that decreased formation of cyclic ADP-ribose (cADPR) results in dysfunction of Ca2+ -induced Ca2+ -release for OT secretion in hypothalamic OT neurons [14]. However, there have been no reports regarding behavior in CD38 knockout (CD38−/− ) pups. In the central nervous system, ADP-ribosyl cyclase activity corresponding to CD38 was detected as early as embryonic day 15 in mouse development [5], and the endogenous brain cADPR content is higher in the developing brain and declines in adults [6]. Therefore, it is interesting to examine the behavior of infant CD38 knockout mice from two points of view regarding brain development and similarity or dissimilarity of infant behavior in OT and OT
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receptor gene knockout mice [27,29]. We examined separation stress-induced motor activity and ultrasonic vocalization (USV) in pups of three genotypes. We also measured plasma OT levels and demonstrated that postnatal day 21 (weaning time for mice) is a time window of differentiating plasma OT in CD38 control and knockout mice. We determined ADP-ribosyl cyclase activity in the developing brain (in the hypothalamus and posterior pituitary) and discuss its relationship with the plasma OT level. CD38+/+ , CD38+/− and CD38−/− mice of both sexes were kept in our laboratory under standard conditions (24 ◦ C; 12-h light/dark cycle, lights on at 8:00 a.m.) and received food and water ad libitum. Obligate litters were obtained by mating homozygous CD38+/+ or CD38−/− parents. Non-obligate litters (n = 16) were obtained by mating CD38 heterozygous male and female mice (n = 2 breeding pairs) and the distribution of three genotypes showed accordance with the Mendelian ratio (4 of CD38-KO; 8 of CD38-HET; 4 of CD38WT). Breeding pairs were maintained separately (1 pair per cage). At 21 days old, offspring were removed and housed in same-sex sibling pairs. Pups aged 1 week to 2 months were used in this study. Eight pups in each cage were weighed. The experiments were carried out in agreement with the guidelines for the care and use of laboratory animals of Kanazawa University. Seven-day-old male pups from CD38−/− , CD38+/+ and CD38+/− breeding pairs were tested 4 h before the dark phase essentially as described previously [27,29] with some modifications. Briefly, the parents were removed from the home cage 20 min before testing. Each pup was placed into the recording chamber (22 cm × 22 cm) for 3 min, and the number of 4.5 cm × 4.5 cm grids crossed during the test was noted. For experiments under mutual interaction, the parents were removed from the home cage 20 min before testing and 4 pups were placed into the recording chamber (22 cm × 22 cm) on four different sides for 20 min. Group behavior was measured using a variant of the “group size” metric [1,2] every 2 min. The overall circumference of aggregation (all pups regardless of whether in contact or not) was measured. Seven-day-old pups of born from CD38+/+ and CD38−/− homozygous pairs or CD38+/− born from mixed pairs were used. Genotyping was performed using three sets of primers: F, 5 -agagagggagagaggcacacaa-3 , R, 5 -acccaaaccactgagcattc-3 ; F, 5 -atcctgtgtcatgatccattctgtg-3 , R, 5 -gggctgactctattgaccaatgaag-3 ; and F, 5 atgaatttagccaggtgtctgggg-3 , R, 5 -gggctgactctattgaccaatgaag-3 , respectively. Before testing, the parents and pups were removed from the animal room to the test room, where the ambient temperature was approximately 24 ◦ C, for 15 min. USV recordings were carried out in an anechoic box (70 cm × 60 cm × 60 cm), which had an ultrasonic microphone mounted in the center of the ceiling that was connected to an amplifier. For pup isolation call recordings, we used the method described by Shu et al. [26]. Each pup was placed in a 500-ml glass beaker in the anechoic box. The microphone was located 5 cm above the pup for the 2-min recording period. Ultrasonic vocalizations were recorded as WAV files and analyzed; the number of calls, frequency, and wave width were measured using a USV monitor (Muromachi Kikai