- Email: [email protected]
Repeated immobilization stress in the early postnatal period increases stress response in adult rats
Physiology & Behavior 93 (2008) 322 – 326
Repeated immobilization stress in the early postnatal period increases stress response in adult rats Toshihiro Yoshihara ⁎, Yasutaka Yawaka Division of Pediatric Dentistry, Department of Oral Functional Science, Hokkaido University Graduate School of Dental Medicine, North 13, West 7, Kita-ku, Sapporo 060-8586, Japan Received 6 June 2007; received in revised form 15 August 2007; accepted 17 September 2007
Abstract Repeated immobilization stress tests in the early postnatal period were performed to determine the effects on the growth of developing rats as well as the response of the HPA axis to subsequent novel stress in adulthood. In addition, effects of maternal deprivation (MD) with the same period of the exposure to immobilization stress were also examined. We used 2 different types of immobilization stress and 2 different types of MD: immobilization stress for 30 min/day from postnatal day 7 (P7) to P13 (IS7-13 group); immobilization stress for 30 min on P7 (IS7 group); MD for 30 min/day from P7 to P13 (MD7-13 group); and MD for 30 min on P7 (MD7 group). Body weights were lower in the IS7-13 group than in the control group from P10 to P50, although body weight gain in the MD7-13 group was only transiently affected. Stress-induced corticosterone levels in the IS7-13 group were higher than in the control group and did not return to baseline levels until at least 120 min after the termination of stress, whereas temporal variations of stress-induced corticosterone levels did not differ between the IS, MD7-13, MD7, and control groups. Repeated immobilization stress in the early postnatal period induced long-term effects on the growth of developing rats and stress response of the HPA axis to the novel stress in adulthood, although a single immobilization stress, periodic MD, or a single MD had little effect. © 2007 Elsevier Inc. All rights reserved. Keywords: Repeated immobilization stress; Maternal deprivation; Hypothalamus–pituitary–adrenocortical axis; Rat
1. Introduction In mature rats, stress activates the hypothalamic-pituitary– adrenal (HPA) axis, resulting in the release of corticotropin releasing hormone (CRH) from the hypothalamic paraventricular nucleus (PVN). CRH causes the anterior pituitary to secrete adrenocorticotropic hormone (ACTH), which in turn stimulates the adrenal cortex to secrete corticosterone [1]. In contrast, mechanisms of hormonal response to stress in immature rats are not fully understood [2]. In particular, mechanisms in early postnatal rats seem to differ from those in mature rats. From postnatal day (P) 4 to P14, rats display a stress hyporesponsive period (SHRP) in the form of a markedly attenuated response of the HPA axis to environmental stress [3,4]. The SHRP is considered important for normal develop⁎ Corresponding author. Tel.: +81 11 706 4292; fax: +81 99 706 4307. E-mail address: [email protected] (T. Yoshihara). 0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2007.09.004
ment of the brain, as high levels of circulating corticosterone during development have been shown to produce developmental deficits [5]. Periodic maternal deprivation (MD) imposed on rats during the SHRP is reported to induce long-term effects on growth of developing rats as well as affect the response of the HPA axis to stress [6,7]. Recently, we reported that rats that had experienced periodic MD from P1 to P6 showed significant reductions in body weight compared with control rats until 9 weeks old and showed increased HPA axis response to novel stress that was subjected at 9 weeks of age [8]. It has been reported that repeated exposure to stress increases the response of the HPA axis to subsequent stress. For example, adult rats that experienced daily 20-minute restraint stress for 8 days show an exaggerated ACTH and corticosterone response to forced swimming, a new stress they were subjected to soon after the termination of restraint stress [9]. In addition, adult rats that experienced daily 3-hour restraint stress for 3 days also
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326
show an exaggerated corticosterone response to 2-dexy glucose injection, a new stress they were subjected to 12 days after the termination of restraint stress [10]. However, no reports appear to have examined the effects of immobilization stress in the early postnatal period on the growth of developing rats and the response of the HPA axis to subsequent new stress in adulthood. When a neonatal rat is immobilized, the rat is removed from the home cage that contains the nursing mother. Thus, exposure to immobilization stress in the early postnatal period might also induce effects associated with MD. The purpose of this study was to determine how immobilization stress in the early postnatal period affects growth of developing rats and the response of the HPA axis to the subsequent new stress in adulthood, taking into account the effects of MD. 2. Materials and methods 2.1. Animals Male Wistar rats were born and reared in our animal facility, in which environmental conditions were controlled as follows: temperature, 22 ± 1 °C; humidity, 60 ± 5%; and a 12-h light:dark cycle (lights on from 06:00 to 18:00). Light intensity at the cage surface was approximately 100 lux. Rats were fed commercial rat chow MF, (Oriental Yeast, Tokyo, Japan; 3.6 kcal/g) and tap water ad libitum, unless otherwise stated. Cyclicity of female rats was examined using vaginal smears. The day on which a female rat was found to be sperm-positive was designated as embryonic day 0, and the day of delivery was designated as postnatal day 0 (P0). Two days before the expected day of delivery, pregnant rats were housed individually in polycarbonate cages (36 × 33 × 17 cm). Just after delivery, body weights of all pups were measured and the members of a litter were rearranged so as to make the mean body weight of a litter equal. The size of each litter was adjusted to 6 males until weaning (P21). After weaning, pups were reared in individual cages (22 × 13 × 5 cm). All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at Kagoshima University Graduate School of Medical and Dental Sciences.
