Post-weaning isolation promotes food intake and body weight gain in rats that experienced neonatal maternal separation

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Post-weaning isolation promotes food intake and body weight gain in rats that experienced neonatal maternal separation Vitaly Ryu a , Sang Bae Yoo a , Dong-Won Kang b , Jong-Ho Lee a,⁎, Jeong Won Jahng a,⁎ a

Department of Oral and Maxillofacial Surgery, Dental Research Institute, Seoul National University School of Dentistry, Seoul, 110-768, Korea Department of Pharmacology, Kwandong University College of Medicine, Gangneung, 210-701, Korea

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Neonatal maternal separation (MS) in rats has been reported to result in permanent

Accepted 3 August 2009

dysfunctions of the hypothalamic-pituitary-adrenal axis and the development of anxiety-

Available online 8 August 2009

and depression-like behaviors later in life. In this study, we examined the effects of postweaning social isolation stress on food intake and body weight gain of rats with MS

Keywords:

experience. MS was performed daily for 180 min during the first 2 weeks of birth and

Maternal separation

nonhandled control (NH) pups were left undisturbed. Weanling male pups were caged either

Social isolation

in a group of three or singly (social isolation), and then subjected to behavioral sessions for

Hyperphagia

anxiety- or depression-like behaviors at 2 months of age. Social isolation following MS

Overweight

experience, but neither MS nor social isolation alone, significantly increased food intake and

Depression

weight gain. MS pups showed increased immobility in forced swim test, compared to NH

Anxiety

pups, regardless of their housing conditions. In elevated plus maze test, group-caged MS pups spent less time in the open arms and more time in the closed arms than group-caged NH pups, but social isolation did not further affect the arm stay of MS pups. However, statistical analyses revealed an interaction between MS and social isolation not only in the time spent in each arms, but also in defecation scores during the ambulatory activity test. These results suggest that post-weaning social isolation may promote hyperphagia and weight gain in young rats that experienced neonatal maternal separation, perhaps, in relation with its impact on the psycho-emotional behaviors of MS pups. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

A number of studies have indicated a strong correlation between traumatic events during early life and the development of behavioral and neuroendocrine abnormalities later in life. For examples, loss of a parent during childhood, a

stressful life event, increased the risk of developing major anxiety disorders (Kendler et al., 1992; Furukawa et al., 1999), and women with histories of childhood abuse displayed abnormal responses of hypothalamic-pituitary-adrenocortical (HPA) axis with signs of depression (Heim et al., 2000, 2001). Neonatal maternal separation (MS) is considered as an animal

⁎ Corresponding authors. J.W. Jahng is to be contacted at Dental Research Institute, Seoul National University School of Dentistry, Seoul, 110-768, Korea. Fax: +82 2 766 4948. J.-H. Lee, Department of Oral and Maxillofacial Surgery, Seoul National University School of Dentistry, Seoul, 110-768, Korea. Fax: +82 2 766 4948. E-mail addresses: [email protected] (J.-H. Lee), [email protected] (J.W. Jahng). Abbreviations: MS, maternal separation; NH, nonhandled; HPA, hypothalamic-pituitary-adrenal; ANOVA, analysis of variance; PND, postnatal day 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.08.006

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model of stressful experience early in life. Many studies have demonstrated its impact both on the activity of HPA axis; i.e., permanent alterations in the characteristics of the HPA response to stress (Ladd et al., 1996; Van Oers et al., 1998; Vazquez et al., 2000; Kim et al., 2005a,b; Ryu et al., 2008) and on the development of depression- (Ladd et al., 2000; Khoury et al., 2006) and anxiety-like behaviors (Kalinichev et al., 2002; Daniels et al., 2004; Lee et al., 2007) later in life. It has been reported that symptoms of anxiety and depression are associated with the pathophysiology of eating disorders (Goossens et al., 2008; see for review), especially with binge-like eating disorders (Grilo et al., 2008; Javaras et al., 2008). In our previous studies, rats that experienced maternal separation repeatedly during the first 2 weeks of birth (MS) showed anxiety- and depression-like behaviors (Lee et al., 2007), and the MS offspring showed binge-like eating when they were challenged with repeated fasting/refeeding cycles during adolescent period (Ryu et al., 2008). Also, it has been reported that dysfunction of the HPA axis is implicated in the pathogenesis of eating disorders (Koo-Loeb et al., 2000; Putignano et al., 2001; Gluck et al., 2004). Our MS model showed alterations in the HPA axis activity responding to stress later in life (Kim et al., 2005a,b) and, their binge-like eating behavior during repeated fasting/refeeding cycles appeared to be related with increased activity of the HPA axis (Ryu et al., 2008). Interestingly, we have found that the basal plasma level of corticosterone was elevated in MS compared with its nonhandled (NH) controls when the weanling pups were singly housed (Noh et al., 2008); however, it did not differ between MS and NH when the pups were housed in groups (Ryu et al., 2008). It has been suggested that increased serum cortisol levels are implicated in anxiety (Albanidou-Farmaki et al., 2008), depression (Heim et al., 2000) and binge eating disorders (Koo-Loeb et al., 2000; Gluck et al., 2004). These studies have led us to hypothesize that increased plasma corticosterone in our single-housed MS pups may be due to increased responsiveness of the HPA axis to post-weaning isolation stress by MS experience, and it may lead to increased food intake and weight gain. Interactions with peers during adolescence are thought to be of principal importance for social development in human adolescents, since individuals spend more time interacting with peers during adolescence than at any other developmental period (Harris, 1995; LaGreca et al., 2001). In rats, early disruption of social interactions, such as isolated rearing, produces profound, long-term neurochemical, endocrinological and behavioral effects (Robbins et al., 1996; Hall, 1998; see for review). Isolation-reared rats showed alterations in the hippocampal neurotransmission (Bickerdike et al., 1993), dysregulation of the HPA axis activation responding to stress (Weiss et al., 2004; Serra et al., 2005) and exhibited anxiety-like behaviors (Wright et al., 1991; Hellemans et al., 2004; Weiss et al., 2004). However, it has been demonstrated that isolation rearing does not significantly affect food intake and weight gain in rats (Hellemans et al., 2004; Weiss et al., 2004; Fone and Porkess, 2008; see for review). Thus, we have hypothesized that post-weaning social isolation stress, when followed after MS experience, may promote hyperphagia and weight gain, and further affect the symptoms of depression- and/or anxiety-like behaviors by MS.