Co., Tokyo, Japan). Click-like sounds ≤4 ms in duration were filtered out. Mice at different ages were anesthetized by intraperitoneal injection of pentobarbital (20 mg/1 kg body weight). The blood samples were collected by cardiac puncture and centrifuged at 1600×g for 15 min at 4 ◦ C. Plasma samples were collected and stored at –80 ◦ C until use. The hypothalamus and posterior pituitary were removed and homogenized in 10 mM Tris–HCl buffer, pH 7.4. The fresh homogenates were used to determine ADP-ribosyl cyclase activity. To determine the concentrations of OT in plasma, immunoassays were performed according to a slight modification of the manu-
Fig. 1. Locomotor activity in mouse pups. (A) CD38−/− pups from obligate and nonobligate litters showed higher locomotor activity than CD38+/− and CD38+/+ pups in the grid crossing test. Data are means ± S.E.M. (n = 14 for obligate and 4 for nonobligate). (B) Group behavior in mouse pups from obligate litters. Smaller areas measured in CD38−/− pups indicate higher locomotor activity, because the pups moved to the center from the corner. Data are means ± S.E.M. (n = 7). *P < 0.05, **P < 0.01 and # P > 0.05 (not significant). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
facturer’s protocol (Assay Designs Inc., Ann Arbor, MI) as reported previously [14]. ADP-ribosyl cyclase activities in the hypothalamus and pituitary were determined using the nicotinamide guanine dinucleotide (NGD) technique as described previously [11]. The specific ADPribosyl cyclase activity was calculated using cyclic guanosine diphosphate ribose (cGDPR) standards, and the results are presented as nM cGDPR per mg protein per minute. All data are shown as means ± S.E.M. To analyze the differences between CD38+/+ and CD38−/− in the obligate group, statistical analysis was conducted with Student’s t-test. The significance of the difference between the three genotypes of the non-obligate group was determined by oneway ANOVA followed by post hoc Tukey-test. P < 0.05 was taken to indicate statistical significance. Both CD38+/+ and CD38−/− mice grew well and gained weight. From the 3rd week of life, CD38−/− mice showed a slightly but not significantly lower body weight as compared with CD38+/+ mice, as reported previously [14]. There was no significant sex-related difference in body weight, suggesting that these mice fed well from the infant stage through the dam’s milk and by solid food after weaning to the adult stage. We examined isolation-induced locomotor behavior and USV of infant males at postnatal day 7 in three genotypes. CD38−/− males from both obligate and non-obligate litters showed significantly higher levels of locomotor activity during the first 3 min after separation from the dam than those of other types (Fig. 1A), when examined individually in the grid crossing test in an observation chamber. Next, the activity of four pups together was measured simultaneously for a much longer period, in which there was no physical contact but some type of interaction, probably with USV
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Fig. 2. Plasma concentration of oxytocin. (A) Time course of concentrations during development in CD38+/+ (circles) and CD38−/− (triangles) mice in obligate litters. The data shown are means ± S.E.M. (n = 4–10). *P < 0.01. (B) Oxytocin concentration in 1week-old pups of three genotypes (n = 4–8). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
as discussed below. The higher locomotor activity in CD38−/− pups (area closed by moving into the center) was persistently observed up to 18 min in the test, although the difference was not marked (Fig. 1B). Both CD38−/− and CD38+/+ pups emitted calls on isolation. The properties of USV were similar. USV mean peak frequency was 61.5 ± 0.57 kHz or 62.7 ± 0.58 kHz and USV duration was 65.7 ± 1.7 ms or 63.4 ± 2.1 ms, respectively, in both CD38+/+ and CD38−/− types (n = 23). However, the calls per 2-min session were significantly less frequent in CD38−/− infants (44 ± 8 calls, n = 10) than in wild-type controls (72 ± 13, n = 