323
2.4. Procedure for exposure to novel stress A rat in an individual cage was transferred to a new cage of the same size, which contained fresh wood chips. For 1 week before the novelty exposure, the home cage was not cleaned. A rat transferred into a new cage immediately showed exploring behaviors such as sniffing, wandering, and standing. 2.5. Blood sampling and determination of corticosterone Blood sampling was undertaken as described previously [11]. A small incision was made at the tip of the freely moving rat tail using a razor blade 1 h before the beginning of blood sampling. About 50 μl of blood was collected from the incision into a heparinized capillary tube. Blood sampling was finished within 2.5 min after the first touch of a rat cage. It was only necessary to snip the tail for the first blood sampling. Separated plasma was frozen at − 80 °C until corticosterone assay. Plasma corticosterone level was determined by the competitive protein binding assay using 10 μl of a sample [12]. The minimum detectable level of corticosterone was 0.125 ng (1.25 μg/dl plasma). Intra- and inter-assay variances were 3.2% and 6.5%, respectively. 2.6. Experimental protocols Thirty male pups born on the same day were divided into the control group (n = 6) and the experimental group (n = 24). The experimental group was divided into 4 sub-groups (n = 6 each) according to single or repeated exposure to immobilization stress or MD; immobilization stress for 30 min/day from P7 to P13 (IS7-13 group), immobilization stress for 30 min on P7 (IS7 group), MD for 30 min/day from P7 to P13 (MD7-13 group), and MD for 30 min on P7 (MD7 group). The control group was not subjected to immobilization stress or MD and was left undisturbed.
2.2. Procedure of immobilization stress Pups were removed from the home cage containing their nursing mother at 10:00 h. Immobilization stress was carried out by taping the body of each rat to a plastic board on its stomach so that the rat was kept immobile for 30 min. Pups were returned to their home cage at 10:30 h. Ambient temperature and humidity were kept at 33 °C and 70–80% during exposure to immobilization stress. 2.3. Procedure of maternal deprivation Pups were removed from the home cage containing their nursing mother and placed in a separation cage (22 × 13 × 5 cm) with woodchip bedding at 10:00 h. Pups were returned to the home cage at 10:30 h. Woodchips in the home cage and separation cage were not changed during MD. Ambient temperature and humidity were kept at 33 °C and 70–80% during the exposure to MD.
Fig. 1. Body weight changes in the rats subjected to the restraint stress or MD of different conditions; IS7-13 (●), IS7 (○), MD 7-13 (■), MD 7 (□) and control group (△). Values are expressed as mean ± standard error of the mean (SEM) (n = 6 in each group). ⁎p b 0.05 and ⁎⁎p b 0.01; IS7-13 group vs. control group. #p b 0.05; MD 7-13 group vs. control group.
324
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326
At 10:00 on the day when pups were considered to have reached adulthood (P60), they were exposed to the novel stress. Blood was sampled at 10:00 (before the exposure to the novel stress, [T0]), 10:30 (T30), 11:00 (T60), 11:30 (T90), 12:00 (T120), 12:30 (T150), and 13:00 (T180). Body weight of each rat was measured every week. 2.7. Statistical analysis The data were analyzed using SPSS Version 10 (SPSS, Inc., Chicago, IL). Two-way analysis of variance with repeated measures and post-hoc Bonferroni testing was used for comparisons between groups. 3. Results 3.1. Effects of immobilization stress on changes in body weight Fig. 1 shows changes in body weight in the IS7-13, IS7, MD7-13, MD7, and control groups. Each group experienced significant variations in body weight (p b 0.01). Body weights were significantly lower in the IS7-13 group than in the control group at P10, P20, P30, P40, and P50 (p = 0.04, p b 0.01, p b 0.01, p = 0.04 and p = 0.04, respectively). Body weights were significantly lower in the MD7-13 group than in the control group at P20 (p = 0.03). No significant differences in body weights were seen between the IS7 and control groups and between the MD7 and control groups. 3.2. Effects of immobilization stress on stress-induced levels of plasma corticosterone in adult rats Fig. 2 shows stress-induced levels of plasma corticosterone in the IS7-13, IS7, MD7-13, MD7, and control groups. Each group experienced significant variations in plasma corticosterone levels (p b 0.01). Plasma corticosterone levels in each
Fig. 2. Basal and stress-induced levels of plasma corticosterone in adult rats subjected to the restraint stress or MD of different conditions; IS7-13 (●), IS7 (○), MD 7-13 (■), MD 7 (□) and control group (△). Values are expressed as mean ± standard error of the mean (SEM) (n = 6 in each group). ⁎p b 0.05; IS713 group vs. control group.