In this study, the effects of post-weaning social isolation on food intake and weight gain of MS rats were examined in parallel with behavioral assessments for depression- and anxiety-like behaviors.

2.

Results

2.1.

Body weight and food intake

MS pups were significantly heavier than NH pups at weaning on PND 22 (P < 0.05, NH-group vs. MS-group) and the weight difference between MS-group and NH-group became nonsignificant from PND 28 (Table 1). Two-way analysis of variance (ANOVA) with repeated measures revealed no effect of maternal separation (MS), near effect of post-weaning isolation (social isolation) [F(1,160)= 3.18, P = 0.084] with interactions between social isolation and PND [F(5,160) = 2.90, P < 0.05] and between MS and social isolation and PND [F(5,160)= 2.82, P < 0.05]. Body weight gain of NH pups caged singly (NH-single) did not differ from NH-group. However, MS-single appeared to gain more weight as they grew, compared to MS-group with statistical significances (P < 0.05, MS-group vs. MS-single) on PND 42, 49 and 56, supporting that social isolation promotes weight gain of MS pups although it may not affect weight gain of NH pups. Twenty-four hour food intake was measured weekly from PND 28 (Table 2). Analysis of food intake with two-way ANOVA with repeated measures revealed no effect of MS, but main effects of social isolation [F(1,80)= 8.17, P < 0.01] and PND [F(4,80)= 168.86, P < 0.0001]. Daily food intake of group-caged MS pups did not differ from group-caged NH pups. Social isolation transiently increased food intake of NH pups on PND 28, a week after weaning (P < 0.05, NH-group vs. NH-single), thereafter the statistical significance between NH-group and NH-single diminished. Food intake of single-caged MS pups did not differ from group-caged MS pups until 2 weeks after weaning (PND 35); however, it became significantly increased (P < 0.05, MSgroup vs. MS-single) on PND 42 and 56, respectively (Table 2).

2.2.

Behavioral assessments

Rats were subjected to the ambulatory activity test at 2 months of age. The ambulatory activity was measured for 60 min as the

Table 1 – Body weight changes during the experimental period (g). PND

22 28 35 42 49 56

NH

MS

Group

Single

Group

Single

60.76 ± 0.79 102.47 ± 1.22 169.88 ± 2.07 243.44 ± 2.60 312.14 ± 3.66 379.21 ± 5.27

60.86 ± 0.90 103.81 ± 2.34 174.92 ± 4.25 247.02 ± 5.14 315.00 ± 6.63 379.79 ± 7.67

64.73 ± 1.37⁎ 106.14 ± 1.87 173.76 ± 2.89 245.48 ± 6.48 313.44 ± 7.42 373.52 ± 10.23

64.32 ± 1.68 107.20 ± 1.94 181.51 ± 3.01 260.04 ± 4.28# 330.08 ± 5.73# 401.56 ± 7.86#

⁎P < 0.05 vs. NH-group, #P < 0.05 vs. MS-group on each day. PND, postnatal days; NH, nonhandled; MS, maternal separation. Data are presented by means ± S.E.

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Table 2 – 24 h food intake on each observed day (g). PND

NH Group

28 35 42 49 56

15.03 ± 0.42 22.24 ± 0.25 25.35 ± 1.50 29.59 ± 0.73 32.28 ± 0.31

MS Single #

17.39 ± 0.48 24.88 ± 0.85 29.17 ± 1.23 32.13 ± 1.08 33.64 ± 0.49

Group

Single

15.22 ± 0.41 23.23 ± 2.33 24.42 ± 1.85 29.72 ± 1.48 31.22 ± 1.26

16.54 ± 0.29 26.21 ± 0.56 31.09 ± 1.19⁎ 32.79 ± 1.33 37.11 ± 2.11⁎

#

P < 0.05 vs. NH-group, ⁎P < 0.05 vs. MS-group on each day. PND, postnatal days; NH, nonhandled; MS, maternal separation. Data are presented by means ± S.E.