13). These results agreed well with previous observations in OT or OT receptor knockout mice [8,27,29]. However, the degree of disruption of infant behavior seemed to be much less marked in the case of CD38−/− in comparison with the two OT-related knockout mouse strains. This observation suggested that CD38−/− pups partially retain the ability to interact socially with others as seen in wild-type controls. This speculation prompted us to measure plasma OT levels to examine whether the OT level is decreased in these pups. We measured the plasma OT levels at five different phases of development (from 1 week to 2 months) in three genotypes of obligate or non-obligate litters (Fig. 2). Surprisingly, the plasma OT concentration in CD38−/− pups at 1–3 weeks of age was similar to those in CD38+/+ and CD38+/− mice of the same age. However, as expected, at 1 and 2 months of age following weaning, we found a significantly lower plasma concentration of OT in CD38−/− than in CD38+/+ mice (291 ± 46 vs. 522 ± 28 pg/ml and 215 ± 20 vs. 440 ± 60 pg/ml, respectively; P < 0.002; Fig. 2). Next, we measured ADP-ribosyl cyclase activity in the brain (hypothalamus and posterior pituitary) in CD38 wild-type and knockout mice during the first two months of life. In the brain regions examined, ADP-ribosyl cyclase activity was significantly higher even in the 1-week-old CD38+/+ mice than in the CD38+/− or CD38−/− mice of the same-age (Fig. 3). From the second week of life to all late development stages, CD38+/+ mice showed a much higher level of ADP-ribosyl cyclase activity in the hypothalamus and pituitary in comparison with CD38−/− mice. In the hypothalamus,
Fig. 3. ADP-ribosyl cyclase activity, measured as the rate of cyclic GDP-ribose formation by whole-cell homogenates isolated from the hypothalamus (A, B) and the posterior pituitary gland (C, D). Data were obtained from pups of different ages from obligate litters (A, C) or 1-week-old pups from non-obligate litters (B, D). The data shown are means ± S.E.M. (n = 4–6). *P < 0.05, **P < 0.01, ***P < 0.001 and # P > 0.05 (not significant). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
ADP-ribosyl cyclase activity increased with age in CD38+/+ mice, but only a slight decrease was observed in CD38−/− mice. ADPribosyl cyclase activity in the pituitary was lower than that in the hypothalamus and showed no increase throughout development. The results of this study indicated that locomotor activity induced by separation from the dam was higher and the number of USV calls was lower in CD38 knockout pups. However, these behaviors were much milder than those observed in OT and OT receptor knockout mice [27,29], suggesting less impairment of social behavior in the CD38−/− pups as compared with the above two knockout lines. Consistent with this finding, the plasma OT levels during development from neonates to 3-week-old juvenile mice remained at the same level between the two genotypes, although ADP-ribosyl cyclase activity was obviously lower in the knockout mice from birth.
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The above decrease after weaning during development observed only in CD38−/− mice seems to indicate an important critical period to distinguish different plasma OT switching from the juvenile to the adult stage. The reason for this is not yet clear. However, as milk is the only food source in lactating pups, we speculated that OT is inevitably taken in from the milk. To confirm this, we performed preliminary quantitative analysis to determine whether OT is present in the mammary gland and milk of lactating dams, according to the methods described previously [10,24]. We found that OT was abundant in the mammary gland tissue and milk in lactating dams of both genotypes. However, it is less likely that this exogenous source can help CD38 knockout pups maintain a plasma OT level similar to that of wild-type pups prior to weaning, because milk OT may not be transported into the bloodstream [20]. In conclusion, as behavioral impairment was observed in CD38 knockout (CD38−/− ) pups, although the plasma OT level was the same between the two types of pup, secretion of OT into the brain may play an important role in the development of social behavior. References [1] J.R. Alberts, Huddling by rat pups: multisensory control of contact behavior, J. Comp. Physiol. Psychol. 