group gradually increased, peaking at T30, then decreased. Plasma corticosterone levels were significantly higher in the IS7-13 group than in the control group at T30, T60, T90, and T120 (p = 0.05, p = 0.03, p = 0.03 and p = 0.04, respectively). No significant differences in plasma corticosterone levels were seen at any time between the IS7 and control groups, between the MD7-13 and control groups, and between the MD7 and control groups. 4. Discussion The present study demonstrates that repeated immobilization stress in early postnatal period induces long-term effects on growth and the response of the HPA axis to novel stress in adulthood. In this study, growth and stress response in rats subjected to MD were compared with rats subjected to immobilization stress to detect whether results were due to the effects of immobilization stress or MD. Rats subjected to repeated MD for 30 min from P7 to P13 (MD7-13 group) showed a significant reduction in body weight that was maintained until at least P20 compared with those in the control group, but did not show a larger HPA response to novel stress than the control group in adulthood. In addition, rats subjected to a single MD for 30 min at P7 (MD7 group) did not show a significant reduction in body weight or increased HPA response compared with control rats. These results suggest that repeated immobilization stress, but not MD, in the early postnatal period induces long-term effects on growth and HPA response to novel stress in adulthood. The reason that repeated or single episodes of MD had no effect on HPA response to novel stress in this study might due to short periods (30 min) of MD. These results are consistent with our previous reports that showed that short periods (less than 3 h) of MD had little effect on growth and HPA response to novel stress [13,14]. An alternative explanation for no effect of MD in this study might be related with age (from P7 to P13) of the pup at the time of MD, since it is known that the effects of MD are most prominent when MD is imposed in the first postnatal week [3,4,6,7]. Rats subjected to repeated immobilization stress from P7 to P13 (IS7-13 group) showed a significant reduction in body weight that was maintained until P50 compared with rats in the other 4 groups. Interestingly, body weight in the MD7-13 group also showed a significant reduction that was maintained until P20 but caught up with that in the control group at P30, although this group was denied maternal care for 30 min for 7 days along with the IS7-13 group. These results indicate that the reduced body weight gain in the IS7-13 group cannot be simply explained by decreased milk intake from the mother, although it is well known that food intake in rats exposed to repeated immobilization stress is transiently inhibited after the termination of stress [10]. An important source of heat in terms of thermoregulation of newborn rats is brown adipose tissue (BAT) [15], as the thermoregulatory system is not fully developed in the early postnatal period [16]. The thermogenetic activity of BAT is regulated by the sympathetic nervous system [17]. Immobilization
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326
stress is reported to increase sympathetic activity [18]. Therefore, increased sympathetic activity could be involved in the long-term effects of repeated immobilization stress on body weight changes in the present study. However, body weight in IS7-13 group should catch up with that of rats in other groups after the termination of immobilization stress due to rebound hyperphagia [19]. This makes it doubtful that the long-term effects on body weight gain result simply from increased metabolism of BAT in the early postnatal period. An alternative hypothesis is that repeated immobilization stress suppresses levels of circulating growth hormones. It is well known that exposure to the stress suppresses feeding [20,21]. Furthermore, increased sympathetic activity induced by stress suppresses feeding and drinking [22]. Malnutrition in the early postnatal period reportedly reduces the concentration of insulinlike growth factor I in adult rats [23]. Lower levels of growth hormones might affect the development of rats subjected to repeated immobilization stress. These changes related to metabolism and/or nutrition induced by repeated immobilization stress might subsequently affect development of the central nervous system, as it is reported that early experiences have profound influences on brain development and thus on adult brain function and behavior [24]. Stress-induced corticosterone levels in adulthood were significantly higher in the IS7-13 group than in other 4 groups at T15 (15 min after the exposure to the novel stress), although there were no significant differences at baseline (T0) between these 5 groups. Furthermore, stress-induced corticosterone levels in the IS7-13 group did not return to baseline levels until at least 120 min after the exposure to the novel stress, although temporal variations in stress-induced corticosterone levels in the IS7 group did not significantly differ from those in the control group. These results suggest that repeated immobilization stress, as opposed to a one-time immobilization stress, in the early postnatal period has a long-term effect on the responsiveness of the HPA axis to novel stress. In the adult rats, stress-related neural circuits in the central nervous system are differentially regulated by repeated stress and a single stress [20,25]. In the early postnatal rats, repeated immobilization stress and a single immobilization stress might also have differential effects on the central nervous system. Although mechanisms for this long-term effect on the stressinduced corticosterone levels by repeated immobilization stress in the early postnatal period are not known, modification of negative feedback regulation of the HPA axis might be involved. Glucocorticoid receptors in the hippocampus mediate the negative feedback effects on the HPA axis [26]. It has been shown that repeated stress produces structural and functional changes in the brain, especially in the hippocampus [27,28]. The long-term effects of repeated immobilization stress in the early postnatal period on the HPA axis may be mediated by increased corticosterone secretion in rats subjected to repeated immobilization stress, modifying the expression of glucocorticoid receptors in the hippocampus and thereby decreasing negative feedback regulation of the HPA axis [29]. In the present study, the later decrease in plasma corticosterone levels from the peak level in the IS7-13 group compared with that in