total counts of beam interruptions in the horizontal sensor during each consecutive 5-min session (Fig. 1). Three-way ANOVA revealed main effects of maternal separation [F(1,384)= 69.66, P < 0.0001] and time points [F(11,384)= 44.09, P < 0.0001]. Main effect of social isolation or an interaction between maternal separation and social isolation was not found. The ambulatory counts of MS-group and MS-single were decreased significantly compared to NH-group (P < 0.05, on 5, 15, 20 and

Fig. 1 – (A) Ambulatory counts of NH and MS pups, which recorded consecutively at every 5 min during 60 min of test period. (B) Total ambulatory counts of the whole session (0–60 min). Rats were subjected to the ambulatory activity test at 2 months of age. *P < 0.05; NH-group vs. MS-group at each time point, #P < 0.05; NH-single vs. MS-single at each time point. NH, nonhandled; MS, maternal separation; min, minutes. Data are presented by means ± S.E.

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45 min time points) and NH-single (P < 0.05, on 5, 15, 25 and 50 min time points), respectively (Fig. 1A). This result reveals that MS pups were less active than NH pups during the ambulatory test, with no effect of housing condition. Total ambulatory counts during 60 min of the test session decreased in MS pups compared with NH pups, regardless of their housing conditions (Fig. 1B). Rearings and defecation behaviors, revealing emotional status of rats, were measured during each ambulation test. Analysis of rearing with two-way ANOVA revealed a near significant effect of maternal separation [F(1,31) = 4.06, P = 0.0526], no effect of social isolation and no interaction between maternal separation and social isolation (Fig. 2A). Post-weaning isolation tended to suppress rearing of NH pups, but a statistical significance between NH-group and NH-single was not found. Rearing numbers of MS-group were reduced significantly compared with NH-group (P < 0.05), and rearing numbers of MS-single did not differ from MS-group (Fig. 2A). Analysis of defecation scores with two-way ANOVA revealed an interaction between maternal separation and social isolation on feces weight [F(1,28) = 4.30, P < 0.05], and near significant interaction on feces number [F(1,28) = 4.10, P = 0.0526]. Experience of maternal separation appeared to suppress defecation behavior of rats; i.e., both the number and weight of feces of MS-group were significantly decreased (P < 0.05) compared to NH-group (Figs. 2B and C). Feces number of NH-single decreased significantly (P < 0.05), and

Fig. 2 – Number of rearings (A) and defecation scores (B and C) measured during the ambulatory test. *P < 0.05, #P = 0.058 vs. NH-group. NH, nonhandled; MS, maternal separation. Data are presented by means ± S.E.

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feces weight nearly decreased (P = 0.058), as compared with NH-group; however, the defecation scores of MS-single tended to be increased without statistical significances, compared to MS-group. In order to assess depression-like behaviors, rats were subjected to forced swim test daily for three consecutive days after the preswim test on day 1. On the first test day, immobility duration of MS pups appeared to be longer than NH pups, regardless of the housing condition (Fig. 3). Twoway ANOVA revealed main effect of maternal separation [F(1,32) = 13.10, P = 0.001], no effect of social isolation, and no interaction between MS and social isolation. Immobility duration was significantly increased in MS-group compared to NH-group (P < 0.05), but it did not differ between NH-group and NH-single (Fig. 3). Post-weaning isolation did not further increase immobility duration of MS pups. The differences in immobility duration between MS and NH pups diminished from the second day of swim test as the immobility of NH pups, but not of MS, increased along the test days (data not shown). Rats were subjected to the elevated plus maze test to assess anxiety-like behaviors (Fig. 4). Analysis of the time spent in the open arms with two-way ANOVA revealed main effect of MS [F(1,32) = 4.46, P < 0.05], no effect of social isolation, and an interaction between MS and social isolation [F(1,32) = 5.37, P < 0.05]. Analysis of the closed arm stay revealed no main effect of MS or social isolation with an interaction between MS and social isolation [F(1,32) = 6.49, P < 0.05]. NH-single spent less time in open arms (P < 0.05), and nearly more time in closed arms (P = 0.086), than NHgroup (Fig. 4A). MS-group spent less time in open arms (P < 0.05), more time in closed arms (P < 0.05), than NH-group, and social isolation did not significantly affect the arm stays of MS pups. Analysis of the percentage of closed arm entry (percent entries into closed arms out of total arm entries) with two-way ANOVA showed main effect of MS [F(1,32) = 4.21, P < 0.05] and no effect of social isolation with near interaction between MS and social isolation [F(1,32) = 3.25, P = 0.081]. Percentage of closed arm entry increased significantly in MS-group (P < 0.05), and nearly increased in NH-

Fig. 3 – Immobility durations of rats during forced swim test. Rats were subjected to Porsolt swim test 3 days after the ambulatory activity test. *P < 0.05 vs. NH-group, #P < 0.05 vs. NH-single. NH, nonhandled; MS, maternal separation; sec, seconds. Data are presented by means ± S.E.