92 (1978) 220–230. [2] J.R. Alberts, Huddling by rat pups: ontogeny of individual and group behavior, Dev. Psychobiol. 49 (2007) 22–32. [3] K.L. Bales, C.S. Carter, Neonatal treatment with oxytocin affects selective sociality in male prairie voles, Horm. Behav. 39 (2001) 324. [4] P.J. Brunton, J.A. Russell, The expectant brain: adapting for motherhood, Nat. Rev. Neurosci. 9 (2008) 11–25. [5] C. Ceni, N. Pochon, V. Brun, H. Muller-Steffner, A. Andrieux, D. Grunwald, F. Schuber, M. DeWaard, F. Lund, M. Villaz, M.J. Moutin, CD38-dependent ADPribosyl cyclase activity in developing and adult mouse brain, Biochem. J. 370 (2003) 175–183. [6] C. Ceni, N. Pochon, M. Villaz, H. Muller-Steffner, F. Schuber, J. Baratier, M. De Waard, M. Ronjat, M.J. Moutin, The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain, Biochem. J. 395 (2006) 417–426. [7] J.N. Ferguson, J.M. Aldag, T.R. Insel, L.J. Young, Oxytocin in the medial amygdala is essential for social recognition in the mouse, J. Neurosci. 21 (2001) 8278–8285. [8] J.N. Ferguson, L.J. Young, E.F. Heam, M.M. Matzuk, T.R. Insel, J.T. Winslow, Social amnesia in mice lacking the oxytocin gene, Nat. Genet. 25 (2000) 284–288. [9] A.B. Fries, T.E. Ziegler, J.R. Kurian, S. Jacoris, S.D. Pollak, Early experience in humans is associated with changes in neuropeptides critical for regulating social behavior, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 17237–17240. [10] H. Grabowski, D. Le Bars, N. Chene, J. Attal, R. Malienou-Ngassa, C. Puissant, L.M. Houdebine, Rabbit whey acidic protein concentration in milk, serum, mammary gland extract, and culture medium, J. Dairy Sci. 74 (1991) 4143–4150. [11] H. Higashida, A. Egorova, C. Higashida, Z.G. Zhong, S. Yokoyama, M. Noda, J.S. Zhang, Sympathetic potentiation of cyclic ADP-ribose formation in rat cardiac myocytes, J. Biol. Chem. 274 (1999) 33348–33354.
[12] E. Hollander, S. Novotny, M. Hanratty, R.Y.C. deCaria, B. Aronowitz, Oxytocin infusion reduces repetitive behaviors in adults with autistic and Asperger’s disorders, Neuropsychopharmacology 28 (2003) 193–198. [13] T.R. Insel, R.D. Fernald, How he brain processes social information: searching for the social brain, Annu. Rev. Neurosci. 27 (2004) 697–722. [14] D. Jin, H.X. Liu, H. Hirai, T. Torashima, T. Nagai, O. Lopatina, N.A. Shnayder, K. Yamada, M. Noda, T. Seike, K. Fujita, S. Takasawa, S. Yokoyama, K. Koizumi, Y. Shiraishi, S. Tanaka, M. Hashii, T. Yoshihara, K. Higashida, M.S. Islam, N. Yamada, K. Hayashi, N. Noguchi, I. Kato, H. Okamoto, A. Matsushima, A. Salmina, T. Munesue, N. Shimizu, S. Mochida, M. Asano, H. Higashida, CD38 is critical for social behaviour by regulating oxytocin secretion, Nature 446 (2007) 41–45. [15] E.B. Keverne, J.P. Curley, Vasopressin, oxytocin and social behavior, Curr. Opin. Neurobiol. 14 (2004) 777–783. [16] M. Kosfeld, M. Heinrichs, P.J. Zak, U. Fischbacher, E. Fehr, Oxytocin increases trust in humans, Nature 435 (2005) 673–676. [17] K.M. Kramer, B.S. Cushing, C.S. Carter, Developmental effects of oxytocin on stress response: single versus repeated exposure, Physiol. Behav. 79 (2003) 775–782. [18] M.M. Lim, L.J. Young, Neuropeptidergic regulation of affiliative behavior and social bonding in animals, Horm. Behav. 50 (2006) 506–517. [19] S.H. Lin, T. Kiyohara, B. Sun, Maternal behavior: activation of the central oxytocin receptor system in parturient rats? Neuroreport 14 (2003) 1439–1444. [20] A. Malek, E. Blann, D. Mattison, Human placental transport of oxytocin, J. Matern. Fetal Med. 5 (1996) 245–255. [21] C. Modahl, L. Green, D. Fein, M. Morris, L. Waterhouse, C. Feinstein, H. Levin, Plasma oxytocin levels in autistic children, Biol. Psychiatry 143 (1998) 270–277. [22] C.A. Pedersen, S.V. Vadlamudi, M.L. Boccia, J.A. Amico, Maternal behavior deficits in nulliparous oxytocin knockout mice, Genes Brain Behav. 