325
the IS7 group might be related to alterations in the negative feedback system, as no significant differences were found between levels of plasma corticosterone at T0 in these 2 groups. In conclusion, repeated immobilization stress from P7 to P13 affected the growth of developing rats and the response of the HPA axis to novel stress in adulthood. Conversely, a single immobilization stress at P7 had little effect. In terms of stress response, modification of negative feedback regulation of the HPA axis might be involved. Acknowledgment This study was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No.18592246). References [1] O'Connor TM, O'Hallman DJ, Shanahan F. The stress response and the hypothalamic-pituitary–adrenal axis: from molecule to melancholia. Q J Med 2000;93:323–33. [2] Dent G, Choic DC, Herman JP, Levine S. GABAergic circuits and the stress hyporesponsive period in the rat: ontogeny of glutamic acid decarboxylase (GAD) 67 mRNA expression in limbic–hypothalamic stress pathways. Brain Res 2007;1138:1–9. [3] Rosenfeld P, Suchecki D, Levine S. Multifactorial regulation of the hypothalamic-pituitary–adrenal axis during development. Neurosci Biobehav Rev 1992;16:553–68. [4] Sapolsky RM, Meaney MJ. Maturation of the adrenocortical stress response in the rat: neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res Rev 1986;11:65–76. [5] Bakker JM, van Bel F, Heijnen CJ. Neonatal glucocorticoids and the developing brain: short-term treatment with long-term consequences? Trends Neurosci 2001;24:649–53. [6] Biagini G, Merlo Pich E, Carani C, Marrama P, Agnati LF. Postnatal maternal separation during the stress hyporesponsive period enhances the adrenocortical response to novelty in adult rats by affecting feedback regulation in the CA1 hippocampal field. Int J Dev Neurosci 1998;16:187–97. [7] Kalinichev M, Easterling KW, Plotsky PM, Holtzman SG. Long-lasting changes in stress-induced corticosterone response and anxiety-like behaviors as a consequence of neonatal maternal separation in Long– Evans rats. Pharmacol Biochem Behav 2002;73:131–40. [8] Matsumoto Y, Yoshihara T, Yamasaki Y. Maternal deprivation in the early versus late postnatal period differentially affects growth and stressinduced corticosterone responses in adolescent rats. Brain Res 2006;1115: 155–61. [9] García A, Martí O, Vallès A, Dal-Zotto S, Armario A. Recovery of the hypothalamic-pituitary–adrenal response to stress: effect of stress intensity, stress duration and previous stress exposure. Neuroendocrinology 2000;72:114–25. [10] Harris RBS, Gu H, Mitchell TD, Endale L, Russo M, Ryan DH. Increased glucocorticoid response to a novel stress in rats that have been restrained. Physiol Behav 2004;81:557–68. [11] Honma K, Honma S, Hiroshige T. Critical role of food amount for prefeeding corticosterone peak in the rats. Am J Physiol 1983;245:R339–44. [12] Murphy BEP. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein binding radioassay. J Clin Endocrinol 1967;27:973–90. [13] Yamazaki A, Ohtsuki Y, Yoshihara T, Honma S, Honma K. Maternal deprivation in rats of different length, time of day and ambient temperature affects the pups growth circadian clock, and stress responsiveness differentially. Physiol Behav 2005;86:136–44. [14] Yoshihara T, Otsuki Y, Yamazaki A, Honma S, Yamasaki Y, Honma K. Enhanced sensitivities of the circadian systems to a light–dark cycle and
326
[15]
[16]
[17]
[18]
[19] [20]
[21]
[22]
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326 periodic feeding in rats deprived of mother in the early postnatal period. Physiol Behav 2005;85:646–54. Fyda DM, Cooper KE, Veale WL. Contribution of brown adipose tissue to central PGE1-evoked hyperthermia in rats. Am J Physiol 1991;260: R59–66. Ibarra GR, Paratcha GC, Rodriguez JA, Napp MI, Azcurra JM. Cold stress related alteration of RNA biosynthesis in brain cortex of mother-deprived newborn rats. Life Sci 1997;60:2165–71. Kikuchi-Utsumi K, Gao B, Ohinata H, Hashimoto M, Yamamoto N, Kuroshima A. Enhanced gene expression of endothelial nitric oxide synthase in brown adipose tissue during cold exposure. Am J Physiol 2002;282: R623–6. Dronjak S, Jezova D, Kvetnansky R. Different effects of novel stressors on sympathoadrenal system activation in rats Exposed to long-term immobilization. Ann N Y Acad Sci 2004;1018:113–23. Iwasaki S, Inoue K, Kiriike N, Hikiji K. Effect of maternal separation on feeding behavior of rats in later life. Physiol Behav 2000;70:551–6. Bhatnagar S, Vining C, Iyer V, Kinni V. Changes in hypothalamic-pituitary– adrenal function, body temperature, body weight and food intake with repeated social stress exposure in rats. J Neuroendocrinol 2006;18:13–24. Dallman MF, La Fleur SE, Pecoraro NC, Gomez F, Houshyar H, Akana SF. Minireview: glucocorticoids — food intake, abdominal obesity, and wealthy nations in 2004. Endocrinology 2004;145:2633–8. Taylor MM, Samson WK. Stress hormone secretion is altered by central administration of intermedin/adrenomedullin-2. Brain Res 2005;1045: 199–205.
[23] Houdijk ME, Engelbregt MT, Popp-Snijders C, Delemarre-van der Waal HA. Long-term effects of early postnatal food restriction on growth hormone secretion in rats. JPNE J Parenter Enteral Nutr 2003;27:260–7. [24] Garoflos E, Panagiotaropoulos T, Pondiki S, Stamatakis A, Philippidis E, Stylianopoulou F. Cellular mechanisms underlying the effects of an early experience on cognitive abilities and affective states. Ann Gen Psychiatry 2005;4:8–19. [25] Berton O, Aguerre S, Sarrieau A, Mormede P, Chaouloff F. Differential effects of social stress on central serotonergic activity and emotional reactivity in Lewis and spontaneously hypertensive rats. Neuroscience 1998;82:147–59. [26] Jacobson L, Sapolsky R. The role of the hippocampus in feedback regulation of the hypothalamic-pituitary–adrenocortical axis. Endocr Rev 1991;12:118–34. [27] Kim JJ, Diamond DM. The stressed hippocampus, synaptic plasticity and lost memories. Nat Rev, Neurosci 2002;3:453–62. [28] McEwen BS. The neurobiology of stress: from serendipity to clinical relevance. Brain Res 2000;886:172–89. [29] Ladd CO, Huot RL, Thrivikraman KV, Nemeroff CB, Meaney MJ, Plotsky PM. Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog Brain Res 2000;122:81–103.