Fig. 4 – Time spent in each arm (A) and percent closed arm entry (B) during elevated plus maze test. The test was performed after a week of recovery period following the forced swim test. *P < 0.05, #P = 0.053 vs. NH-group. NH, nonhandled; MS, maternal separation; sec, seconds. Data are presented by means ± S.E.

single (P = 0.053), as compared with NH-group (Fig. 4B). Social isolation did not alter the % closed arm entries of MS pups.

3.

Discussion

3.1.

Effects of post-weaning isolation in NH rats

Early disruption of social interactions between conspecifics, such as isolated rearing, may affect brain development, leading to prolonged aberrant behavior (Harlow and Harlow, 1965; Heim et al., 2004; Rapoport et al., 2005). It has been reported that social isolation in rats may lead to the development of anxiety-like behaviors (Wright et al., 1991; Bickerdike et al., 1993; Hellemans et al., 2004; Weiss et al., 2004). In this study, we have demonstrated that single-caged NH pups (isolates) spent less time in the open arms of elevated plus maze, and their percentage of closed arm entry increased with a near significance (P = 0.053), compared to group-caged NH pups, supporting the anxiogenic property of post-weaning social isolation. Also, we have found that defecation activities of the NH isolates are decreased, as compared to the group caged ones. Although many studies

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have reported that decreased defecation may reveal an anxiolytic property (Martín-García and Pallarès, 2005; Ghisleni et al., 2008), decreased defecation in adolescent rats was suggested to be an anxiety-related behavior (Aitchison and Hughes, 2006). In both rats and humans the adolescent brain is not fully mature and represents “a brain in transition” differing both anatomically and neurochemically from the adult brain (Spear, 2000). Indeed, Gentsch and colleagues (1981, 1982) have reported that isolated rearing affects defecation scores in rats, and explained that decreased defecations by social isolation could be due to an increased fearfulness of the isolated rats, so interpreting as a ‘frozen behavior.’ Thus, it is concluded that decreased defecation scores in the NH isolates may further support the anxiogenic property of social isolation in NH pups. Whilst the NH isolates showed anxiety-like behaviors in this study, they did not exhibit depression-like behaviors; i.e., the immobility duration of NH-single during forced swim test did not differ from NH-group. This result is in agreement with previous studies reporting no significant effect of post-weaning isolation in rodents on the immobility or struggling time in Porsolt swim test (Hilakivi et al., 1989; Hall et al., 1998, 2001). The ambulatory activities of NH isolates did not differ from NH-group, in accordance with previous reports that postweaning isolation may not affect the ambulatory activities of rats (Geyer et al., 1993; Weiss et al., 2000, 2001, 2004). Lastly, food intake, a motivational behavior, of NH-single did not differ from NH-group, except a transient increase in the isolates shortly after the weaning procedure. Also, body weight gain of NH-single did not differ from NH-group. These results concur with previous reports that isolation rearing may not significantly affect food intake and weight gain of rats (Hellemans et al., 2004; Weiss et al., 2004; Fone and Porkess, 2008; see for review). Thus, it is concluded that postweaning isolation in NH rats may lead to the development of anxiety-like behaviors, although it may not induce behavioral depressions nor affect food intake.

3.2. Effects of maternal separation: group-caged NH vs. group-caged MS Many studies have demonstrated that experience of neonatal maternal separation may lead to the development of depression- and anxiety-like behaviors later in life (Ladd et al., 2000; Kalinichev et al., 2002; Daniels et al., 2004; Khoury et al., 2006; Lee et al., 2007). In this study, the ambulatory activities of group-caged young adult MS rats were suppressed significantly compared with group-caged NH rats, suggesting that depression and/or anxiety symptoms may be developed by experience of maternal separation. Indeed, group-caged MS rats showed behavioral depression in Porsolt swim test, and anxiety-like behaviors in elevated plus maze test, compared to group-caged NH rats. Decreased number of rearings and defecation scores in group-caged MS, compared to group-caged NH, further supported the anxiogenic effect of maternal separation. It has been reported that symptoms of anxiety and depression are associated with the pathophysiology of eating disorders (Goossens et al., 2008; see for review). However, food intake and weight gain of groupcaged MS pups did not differ from group-caged NH pups,

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except a transient slight increase in weight gain in MS pups shortly after weaning. This result is in accordance with previous studies carried on similar experimental paradigms; i.e., no significant effect of neonatal maternal separation was found on food intake and weight gain of the offspring when they were group-reared at weaning following 3 hours of daily MS during the first 2 weeks of birth (McIntosh et al., 1999; Iwasaki et al., 2000; Kalinichev et al., 2002; Kim et al., 2005a). Taken all together, it is concluded that repeated experience of maternal separation during the first 2 weeks of birth may not permanently affect food intake and body weight gain of the offspring as long as the pups are reared in a group, although it may still lead to the development of anxiety- and depression-like behaviors.

3.3.