3 (2006) 274–281. [23] P. Popik, J. Vetulani, J.M. van Ree, Low doses of oxytocin facilitate social recognition in rats, Psychopharmacology (Berl) 106 (1992) 71–74. [24] B.L. Reis, C.L. Keen, B. Lonnerdal, L.S. Hurley, Longitudinal changes in the mineral composition of mouse milk and the relationship to zinc metabolism of the suckling neonate, J. Nutr. 121 (1991) 687–699. [25] L.M. Simone, R.B. Valerie, E. Gkoumassi, W.F. van Leeuwen, A.J. Douglas, Dynamic changes in oxytocin receptor expression and activation at parturition in the rat brain, Endocrinology 148 (2007) 5095–5104. [26] W. Shu, J.Y. Cho, Y. Jiang, M. Zhang, D. Weisz, G.A. Elder, J. Schmeidler, R. De Gasperi, M.A. Sosa, D. Rabidou, A.C. Santucci, D. Perl, E. Morrisev, J.D. Buxbaum, Altered ultrasonic vocalization in mice with a disruption in the Foxp2 gene, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 9643–9648. [27] Y. Takayanagi, M. Yoshida, I.F. Bielsky, H.E. Ross, M. Kawamata, T. Onaka, T. Yanagisawa, T. Kimura, M.M. Matzuk, L.J. Young, K. Nishimori, Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 16096–160101. [28] Y. Tanoue, S. Oda, Weaning time of children with infantile autism, J. Autism Dev. Disord. 19 (1989) 425–434. [29] J.T. Winslow, E.F. Hearn, J. Ferguson, L.J. Young, M.M. Matzuk, T.R. Insel, Infant vocalization, adult aggression, and fear behavior of an oxytocin null mutant mouse, Horm. Behav. 37 (2000) 145–155. [30] L.J. Young, Z. Wang, The neurobiology of pair bonding, Nat. Neurosci. 7 (2004) 1048–1054. [31] P.J. Zak, A.A. Stanton, S. Ahmadi, Oxytocin increases generosity in humans, PLoS ONE 2 (2007) e1128.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Locomotor activity, ultrasonic vocalization and oxytocin levels in infant CD38 knockout mice Hong-Xiang Liu a,b,1 , Olga Lopatina a,b,1 , Chiharu Higashida a , Takahiro Tsuji a , Ichiro Kato c , Shin Takasawa d , Hiroshi Okamoto e , Shigeru Yokoyama a,b , Haruhiro Higashida a,b,∗ a
Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan The Kanazawa University 21st Century COE Program on Innovative Brain Science for Development, Learning and Memory, Kanazawa 920-8640, Japan Department of Biochemistry, Toyama University School of Medicine, Toyama 930-0194, Japan d Department of Biochemistry, Nara Medical University, Nara 634-8521, Japan e Department of Advanced Biological Science for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan b c
a r t i c l e
i n f o
Article history: Received 11 July 2008 Received in revised form 22 September 2008 Accepted 24 September 2008 Keywords: Oxytocin Social behavior Ultrasonic vocalization CD38 ADP-ribosyl cyclase Development
a b s t r a c t Oxytocin (OT), a neurohormone involved in reproduction, plays a critical role in social behavior in a wide range of mammalian species from rodents to humans. The role of CD38 in regulating OT secretion for social behavior has been demonstrated in adult mice, but has not been examined in pups or during development. Separation from the dam induces stress in 7-day-old mouse pups. During such isolation, locomotor activity was higher in CD38 knockout (CD38−/− ) pups than in wild-type (CD38+/+ ) or heterozygous (CD38+/− ) controls. The number of ultrasonic vocalizations was lower in CD38−/− pups than in CD38+/+ pups. However, the difference between the two genotypes was less severe than that in OT knockout or OT receptor knockout mice. To explain this, we measured plasma OT levels. The level was not lower in CD38−/− pups during the period 1–3 weeks after birth, but was significantly reduced after weaning (>3 weeks). ADP-ribosyl cyclase activities in the hypothalamus and pituitary were markedly lower from 1 week after birth in CD38−/− mice and were consistently lower thereafter to the adult stage (2 months old). These results showed that the reduced severity of behavioral abnormalities in CD38−/− pups was due to partial compensation by the high level of plasma OT. © 2008 Elsevier Ireland Ltd. All rights reserved.