Repeated immobilization stress in the early postnatal period increases stress response in adult rats Toshihiro Yoshihara ⁎, Yasutaka Yawaka Division of Pediatric Dentistry, Department of Oral Functional Science, Hokkaido University Graduate School of Dental Medicine, North 13, West 7, Kita-ku, Sapporo 060-8586, Japan Received 6 June 2007; received in revised form 15 August 2007; accepted 17 September 2007
Abstract Repeated immobilization stress tests in the early postnatal period were performed to determine the effects on the growth of developing rats as well as the response of the HPA axis to subsequent novel stress in adulthood. In addition, effects of maternal deprivation (MD) with the same period of the exposure to immobilization stress were also examined. We used 2 different types of immobilization stress and 2 different types of MD: immobilization stress for 30 min/day from postnatal day 7 (P7) to P13 (IS7-13 group); immobilization stress for 30 min on P7 (IS7 group); MD for 30 min/day from P7 to P13 (MD7-13 group); and MD for 30 min on P7 (MD7 group). Body weights were lower in the IS7-13 group than in the control group from P10 to P50, although body weight gain in the MD7-13 group was only transiently affected. Stress-induced corticosterone levels in the IS7-13 group were higher than in the control group and did not return to baseline levels until at least 120 min after the termination of stress, whereas temporal variations of stress-induced corticosterone levels did not differ between the IS, MD7-13, MD7, and control groups. Repeated immobilization stress in the early postnatal period induced long-term effects on the growth of developing rats and stress response of the HPA axis to the novel stress in adulthood, although a single immobilization stress, periodic MD, or a single MD had little effect. © 2007 Elsevier Inc. All rights reserved. Keywords: Repeated immobilization stress; Maternal deprivation; Hypothalamus–pituitary–adrenocortical axis; Rat
1. Introduction In mature rats, stress activates the hypothalamic-pituitary– adrenal (HPA) axis, resulting in the release of corticotropin releasing hormone (CRH) from the hypothalamic paraventricular nucleus (PVN). CRH causes the anterior pituitary to secrete adrenocorticotropic hormone (ACTH), which in turn stimulates the adrenal cortex to secrete corticosterone [1]. In contrast, mechanisms of hormonal response to stress in immature rats are not fully understood [2]. In particular, mechanisms in early postnatal rats seem to differ from those in mature rats. From postnatal day (P) 4 to P14, rats display a stress hyporesponsive period (SHRP) in the form of a markedly attenuated response of the HPA axis to environmental stress [3,4]. The SHRP is considered important for normal develop⁎ Corresponding author. Tel.: +81 11 706 4292; fax: +81 99 706 4307. E-mail address: [email protected] (T. Yoshihara). 0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2007.09.004
ment of the brain, as high levels of circulating corticosterone during development have been shown to produce developmental deficits [5]. Periodic maternal deprivation (MD) imposed on rats during the SHRP is reported to induce long-term effects on growth of developing rats as well as affect the response of the HPA axis to stress [6,7]. Recently, we reported that rats that had experienced periodic MD from P1 to P6 showed significant reductions in body weight compared with control rats until 9 weeks old and showed increased HPA axis response to novel stress that was subjected at 9 weeks of age [8]. It has been reported that repeated exposure to stress increases the response of the HPA axis to subsequent stress. For example, adult rats that experienced daily 20-minute restraint stress for 8 days show an exaggerated ACTH and corticosterone response to forced swimming, a new stress they were subjected to soon after the termination of restraint stress [9]. In addition, adult rats that experienced daily 3-hour restraint stress for 3 days also
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326
show an exaggerated corticosterone response to 2-dexy glucose injection, a new stress they were subjected to 12 days after the termination of restraint stress [10]. However, no reports appear to have examined the effects of immobilization stress in the early postnatal period on the growth of developing rats and the response of the HPA axis to subsequent new stress in adulthood. When a neonatal rat is immobilized, the rat is removed from the home cage that contains the nursing mother. Thus, exposure to immobilization stress in the early postnatal period might also induce effects associated with MD. The purpose of this study was to determine how immobilization stress in the early postnatal period affects growth of developing rats and the response of the HPA axis to the subsequent new stress in adulthood, taking into account the effects of MD. 2. Materials and methods 2.1. Animals Male Wistar rats were born and reared in our animal facility, in which environmental conditions were controlled as follows: temperature, 22 ± 1 °C; humidity, 60 ± 5%; and a 12-h light:dark cycle (lights on from 06:00 to 18:00). Light intensity at the cage surface was approximately 100 lux. Rats were fed commercial rat chow MF, (Oriental Yeast, Tokyo, Japan; 3.6 kcal/g) and tap water ad libitum, unless otherwise stated. Cyclicity of female rats was examined using vaginal smears. The day on which a female rat was found to be sperm-positive was designated as embryonic day 0, and the day of delivery was designated as postnatal day 0 (P0). Two days before the expected day of delivery, pregnant rats were housed individually in polycarbonate cages (36 × 33 × 17 cm). Just after delivery, body weights of all pups were measured and the members of a litter were rearranged so as to make the mean body weight of a litter equal. The size of each litter was adjusted to 6 males until weaning (P21). After weaning, pups were reared in individual cages (22 × 13 × 5 cm). All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at Kagoshima University Graduate School of Medical and Dental Sciences.