Effects of post-weaning isolation in MS rats

In this study, post-weaning isolation appeared to promote hyperphagia in MS pups; i.e., food intake and weight gain of MS-single were increased in comparison to MS-group. Previous studies have reported that binge eating disorders are frequently occurred concurrently with mood and anxiety disorders (Grilo et al., 2008; Javaras et al., 2008). In this study, the behavioral scores of MS rats, such as ambulatory counts, rearings, defecation scores, immobility duration during the swim test, and the arm stays and entries of elevated plus maze test, did not seem to be further worsened by isolation rearing; i.e., those scores of MS-single did not differ from MSgroup per se. However, statistical analyses of the behavioral scores with two-way ANOVA revealed an interaction between maternal separation and post-weaning isolation not only in the open arms stay, but also in the closed arms stay of elevated plus maze test, suggesting impacts of post-weaning isolation on anxiety-like behaviors of MS pups. Furthermore, the statistical analysis of defecation scores, representing emotional status (Gentsch et al., 1981, 1982; Aitchison and Hughes, 2006), also revealed an interaction between maternal separation and isolation rearing. Thus, it is concluded that post-weaning isolation may promote hyperphagia and weight gain in MS pups, perhaps, in relation with its impact on the psychoemotional behaviors representing anxiety. It should be noticed that the effect of isolation rearing on weight gain of MS pups seemed to be stronger than the effect on food intake, and the statistical analysis revealed an interaction between maternal separation and isolation rearing and PND in weight gain, but not in food intake, of the MS isolates. This result suggests that increased weight gain in MS isolates may be attributed by both increased food intake and decreased metabolic rates, and the psychoemotional impacts of social isolation on MS pups may be related, if so, more strongly to weight gain than to food intake. Tentative effect of social isolation on the metabolic rates of MS pups remains to be elucidated. In conclusion, post-weaning social isolation may promote hyperphagia and weight gain in young rats that experienced neonatal maternal separation, perhaps, in relation with its impact on anxiety-like behaviors by maternal separation. Further studies are required to define the neurochemical mechanism underlying the increased food intake and weight gain in MS isolates.

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4.

Experimental procedure

4.1.

Animals

Sprague-Dawley rats were purchased (Samtako Bio, Osan, Korea) and cared for in a specific-pathogen-free barrier area with constant control of temperature (22 ± 1 °C), humidity (55%), and a 12:12 h light/dark cycle (lights on at 07:00 AM). Standard laboratory food (Purina Rodent Chow, Purina Co., Seoul, Korea) and membrane filtered purified water were available ad libitum. Animals were cared according to the Guideline for Animal Experiments, 2000, edited by the Korean Academy of Medical Sciences, which is consistent with the NIH Guidelines for the Care and Use of Laboratory Animals, revised 1996. All animal experiments were approved by the Committee for the Care and Use of Laboratory Animals at Seoul National University. Nulliparous females and proven breeder males were used for breeding in the laboratory of the animal facility, and the pups were reared in a controlled manner to minimize and standardize unwanted environmental stimulation from in utero life. Twelve hours after confirming delivery (PND 1), pups were culled to five males and five females per litter. Each litter was assigned either for the maternal separation (MS) group or for the nonhandled (NH) group. MS was performed as we previously described (Kim et al., 2005a; Lee et al., 2007; Ryu et al., 2008). In brief, MS pups were removed from their dam and home cage and placed closely together in a new cage bedded with woodchips for 180 min, and then returned to their home cage and dam. MS was performed at room temperature; i.e., no additional treatment to keep the pups warm during the separation period, other than placing them closely together, was offered and pup-cooling during MS was expected. MS was performed during 9:00 h–12:00 h daily from PND 1 through 14, and then the pups were left with their dam undisturbed until weaning on PND 22. The NH group remained undisturbed until weaning except for routine cage cleaning. On weaning day (PND 22), male pups were weighed and caged either in groups of three littermate pups (NH-group or MS-group) or singly (NHsingle or MS-single). Female pups were excluded from the study, because our previous studies supporting the rationale to plan the present study had been performed with male offspring. Body weight gain and 24 h food intake were measured weekly from PND 28. On each weekly measuring day, pups were weighed, and provided with weighed amounts of chow and allowed to eat ad libitum for 24 h. Then the left over chow was carefully collected and weighed, and the value was subtracted from the value of chow provided on the previous day. For the group-caged pups, the measured value was divided by three, and each cage of three pups was considered n = 1. The neurobehavioral tests were performed at two months of age.

4.2.

Ambulatory activity

Rats were subjected to the ambulatory test at 2 months of age; i.e. between PND 58 and 61 (n = 9 per each group, total 36 rats from 10 different liters). On each trial, the rat was placed in the center of the activity chamber (43.2 cm in length, 42.2 cm in

width, and 30.5 cm in height, MED Associates, VT, USA), a transparent acryl chamber was equipped with two horizontal planes of 16 infrared photocell-detector pairs placed in x, y dimension and spaced 2.5 cm apart, and its ambulatory activity was monitored by the computerized system for 60 min. Light condition of the test room was maintained in the same intensity with animal rooms under daylight condition. Ambulatory activity was measured as the total counts of beam interruptions in the horizontal sensor during each consecutive 5-min session. During each ambulation test, rearing (repetitive standing with the forepaws up) and the defecation activity of each rat (number and weight of boluses) were measured as well. The activity chamber was cleaned with 70% ethanol after each use to eliminate any olfactory cues of the previously tested rat.

