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Interaction between isolation rearing and social development on exploratory behavior in male rats
Behavioural Processes 70 (2005) 223–234
Interaction between isolation rearing and social development on exploratory behavior in male rats Hiroyuki Arakawa ∗ Department of Psychology, Graduate School of Letters, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Received 13 January 2005; received in revised form 10 July 2005; accepted 10 July 2005
Abstract The effect of isolation on exploratory behavior has been shown to differ depending on the developmental stages of male rats. However, there has been little systematic comparison of the frequencies and the patterns of exploratory behavior across the developmental stages. The present study assessed the frequencies of exploration using the emergence test and exploratory patterns in the open-field test in three developmental stages of male rats: juvenile, post-puberty, and adult. A lower propensity for exploration was observed in rats isolated during the juvenile stage, as assessed by increased latency and decreased duration of exploratory behaviors compared to pair-reared rats, and this tendency was maintained in adulthood. Altered patterns of exploratory behavior were demonstrated both in rats isolated in adulthood, who showed an increased active pattern, and those pair-reared following puberty, who shifted to a more passive pattern. However, rats isolated during the juvenile stage did not change their exploratory patterns following puberty. These results suggest that the changes in the exploratory pattern, which can be observed in adulthood, are associated with the emergence of adult-like dominance relationships. Juvenile-isolated rats did not show these changes following puberty, suggesting the importance of social interaction as juveniles for the ontogenetic emergence of behavioral flexibility implicated in the regulation of exploratory patterns. © 2005 Elsevier B.V. All rights reserved. Keywords: Isolation rearing; Exploratory behavior; Social development; Anxiety; Rats
1. Introduction Isolation rearing of animals after weaning is considered to be one model of social stress (Blanchard et al., 1998; Palanza, 2001; Sutanto and de Kloet, 1994). Animals reared in isolation have been found ∗ Present address: School of Psychology, Chukyo University, 1012 Yagoto-Honmachi, Showa-ku, Nagoya 466-8666, Japan. Tel.: +81 52 789 2263; fax: +81 52 835 7144. E-mail address: [email protected].
0376-6357/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2005.07.002
to demonstrate a variety of behavioral and physiological changes in comparison to those reared in groups (Hall, 1998; Palanza, 2001). A previous study by the author (Arakawa, 2003) suggested that one particularly important factor is the age of the subjects during isolation. Specifically, that study demonstrated that rats isolated during the juvenile stage demonstrated diminished exploratory behavior, as indicated by a decrease in duration of stay at the center area of an open-field, compared to rats reared in pairs. In contrast, rats isolated during the pubertal stage showed no reduction in
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exploratory behavior, while rats isolated in adulthood demonstrated increased exploratory behavior. One possible explanation of these findings is that developmental changes in social behavior of rats are responsible for this age-dependent effect of isolation (Arakawa, 2002, 2003; Von Frijtag et al., 2002). For rats, the frequency of social contacts changes with age (Pellis and Pellis, 1998a). As such, the effect of isolation, in which rats are deprived of social contacts with conspecifics (Hall, 1998; Holloway and Suter, 2004), would also be expected to differ as a function of the age of the animals at the time of isolation. However, another possibility is plausible. Based on the analysis of individual differences in behavioral patterns associated with social stress, Blanchard et al. (1998, 2001) found that rats exhibited two patterns of exploratory behavior in a novel environment: an active pattern in which rats approach a novel stimulus, indicated by the amount of locomotion or the duration of stay at the center area of an open-field, and a passive pattern (risk assessment behavior) in which rats participate in behaviors that allow the gathering of information about potential threats, including behaviors such as sniffing or stretching towards the phobic area without ambulation (see also Arakawa, 2005; Haller and Hal´asz, 1999). Thus it is also possible to attribute the age-dependent effect of isolation not to the developmental changes in the frequency of social activity that affect exploratory behavior, but to the characteristics of exploratory behavior that are affected by isolation. However, a simple open-field composed of a dimly lit undifferentiated square floor is not a suitable apparatus for determining whether isolation-reared rats exhibit changes in the frequency or pattern of exploratory behavior. For example, it is often difficult to determine whether the responses of animals staying motionlessly in a corner of the field reflect a decrease in activity or an increase in anxiety. In other words, it is unavoidable that in such testing conditions a behavioral index might reflect a number of factors such as activity level, anxiety level, and exploratory patterns (De Passill´e et al., 1995; Ramos and Morm`ede, 1998; Rushen, 2000). In order to further elucidate the exploratory behavior of rats in a novel environment, it is necessary to use a test apparatus that includes a less phobic area into which animals can escape freely from a more phobic area, and to observe both the level