Oxytocin (OT), a nonapeptide involved in reproduction, is synthesized in the paraventricular nucleus and supraoptic nucleus of the hypothalamus and travels down neuronal axons to the posterior pituitary [4]. It is secreted into the general circulation from the nerve endings of the neurohypophysis and into the brain from dendrites [4]. It is well known that OT is linked to complex social behavior [9,12,13,21,28]. In humans, intranasal OT may promote trust [16] and infusion with OT can increase generosity [31]. In rodents, OT is highly involved in social interaction, social recognition, pair bonding, and maternal behavior [8,15,17,18,19,22,25,30]. In addition, animal studies have shown that increased levels of OT in the early postnatal period may affect behavior and last into adulthood [3,17], and that subcutaneous administration of low doses of OT facilitates social recognition [23]. Two types of OT and OT recep-
∗ Corresponding author. Department of Biophysical Genetics, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. Tel.: +81 76 265 2455; fax: +81 76 265 2455. E-mail address: [email protected] (H. Higashida). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.09.084
tor gene knockout mice show profound social amnesia that can be fully rescued by injection of OT into the medial amygdala [7,27,29]. In these knockout mice, impairment of social behavior is clearly observed even in pups. These observations suggest that OT plays an important role in social behavior during brain development throughout the juvenile to adult stages. We demonstrated previously that adult mice with a null mutation in CD38, a “niceness” protein with ADP-ribosyl cyclase activity, showed deficiency in social behavior due to abnormalities in central and peripheral OT secretion [14]. We also showed that decreased formation of cyclic ADP-ribose (cADPR) results in dysfunction of Ca2+ -induced Ca2+ -release for OT secretion in hypothalamic OT neurons [14]. However, there have been no reports regarding behavior in CD38 knockout (CD38−/− ) pups. In the central nervous system, ADP-ribosyl cyclase activity corresponding to CD38 was detected as early as embryonic day 15 in mouse development [5], and the endogenous brain cADPR content is higher in the developing brain and declines in adults [6]. Therefore, it is interesting to examine the behavior of infant CD38 knockout mice from two points of view regarding brain development and similarity or dissimilarity of infant behavior in OT and OT
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receptor gene knockout mice [27,29]. We examined separation stress-induced motor activity and ultrasonic vocalization (USV) in pups of three genotypes. We also measured plasma OT levels and demonstrated that postnatal day 21 (weaning time for mice) is a time window of differentiating plasma OT in CD38 control and knockout mice. We determined ADP-ribosyl cyclase activity in the developing brain (in the hypothalamus and posterior pituitary) and discuss its relationship with the plasma OT level. CD38+/+ , CD38+/− and CD38−/− mice of both sexes were kept in our laboratory under standard conditions (24 ◦ C; 12-h light/dark cycle, lights on at 8:00 a.m.) and received food and water ad libitum. Obligate litters were obtained by mating homozygous CD38+/+ or CD38−/− parents. Non-obligate litters (n = 16) were obtained by mating CD38 heterozygous male and female mice (n = 2 breeding pairs) and the distribution of three genotypes showed accordance with the Mendelian ratio (4 of CD38-KO; 8 of CD38-HET; 4 of CD38WT). Breeding pairs were maintained separately (1 pair per cage). At 21 days old, offspring were removed and housed in same-sex sibling pairs. Pups aged 1 week to 2 months were used in this study. Eight pups in each cage were weighed. The experiments were carried out in agreement with the guidelines for the care and use of laboratory animals of Kanazawa University. Seven-day-old male pups from CD38−/− , CD38+/+ and CD38+/− breeding pairs were tested 4 h before the dark phase essentially as described previously [27,29] with some modifications. Briefly, the parents were removed from the home cage 20 min before testing. Each pup was placed into the recording chamber (22 cm × 22 cm) for 3 min, and the number of 4.5 cm × 4.5 cm grids crossed during the test was noted. For experiments