4.3.

Forced swim test

Three days after the ambulatory activity test, rats were subjected to the forced swim test, according to the method previously described (Porsolt et al., 1977). Each rat was allowed to swim in a glass cylinder (54 cm in height and 24 cm in diameter) filled with water in 40 cm of depth (23–25 °C) for 15 min, as preswim trials, and then removed from the cylinder, dried with paper towel and returned to their home cage. From the next day (PND 64), rats were subjected to 5 min of swim test daily for three consecutive days to include analysis of learned helplessness behavior. All test sessions were recorded by a video camera from the side of the cylinder. Duration of rat's immobility in the water was scored from videotapes by a trained observer who was blinded to the experimental conditions. Immobility was defined as the state in which rats were judged to be making only the movements necessary to keep their head above the surface. Comparisons among the groups were made with the data obtained on the test day 1. After the end session of swim test, rats were allowed to rest in their home cages for 7 days to minimize any effects of previous stress, and then subjected to the elevated plus maze test.

4.4.

Elevated plus maze

After a week of recovery period following the swim test, rats were subjected to the behavioral assessment in an elevated plus maze, a plus shaped acryl maze with two opposite open arms (50 cm in length and 10 cm in width) and two opposite closed arms (50 cm in length, 10 cm in width, and 31 cm in height), extending out from a central platform (10 cm × 10 cm). The whole apparatus was elevated 50 cm above the floor. The test procedure was followed as previously described (Daniels et al., 2004). Each rat was placed in the center of the maze facing one of the open arms, and then allowed to explore the open or closed arms of the maze for 5 min. The time spent in the different arms was recorded, respectively. Four paws had to be inside the entrance line to each arm, which signaled the start of the time spent in the specific arm, and then the end time was recorded when all four paws were outside the line again. The maze was cleaned with 70% ethanol after each test to prevent influences of the previously tested rat.

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Rats were placed in the test room at least 2 h prior to each test to minimize unwanted stress effects, and all behavioral assessments were performed between 9:00 AM and 12:00 PM of the day to avoid the influences of circadian variances.

4 5.

Statistical analysis

Data were analyzed by analysis of variance (ANOVA), using StatView software (Abacus, Berkeley, CA). Body weight and food intake data were analyzed by two-way ANOVA with repeated measures (maternal separation x post-weaning rearing with PND as the repeated measures), and ambulatory activity by three-way ANOVA (maternal separation x postweaning rearing x time points). Rearing, defecation, elevated plus maze and forced swim tests data were analyzed by twoway ANOVA (maternal separation x post-weaning rearing). Significant interactions were followed by one-way ANOVA and preplanned comparisons between groups performed by post hoc Fisher's PLSD test. Significance was set at P < 0.05, and all values were presented as means ± S.E.

Acknowledgments Authors thank Prof. Nori D. Geary for critical comments on manuscript, Dr. S. Lee for helpful comments on behavioral measurements, and Mr. J.Y. Kim for sincere assistance. This work was supported by R&D Grant funded by KHIDI given to JWJ (A080918). First two authors (V.R. and S.B.Y.) contributed equally.

REFERENCES

Aitchison, L.K., Hughes, R.N., 2006. Treatment of adolescent rats with 1-benzylpiperazine: a preliminary study of subsequent behavioral effects. Neurotoxicol. Teratol. 28, 453–458. Albenidou-Farmaki, E., Poulopoulos, A.K., Epivatianos, A., Farmakis, K., Karamouzis, M., Antoniades, D., 2008. Increased anxiety level and high salivary and serum cortisol concentrations in patients with recurrent aphthous stomatitis. Tohoku J. Exp. Med. 214, 291–296. Bickerdike, M.J., Wright, I.K., Marsden, C.A., 1993. Social isolation attenuates rat forebrain 5-HT release induced by KCl stimulation and exposure to a novel environment. Behav. Pharmacol. 4, 231–236. Daniels, W.M., Pietersen, C.Y., Carstens, M.E., Stein, D.J., 2004. Maternal separation in rats lead to anxiety-like behavior and a blunted ACTH response and altered neurotransmitter levels in response to a subsequent stressor. Metab. Brain Dis. 19, 3–14. Fone, K.C.F., Porkess, M.V., 2008. Behavioural and neurochemical effects of post-weaning social isolation in rodents — relevance to developmental neuropsychiatric disorders. Neurosci. Biobehav. Rev. 32, 1087–1102. Furukawa, T.A., Ogura, A., Hirai, T., Fujihara, S., Kitamura, T., Takahashi, K., 1999. Early parental separation experiences among patients with bipolar and major depression: a case-control study. J. Affect. Dis. 52, 85–91. Gentsch, C., Lichtsteiner, M., Feer, H., 1981. Locomotor activity, defecation score and corticosterone levels during an openfield exposure: a comparison among individually and group-housed rats and genetically selected rat lines. Physiol. Behav. 27, 183–186.