323
2.4. Procedure for exposure to novel stress A rat in an individual cage was transferred to a new cage of the same size, which contained fresh wood chips. For 1 week before the novelty exposure, the home cage was not cleaned. A rat transferred into a new cage immediately showed exploring behaviors such as sniffing, wandering, and standing. 2.5. Blood sampling and determination of corticosterone Blood sampling was undertaken as described previously [11]. A small incision was made at the tip of the freely moving rat tail using a razor blade 1 h before the beginning of blood sampling. About 50 μl of blood was collected from the incision into a heparinized capillary tube. Blood sampling was finished within 2.5 min after the first touch of a rat cage. It was only necessary to snip the tail for the first blood sampling. Separated plasma was frozen at − 80 °C until corticosterone assay. Plasma corticosterone level was determined by the competitive protein binding assay using 10 μl of a sample [12]. The minimum detectable level of corticosterone was 0.125 ng (1.25 μg/dl plasma). Intra- and inter-assay variances were 3.2% and 6.5%, respectively. 2.6. Experimental protocols Thirty male pups born on the same day were divided into the control group (n = 6) and the experimental group (n = 24). The experimental group was divided into 4 sub-groups (n = 6 each) according to single or repeated exposure to immobilization stress or MD; immobilization stress for 30 min/day from P7 to P13 (IS7-13 group), immobilization stress for 30 min on P7 (IS7 group), MD for 30 min/day from P7 to P13 (MD7-13 group), and MD for 30 min on P7 (MD7 group). The control group was not subjected to immobilization stress or MD and was left undisturbed.
2.2. Procedure of immobilization stress Pups were removed from the home cage containing their nursing mother at 10:00 h. Immobilization stress was carried out by taping the body of each rat to a plastic board on its stomach so that the rat was kept immobile for 30 min. Pups were returned to their home cage at 10:30 h. Ambient temperature and humidity were kept at 33 °C and 70–80% during exposure to immobilization stress. 2.3. Procedure of maternal deprivation Pups were removed from the home cage containing their nursing mother and placed in a separation cage (22 × 13 × 5 cm) with woodchip bedding at 10:00 h. Pups were returned to the home cage at 10:30 h. Woodchips in the home cage and separation cage were not changed during MD. Ambient temperature and humidity were kept at 33 °C and 70–80% during the exposure to MD.
Fig. 1. Body weight changes in the rats subjected to the restraint stress or MD of different conditions; IS7-13 (●), IS7 (○), MD 7-13 (■), MD 7 (□) and control group (△). Values are expressed as mean ± standard error of the mean (SEM) (n = 6 in each group). ⁎p b 0.05 and ⁎⁎p b 0.01; IS7-13 group vs. control group. #p b 0.05; MD 7-13 group vs. control group.
324
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326
At 10:00 on the day when pups were considered to have reached adulthood (P60), they were exposed to the novel stress. Blood was sampled at 10:00 (before the exposure to the novel stress, [T0]), 10:30 (T30), 11:00 (T60), 11:30 (T90), 12:00 (T120), 12:30 (T150), and 13:00 (T180). Body weight of each rat was measured every week. 2.7. Statistical analysis The data were analyzed using SPSS Version 10 (SPSS, Inc., Chicago, IL). Two-way analysis of variance with repeated measures and post-hoc Bonferroni testing was used for comparisons between groups. 3. Results 3.1. Effects of immobilization stress on changes in body weight Fig. 1 shows changes in body weight in the IS7-13, IS7, MD7-13, MD7, and control groups. Each group experienced significant variations in body weight (p b 0.01). Body weights were significantly lower in the IS7-13 group than in the control group at P10, P20, P30, P40, and P50 (p = 0.04, p b 0.01, p b 0.01, p = 0.04 and p = 0.04, respectively). Body weights were significantly lower in the MD7-13 group than in the control group at P20 (p = 0.03). No significant differences in body weights were seen between the IS7 and control groups and between the MD7 and control groups. 3.2. Effects of immobilization stress on stress-induced levels of plasma corticosterone in adult rats Fig. 2 shows stress-induced levels of plasma corticosterone in the IS7-13, IS7, MD7-13, MD7, and control groups. Each group experienced significant variations in plasma corticosterone levels (p b 0.01). Plasma corticosterone levels in each
Fig. 2. Basal and stress-induced levels of plasma corticosterone in adult rats subjected to the restraint stress or MD of different conditions; IS7-13 (●), IS7 (○), MD 7-13 (■), MD 7 (□) and control group (△). Values are expressed as mean ± standard error of the mean (SEM) (n = 6 in each group). ⁎p b 0.05; IS713 group vs. control group.