under mutual interaction, the parents were removed from the home cage 20 min before testing and 4 pups were placed into the recording chamber (22 cm × 22 cm) on four different sides for 20 min. Group behavior was measured using a variant of the “group size” metric [1,2] every 2 min. The overall circumference of aggregation (all pups regardless of whether in contact or not) was measured. Seven-day-old pups of born from CD38+/+ and CD38−/− homozygous pairs or CD38+/− born from mixed pairs were used. Genotyping was performed using three sets of primers: F, 5 -agagagggagagaggcacacaa-3 , R, 5 -acccaaaccactgagcattc-3 ; F, 5 -atcctgtgtcatgatccattctgtg-3 , R, 5 -gggctgactctattgaccaatgaag-3 ; and F, 5 atgaatttagccaggtgtctgggg-3 , R, 5 -gggctgactctattgaccaatgaag-3 , respectively. Before testing, the parents and pups were removed from the animal room to the test room, where the ambient temperature was approximately 24 ◦ C, for 15 min. USV recordings were carried out in an anechoic box (70 cm × 60 cm × 60 cm), which had an ultrasonic microphone mounted in the center of the ceiling that was connected to an amplifier. For pup isolation call recordings, we used the method described by Shu et al. [26]. Each pup was placed in a 500-ml glass beaker in the anechoic box. The microphone was located 5 cm above the pup for the 2-min recording period. Ultrasonic vocalizations were recorded as WAV files and analyzed; the number of calls, frequency, and wave width were measured using a USV monitor (Muromachi Kikai Co., Tokyo, Japan). Click-like sounds ≤4 ms in duration were filtered out. Mice at different ages were anesthetized by intraperitoneal injection of pentobarbital (20 mg/1 kg body weight). The blood samples were collected by cardiac puncture and centrifuged at 1600×g for 15 min at 4 ◦ C. Plasma samples were collected and stored at –80 ◦ C until use. The hypothalamus and posterior pituitary were removed and homogenized in 10 mM Tris–HCl buffer, pH 7.4. The fresh homogenates were used to determine ADP-ribosyl cyclase activity. To determine the concentrations of OT in plasma, immunoassays were performed according to a slight modification of the manu-
Fig. 1. Locomotor activity in mouse pups. (A) CD38−/− pups from obligate and nonobligate litters showed higher locomotor activity than CD38+/− and CD38+/+ pups in the grid crossing test. Data are means ± S.E.M. (n = 14 for obligate and 4 for nonobligate). (B) Group behavior in mouse pups from obligate litters. Smaller areas measured in CD38−/− pups indicate higher locomotor activity, because the pups moved to the center from the corner. Data are means ± S.E.M. (n = 7). *P < 0.05, **P < 0.01 and # P > 0.05 (not significant). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
facturer’s protocol (Assay Designs Inc., Ann Arbor, MI) as reported previously [14]. ADP-ribosyl cyclase activities in the hypothalamus and pituitary were determined using the nicotinamide guanine dinucleotide (NGD) technique as described previously [11]. The specific ADPribosyl cyclase activity was calculated using cyclic guanosine diphosphate ribose (cGDPR) standards, and the results are presented as nM cGDPR per mg protein per minute. All data are shown as means ± S.E.M. To analyze the differences between CD38+/+ and CD38−/− in the obligate group, statistical analysis was conducted with Student’s t-test. The significance of the difference between the three genotypes of the non-obligate group was determined by oneway ANOVA followed by post hoc Tukey-test. P < 0.05 was taken to indicate statistical significance. Both CD38+/+ and CD38−/− mice grew well and gained weight. From the 3rd week of life, CD38−/− mice showed a slightly but not significantly lower body weight as compared with CD38+/+ mice, as reported previously [14]. There was no significant sex-related difference in body weight, suggesting that these mice fed well from the infant stage through the dam’s milk and by solid food after weaning to the adult stage. We examined isolation-induced locomotor behavior and USV of infant males at postnatal day 7 in three genotypes. CD38−/− males from both obligate and non-obligate litters showed significantly higher levels of locomotor activity during the first 3 min after separation from the dam than those of other types (Fig. 1A), when examined individually in the grid crossing test in an observation chamber. Next, the activity of four pups together was measured simultaneously for a much longer period, in which there was no physical contact but some type of interaction, probably with USV