133

Gentsch, C., Lichtsteiner, M., Kraeuchi, K., Feer, H., 1982. Different reaction patterns in individually and socially reared rats during exposures to novel environments. Behav. Brain Res. 4, 45–54. Geyer, M.A., Wilkinson, L.S., Humby, T., Robbins, T.W., 1993. Isolation rearing of rats produces a deficit in prepulse inhibition of acoustic startle similar to that in schizophrenia. Biol. Psychiatry 34, 361–372. Ghisleni, G., Kazlauckas, V., Both, F.L., Pagnussat, N., Mioranzza, S., Rocha, J.B.T., Souza, D.O., Porciúncula, L.O., 2008. Diphenyl diselenide exerts anxiolytic-like effect in Wistar rats: putative roles of GABAA and 5HT receptors. Prog. Neuro-sychopharmacol. Biol. Psychiatr. 32, 1508–1515. Gluck, M.E., Geliebter, A., Lorence, M., 2004. Cortisol stress response is positively correlated with central obesity in obese women with binge eating disorder (BED) before and after cognitive-behavioral treatment. Ann. N.Y. Acad. Sci. 1032, 202–207. Goossens, L., Braet, C., Van Vlierberghe, L., Mels, S., 2008. Loss of control over eating in overweight youngsters: the role of anxiety, depression and emotional eating. Eur. Eat. Disord. Rev. doi:10.1002/erv.892. Published on-line. Grilo, C.M., White, M.A., Masheb, R.M., 2008. DSM-IV psychiatric disorder comorbidity and its correlates in binge eating disorder. Int. J. Eat. Disord. doi:10.1002/eat.20599. Published on-line. Hall, F.S., 1998. Social deprivation of neonatal, adolescent, and adult rats has distinct neurochemical and behavioral consequences. Crit. Rev. Neurobiol. 12, 129–162. Hall, F.S., Huang, S., Fong, G.F., Pert, A., 1998. The effects of social isolation on the forced swim test in Fawn hooded and Wistar rats. J. Neurosci. Methods 79, 47–51. Hall, F.S., Sundstrom, J.M., Lerner, J., Perth, A., 2001. Enhanced corticosterone release after amodified forced swim test in Fawn hooded rats is independent of rearing experience. Pharmacol. Biochem. Behav. 69, 629–634. Harlow, H.F., Harlow, M.K., 1965. The effect of rearing conditions on behavior. Int. J. Psychiatry 1, 43–51. Harris, R.J., 1995. Where is the child's environment? A group socialization theory of development. Psychol. Rev. 102, 458–489. Heim, C., Newport, D.J., Heit, S., Graham, Y.P., Wilcox, M., Bonsall, R., et al., 2000. Pituitary-adrenal and autonomic responses to stress in women after sexual and physical abuse in childhood. JAMA 284, 593–597. Heim, C., Newport, D.J., Bonsall, R., Miller, A.H., Nemeroff, C.B., 2001. Altered pituitary-adrenal axis responses to provocative challenge tests in adult survivors of childhood abuse. Am. J. Psychiatry 158, 575–581. Heim, C., Plotsky, P.M., Nemeroff, C.B., 2004. Importance of studying the contributions of early adverse experience to neurobiological findings in depression. Neuropsychopharmacology 29, 641–648. Hellemans, K.G., Benge, L.C., Olmstead, M.C., 2004. Adolescent enrichment partially reverses the social isolation syndrome. Dev. Brain Res. 150, 103–115. Hilakivi, L.A., Ota, M., Lister, R.G., 1989. Effect of isolation on brain monoamines and the behavior of mice in tests of exploration, locomotion, anxiety and behavioral despair. Pharmacol. Biochem. Behav. 33, 371–374. Iwasaki, S., Inoue, K., Kriike, N., Hikiji, K., 2000. Effect of maternal separation on feeding behavior of rats in later life. Physiol. Behav. 70, 551–556. Javaras, K.N., Pope, H.G., Lalonde, J.K., Roberts, J.L., Nillni, Y.I., Laird, N.M., et al., 2008. Co-occurrence of binge eating disorder with psychiatric and mental disorders. J. Clin. Psychiatry 69, 266–273. Kalinichev, M., Easterling, K.W., Plotsky, P.M., Holtzman, S.G., 2002. Long-lasting changes in stress-induced corticosterone response and anxiety-like behaviors as a consequence of