group gradually increased, peaking at T30, then decreased. Plasma corticosterone levels were significantly higher in the IS7-13 group than in the control group at T30, T60, T90, and T120 (p = 0.05, p = 0.03, p = 0.03 and p = 0.04, respectively). No significant differences in plasma corticosterone levels were seen at any time between the IS7 and control groups, between the MD7-13 and control groups, and between the MD7 and control groups. 4. Discussion The present study demonstrates that repeated immobilization stress in early postnatal period induces long-term effects on growth and the response of the HPA axis to novel stress in adulthood. In this study, growth and stress response in rats subjected to MD were compared with rats subjected to immobilization stress to detect whether results were due to the effects of immobilization stress or MD. Rats subjected to repeated MD for 30 min from P7 to P13 (MD7-13 group) showed a significant reduction in body weight that was maintained until at least P20 compared with those in the control group, but did not show a larger HPA response to novel stress than the control group in adulthood. In addition, rats subjected to a single MD for 30 min at P7 (MD7 group) did not show a significant reduction in body weight or increased HPA response compared with control rats. These results suggest that repeated immobilization stress, but not MD, in the early postnatal period induces long-term effects on growth and HPA response to novel stress in adulthood. The reason that repeated or single episodes of MD had no effect on HPA response to novel stress in this study might due to short periods (30 min) of MD. These results are consistent with our previous reports that showed that short periods (less than 3 h) of MD had little effect on growth and HPA response to novel stress [13,14]. An alternative explanation for no effect of MD in this study might be related with age (from P7 to P13) of the pup at the time of MD, since it is known that the effects of MD are most prominent when MD is imposed in the first postnatal week [3,4,6,7]. Rats subjected to repeated immobilization stress from P7 to P13 (IS7-13 group) showed a significant reduction in body weight that was maintained until P50 compared with rats in the other 4 groups. Interestingly, body weight in the MD7-13 group also showed a significant reduction that was maintained until P20 but caught up with that in the control group at P30, although this group was denied maternal care for 30 min for 7 days along with the IS7-13 group. These results indicate that the reduced body weight gain in the IS7-13 group cannot be simply explained by decreased milk intake from the mother, although it is well known that food intake in rats exposed to repeated immobilization stress is transiently inhibited after the termination of stress [10]. An important source of heat in terms of thermoregulation of newborn rats is brown adipose tissue (BAT) [15], as the thermoregulatory system is not fully developed in the early postnatal period [16]. The thermogenetic activity of BAT is regulated by the sympathetic nervous system [17]. Immobilization
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326
stress is reported to increase sympathetic activity [18]. Therefore, increased sympathetic activity could be involved in the long-term effects of repeated immobilization stress on body weight changes in the present study. However, body weight in IS7-13 group should catch up with that of rats in other groups after the termination of immobilization stress due to rebound hyperphagia [19]. This makes it doubtful that the long-term effects on body weight gain result simply from increased metabolism of BAT in the early postnatal period. An alternative hypothesis is that repeated immobilization stress suppresses levels of circulating growth hormones. It is well known that exposure to the stress suppresses feeding [20,21]. Furthermore, increased sympathetic activity induced by stress suppresses feeding and drinking [22]. Malnutrition in the early postnatal period reportedly reduces the concentration of insulinlike growth factor I in adult rats [23]. Lower levels of growth hormones might affect the development of rats subjected to repeated immobilization stress. These changes related to metabolism and/or nutrition induced by repeated immobilization stress might subsequently affect development of the central nervous system, as it is reported that early experiences have profound influences on brain development and thus on adult brain function and behavior [24]. Stress-induced corticosterone levels in adulthood were significantly higher in the IS7-13 group than in other 4 groups at T15 (15 min after the exposure to the novel stress), although there were no significant differences at baseline (T0) between these 5 groups. Furthermore, stress-induced corticosterone levels in the IS7-13 group did not return to baseline levels until at least 120 min after the exposure to the novel stress, although temporal variations in stress-induced corticosterone levels in the IS7 group did not significantly differ from those in the control group. These results suggest that repeated immobilization stress, as opposed to a one-time immobilization stress, in the early postnatal period has a long-term effect on the responsiveness of the HPA axis to novel stress. In the adult rats, stress-related neural circuits in the central nervous system are differentially regulated by repeated stress and a single stress [20,25]. In the early postnatal rats, repeated immobilization stress and a single immobilization stress might also have differential effects on the central nervous system. Although mechanisms for this long-term effect on the stressinduced corticosterone levels by repeated immobilization stress in the early postnatal period are not known, modification of negative feedback regulation of the HPA axis might be involved. Glucocorticoid receptors in the hippocampus mediate the negative feedback effects on the HPA axis [26]. It has been shown that repeated stress produces structural and functional changes in the brain, especially in the hippocampus [27,28]. The long-term effects of repeated immobilization stress in the early postnatal period on the HPA axis may be mediated by increased corticosterone secretion in rats subjected to repeated immobilization stress, modifying the expression of glucocorticoid receptors in the hippocampus and thereby decreasing negative feedback regulation of the HPA axis [29]. In the present study, the later decrease in plasma corticosterone levels from the peak level in the IS7-13 group compared with that in