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Fig. 2. Plasma concentration of oxytocin. (A) Time course of concentrations during development in CD38+/+ (circles) and CD38−/− (triangles) mice in obligate litters. The data shown are means ± S.E.M. (n = 4–10). *P < 0.01. (B) Oxytocin concentration in 1week-old pups of three genotypes (n = 4–8). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
as discussed below. The higher locomotor activity in CD38−/− pups (area closed by moving into the center) was persistently observed up to 18 min in the test, although the difference was not marked (Fig. 1B). Both CD38−/− and CD38+/+ pups emitted calls on isolation. The properties of USV were similar. USV mean peak frequency was 61.5 ± 0.57 kHz or 62.7 ± 0.58 kHz and USV duration was 65.7 ± 1.7 ms or 63.4 ± 2.1 ms, respectively, in both CD38+/+ and CD38−/− types (n = 23). However, the calls per 2-min session were significantly less frequent in CD38−/− infants (44 ± 8 calls, n = 10) than in wild-type controls (72 ± 13, n = 13). These results agreed well with previous observations in OT or OT receptor knockout mice [8,27,29]. However, the degree of disruption of infant behavior seemed to be much less marked in the case of CD38−/− in comparison with the two OT-related knockout mouse strains. This observation suggested that CD38−/− pups partially retain the ability to interact socially with others as seen in wild-type controls. This speculation prompted us to measure plasma OT levels to examine whether the OT level is decreased in these pups. We measured the plasma OT levels at five different phases of development (from 1 week to 2 months) in three genotypes of obligate or non-obligate litters (Fig. 2). Surprisingly, the plasma OT concentration in CD38−/− pups at 1–3 weeks of age was similar to those in CD38+/+ and CD38+/− mice of the same age. However, as expected, at 1 and 2 months of age following weaning, we found a significantly lower plasma concentration of OT in CD38−/− than in CD38+/+ mice (291 ± 46 vs. 522 ± 28 pg/ml and 215 ± 20 vs. 440 ± 60 pg/ml, respectively; P < 0.002; Fig. 2). Next, we measured ADP-ribosyl cyclase activity in the brain (hypothalamus and posterior pituitary) in CD38 wild-type and knockout mice during the first two months of life. In the brain regions examined, ADP-ribosyl cyclase activity was significantly higher even in the 1-week-old CD38+/+ mice than in the CD38+/− or CD38−/− mice of the same-age (Fig. 3). From the second week of life to all late development stages, CD38+/+ mice showed a much higher level of ADP-ribosyl cyclase activity in the hypothalamus and pituitary in comparison with CD38−/− mice. In the hypothalamus,
Fig. 3. ADP-ribosyl cyclase activity, measured as the rate of cyclic GDP-ribose formation by whole-cell homogenates isolated from the hypothalamus (A, B) and the posterior pituitary gland (C, D). Data were obtained from pups of different ages from obligate litters (A, C) or 1-week-old pups from non-obligate litters (B, D). The data shown are means ± S.E.M. (n = 4–6). *P < 0.05, **P < 0.01, ***P < 0.001 and # P > 0.05 (not significant). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
ADP-ribosyl cyclase activity increased with age in CD38+/+ mice, but only a slight decrease was observed in CD38−/− mice. ADPribosyl cyclase activity in the pituitary was lower than that in the hypothalamus and showed no increase throughout development. The results of this study indicated that locomotor activity induced by separation from the dam was higher and the number of USV calls was lower in CD38 knockout pups. However, these behaviors were much milder than those observed in OT and OT receptor knockout mice [27,29], suggesting less impairment of social behavior in the CD38−/− pups as compared with the above two knockout lines. Consistent with this finding, the plasma OT levels during development from neonates to 3-week-old juvenile mice remained at the same level between the two genotypes, although ADP-ribosyl cyclase activity was obviously lower in the knockout mice from birth.
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