134

BR A I N R ES E A RC H 1 2 9 5 ( 2 00 9 ) 1 2 7 –13 4

neonatal maternal separation in Long-Evans rats. Pharm. Biochem. Behav. 73, 131–140. Kendler, K.S., Neale, M.C., Kessler, R.C., Heath, A.C., Eaves, L.J., 1992. Childhood parental loss and adult psychopathology in women. A twin study perspective. Arch. Gen. Psychiatry 49, 109–116. Khoury, A.E., Gruber, S.H.M., Mork, A., Mathe, A.A., 2006. Adult life behavioral consequences of early maternal separation are alleviated by escitalopram treatment in a rat model of depression. Prog. Neuro-Psychopharmacol. Biol. Psychiatr. 30, 535–540. Kim, H.J., Lee, J.H., Choi, S.H., Lee, Y.S., Jahng, J.W., 2005a. Fasting-induced increases of arcuate NPY mRNA and plasma corticosterone are blunted in the rat experienced neonatal maternal separation. Neuropeptides 39, 587–594. Kim, H.J., Yoo, S.B., Ryu, V., Kim, G.T., Jahng, J.W., 2005b. The hypothalamic expression of corticotropin-releasing hormone by stress is blunted in the rat experienced maternal separation during neonatal period. Soc. Neurosci. Abstr. 527.10. Koo-Loeb, J.H., Costello, N., Light, K.C., Girdler, S.S., 2000. Women with eating disorder tendencies display altered cardiovascular, neuroendocrine, and physiological profiles. Psychosom. Med. 62, 539–548. Ladd, C.O., Owens, M.J., Nemeroff, C.B., 1996. Persistant changes in corticotropin-releasing factor neuronal systems induced by maternal deprivation. Endocrinology 137, 1212–1218. Ladd, C.O., Huot, R.L., Thrivikraman, K.V., Nemeroff, C.B., Meaney, M.J., Plotsky, P.M., 2000. Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog. Brain Res. 122, 81–103. LaGreca, A.M., Prinstein, M.J., Fetter, M.D., 2001. Adolescent peer crow affiliation: linkages with health-risk behaviors and close friendships. J. Pediatric Psychol. 26, 131–143. Lee, J.H., Kim, H.J., Kim, J.G., Ryu, V., Kim, B.T., Kang, D.W., Jahng, J.W., 2007. Depressive behaviors and decreased expression of serotonin reuptake transporter in rats that experienced neonatal maternal separation. Neurosci. Res. 58, 32–39. Martín-García, E., Pallarès, M., 2005. Intrahippocampal nicotine and neurosteroids effects on the anxiety-like behaviour in voluntary and chronic alcohol-drinking rats. Behav. Brain Res. 164, 117–127. McIntosh, J., Animan, H., Merali, Z., 1999. Short- and long periods of neonatal maternal separation differentially affect anxiety and feeding in adult rats: gender-dependent effects. Brain Res. Dev. Brain Res. 113, 97–106. Noh, S.J., Ryu, V., Yoo, S.B., Lee, J.H., Min, B.M., Jahng, J.W., 2008. Suppressed intake of highly palatable food and dysfunction of the HPA axis activity responding to restraint stress in

adolescent rats that experienced neonatal maternal separation. Appetite 51, 388. Porsolt, R.D., Le Pichon, M., Jalfre, M., 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266, 730–732. Putignano, P., Dubini, A., Toja, P., Invitti, C., Bonfanti, S., Redaelli, G., Zappulli, D., Cavagnini, F., 2001. Salivary cortisol measurement in normal-weight, obese and anorexic women: comparison with plasma cortisol. Eur. J. Endocrinol. 145, 165–171. Rapoport, J.L., Addington, A.M., Frangou, S., Psych, M., 2005. The neurodevelopmental model of schizophrenia: update 2005. Mol. Psychiatry 10, 434–449. Robbins, T.W., Jones, G.H., Wilkinson, L.S., 1996. Behavioural and neurochemical effects of early social deprivation in the rat. J. Psychopharmacol. 10, 39–47. Ryu, V., Lee, J.H., Yoo, S.B., Gu, X.F., Moon, Y.W., Jahng, J.W., 2008. Sustained hyperphagia in adolescent rats that experienced neonatal maternal separation. Int. J. Obes. 32, 1355–1362. Serra, M., Pisu, M.G., Floris, I., Biggio, G., 2005. Social isolation-induced changes in the hypothalamic-pituitary-adrenal axis in the rat. Stress 8, 259–264. Spear, L.P., 2000. The adolescent brain and age-related behavioral manifestations. Neurosci. Biobehav. Rev. 24, 417–463. Van Oers, H.J., de Kloet, E.R., Levins, S., 1998. Early vs. late maternal deprivation differentially alters the endocrine and hypothalamic responses to stress. Brain Res. Dev. Brain Res. 111, 245–252. Vazquez, D.M., Lopez, J.F., van Hoers, H., Watson, S.J., Levin, S., 2000. Maternal deprivation regulates serotonin 1A and 2A receptors in the infant rat. Brain Res. 855, 76–82. Weiss, I.C., Di Iorio, L., Feldon, J., Domeney, A.M., 2000. Strain differences in the isolation-induced effects on prepulse inhibition of the acoustic startle response and on locomotor activity. Behav. Neurosci. 114, 364–373. Weiss, I.C., Domeney, A.M., Moreau, J.-L., Russig, H., Feldon, J., 2001. Dissociation between the effects of pre-weaning and/or post-weaning social isolation on prepulse inhibition and latent inhibition in adult Sprague–Dawley rats. Behav. Brain Res. 121, 207–218. Weiss, I.C., Pryce, C.R., Jongen-Relo, A.L., Nanz-Bahr, N.I., Feldon, J., 2004. Effect of social isolation on stress-induced behavioral and neuroendocrine state in the rat. Behav. Brain Res. 152, 279–295. Wright, I.K., Upton, N., Marsden, C.A., 1991. Resocialisation of isolation-reared rats does not alter their anxiogenic profile on the elevated X-maze model of anxiety. Physiol. Behav. 50, 1129–1132.