325
the IS7 group might be related to alterations in the negative feedback system, as no significant differences were found between levels of plasma corticosterone at T0 in these 2 groups. In conclusion, repeated immobilization stress from P7 to P13 affected the growth of developing rats and the response of the HPA axis to novel stress in adulthood. Conversely, a single immobilization stress at P7 had little effect. In terms of stress response, modification of negative feedback regulation of the HPA axis might be involved. Acknowledgment This study was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No.18592246). References [1] O'Connor TM, O'Hallman DJ, Shanahan F. The stress response and the hypothalamic-pituitary–adrenal axis: from molecule to melancholia. Q J Med 2000;93:323–33. [2] Dent G, Choic DC, Herman JP, Levine S. GABAergic circuits and the stress hyporesponsive period in the rat: ontogeny of glutamic acid decarboxylase (GAD) 67 mRNA expression in limbic–hypothalamic stress pathways. Brain Res 2007;1138:1–9. [3] Rosenfeld P, Suchecki D, Levine S. Multifactorial regulation of the hypothalamic-pituitary–adrenal axis during development. Neurosci Biobehav Rev 1992;16:553–68. [4] Sapolsky RM, Meaney MJ. Maturation of the adrenocortical stress response in the rat: neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res Rev 1986;11:65–76. [5] Bakker JM, van Bel F, Heijnen CJ. Neonatal glucocorticoids and the developing brain: short-term treatment with long-term consequences? Trends Neurosci 2001;24:649–53. [6] Biagini G, Merlo Pich E, Carani C, Marrama P, Agnati LF. Postnatal maternal separation during the stress hyporesponsive period enhances the adrenocortical response to novelty in adult rats by affecting feedback regulation in the CA1 hippocampal field. Int J Dev Neurosci 1998;16:187–97. [7] Kalinichev M, Easterling KW, Plotsky PM, Holtzman SG. Long-lasting changes in stress-induced corticosterone response and anxiety-like behaviors as a consequence of neonatal maternal separation in Long– Evans rats. Pharmacol Biochem Behav 2002;73:131–40. [8] Matsumoto Y, Yoshihara T, Yamasaki Y. Maternal deprivation in the early versus late postnatal period differentially affects growth and stressinduced corticosterone responses in adolescent rats. Brain Res 2006;1115: 155–61. [9] García A, Martí O, Vallès A, Dal-Zotto S, Armario A. Recovery of the hypothalamic-pituitary–adrenal response to stress: effect of stress intensity, stress duration and previous stress exposure. Neuroendocrinology 2000;72:114–25. [10] Harris RBS, Gu H, Mitchell TD, Endale L, Russo M, Ryan DH. Increased glucocorticoid response to a novel stress in rats that have been restrained. Physiol Behav 2004;81:557–68. [11] Honma K, Honma S, Hiroshige T. Critical role of food amount for prefeeding corticosterone peak in the rats. Am J Physiol 1983;245:R339–44. [12] Murphy BEP. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein binding radioassay. J Clin Endocrinol 1967;27:973–90. [13] Yamazaki A, Ohtsuki Y, Yoshihara T, Honma S, Honma K. Maternal deprivation in rats of different length, time of day and ambient temperature affects the pups growth circadian clock, and stress responsiveness differentially. Physiol Behav 2005;86:136–44. [14] Yoshihara T, Otsuki Y, Yamazaki A, Honma S, Yamasaki Y, Honma K. Enhanced sensitivities of the circadian systems to a light–dark cycle and
326
[15]
[16]
[17]
[18]
[19] [20]
[21]
[22]
T. Yoshihara, Y. Yawaka / Physiology & Behavior 93 (2008) 322–326 periodic feeding in rats deprived of mother in the early postnatal period. Physiol Behav 2005;85:646–54. Fyda DM, Cooper KE, Veale WL. Contribution of brown adipose tissue to central PGE1-evoked hyperthermia in rats. Am J Physiol 1991;260: R59–66. Ibarra GR, Paratcha GC, Rodriguez JA, Napp MI, Azcurra JM. Cold stress related alteration of RNA biosynthesis in brain cortex of mother-deprived newborn rats. Life Sci 1997;60:2165–71. Kikuchi-Utsumi K, Gao B, Ohinata H, Hashimoto M, Yamamoto N, Kuroshima A. Enhanced gene expression of endothelial nitric oxide synthase in brown adipose tissue during cold exposure. Am J Physiol 2002;282: R623–6. Dronjak S, Jezova D, Kvetnansky R. Different effects of novel stressors on sympathoadrenal system activation in rats Exposed to long-term immobilization. Ann N Y Acad Sci 2004;1018:113–23. Iwasaki S, Inoue K, Kiriike N, Hikiji K. Effect of maternal separation on feeding behavior of rats in later life. Physiol Behav 2000;70:551–6. Bhatnagar S, Vining C, Iyer V, Kinni V. Changes in hypothalamic-pituitary– adrenal function, body temperature, body weight and food intake with repeated social stress exposure in rats. J Neuroendocrinol 2006;18:13–24. Dallman MF, La Fleur SE, Pecoraro NC, Gomez F, Houshyar H, Akana SF. Minireview: glucocorticoids — food intake, abdominal obesity, and wealthy nations in 2004. Endocrinology 2004;145:2633–8. Taylor MM, Samson WK. Stress hormone secretion is altered by central administration of intermedin/adrenomedullin-2. Brain Res 2005;1045: 199–205.
[23] Houdijk ME, Engelbregt MT, Popp-Snijders C, Delemarre-van der Waal HA. Long-term effects of early postnatal food restriction on growth hormone secretion in rats. JPNE J Parenter Enteral Nutr 2003;27:260–7. [24] Garoflos E, Panagiotaropoulos T, Pondiki S, Stamatakis A, Philippidis E, Stylianopoulou F. Cellular mechanisms underlying the effects of an early experience on cognitive abilities and affective states. Ann Gen Psychiatry 2005;4:8–19. [25] Berton O, Aguerre S, Sarrieau A, Mormede P, Chaouloff F. Differential effects of social stress on central serotonergic activity and emotional reactivity in Lewis and spontaneously hypertensive rats. Neuroscience 1998;82:147–59. [26] Jacobson L, Sapolsky R. The role of the hippocampus in feedback regulation of the hypothalamic-pituitary–adrenocortical axis. Endocr Rev 1991;12:118–34. [27] Kim JJ, Diamond DM. The stressed hippocampus, synaptic plasticity and lost memories. Nat Rev, Neurosci 2002;3:453–62. [28] McEwen BS. The neurobiology of stress: from serendipity to clinical relevance. Brain Res 2000;886:172–89. [29] Ladd CO, Huot RL, Thrivikraman KV, Nemeroff CB, Meaney MJ, Plotsky PM. Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog Brain Res 2000;122:81–103.