and the pattern of approach behavior to the phobic area (Blanchard et al., 1998; Fujita et al., 1994). The present study, which investigated whether the age-dependent effect of isolation found in the previous study (Arakawa, 2003) was ascribed to changes in the frequency or in the pattern of exploratory behavior, addressed these issues by using two types of apparatus and an appropriate procedure designed to minimize such confounds. Specifically, an emergence test was conducted in order to measure the level of anxiety. In this test, rats were first placed in a small and dimly lit box for several minutes and then were allowed to explore a phobic area (i.e. a large and bright area) outside it. Rats show several exploratory behaviors including active and passive patterns for searching a phobic area. Then the indices of anxiety included latency and duration spent to search the phobic area (Crawley and Goodwin, 1980; Einon and Morgan, 1977; Pijlman et al., 2002). Patterns of exploratory behavior were measured using an open-field apparatus that was divided by an inner wall into a brightly lit center square and a dimly lit peripheral alley. Exploratory patterns of rats were identified according to the type of approach behavior to the center square (Ossenkopp et al., 1994; Roth and Katz, 1979; Seliger, 1977). Both the number of area entries (representing the active pattern of exploratory behavior) and the number of stretch-attend postures for the center area (representing the passive pattern of exploratory behavior) were counted. The ratio of the number of stretch-attend postures to the total number of exploratory behaviors was defined as the risk assessment ratio. The frequency of play fighting in rats peaks during juvenile stage, then declines with the onset of puberty (Pellis and Pellis, 1990, 1991; Takahashi and Lore, 1983; Thor and Holloway, 1984). Aggressive fighting with conspecifics increases gradually with puberty, and peaks in adult rats after the age of 100 days (Pellis and Pellis, 1987; Takahashi and Lore, 1982). Thus, male rats exhibit a transition between these social behaviors at puberty (Matuszcayk et al., 1994; Meaney and Stewart, 1981; S¨odersten et al., 1977). The animals in the present experiments were tested at 40, 65, or 130 days of age (Arakawa, 2002, 2003), which represent the juvenile, post-pubertal and adult stages in which they were isolated, respectively. The period of isolation for the different developmental stages tested was the 14
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days that immediately preceded the corresponding test age (Arakawa, 2002).
2. Experiment 1 Experiment 1 examined the effect of isolation rearing on exploratory behavior of rats in the three developmental stages. The emergence test and the open-field test were conducted by a two-way factorial design with rearing condition (isolation or pair) and age (40, 65, or 130 days) as the between-subjects factors. 2.1. Materials and methods 2.1.1. Subjects and rearing conditions Forty-eight male Wistar rats, bred and reared in the colony room of the Department of Psychology at Nagoya University, were used as subjects. They were obtained from 12 Wistar dams (litter size = 4–12), and were undisturbed and housed until weaning with the parent and siblings in standard opaque plastic cages (36 cm × 30 cm × 18 cm, l × w × h) with pine shavings as bedding material. Immediately after weaning at the age of 23 days, male rats were housed in pairs with same-sex littermates. At the age of 26 days, they were randomly divided into one of six groups (N = 8 for each group). Then each animal obtained from a litter of the Wistar dams were assigned into other groups as possible. Of these, three groups of rats were in the isolated rearing condition (Groups IS) and each group was tested after a 14-day isolation period. Thus, rats tested at 40 days old (IS-40) were isolated at 26 days, rats tested at 65 days old (IS-65) were isolated at 51 days, and rats tested at 130 days old (IS-130) were isolated at 116 days, respectively. The other three groups of animals were in the paired rearing condition (Groups PS-40, PS-65, and PS-130), and were reared in pairs with a littermate throughout the experiment. All subjects were reared in opaque plastic cages (36 cm × 30 cm × 18 cm, l × w × h) with lids of stainless steel wire grid. The floors of the cage were covered with pine shavings, which was changed every week. The colony room was illuminated on a 12:12 light–dark cycle with light from 07:00 to 19:00 h. The temperature and humidity were maintained at 23 ± 1 ◦ C and 60%, respectively. Subjects were allowed access to food and water freely throughout the experiment. All animal pro-
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tocols were in accordance with the Nagoya University guidelines for the use of animals. 2.1.2. Apparatus 2.1.2.1. Emergence test. This test apparatus was composed of a simple open-field (90 cm × 90 cm × 40 cm, l × w × h), in which a gray plastic start box (20 cm × 20 cm × 20 cm, l × w × h) with a door (10 cm × 10 cm), was placed in the corner. The open-field was made of opaque gray plastic with the floor divided by black lines into 81 equal squares (10 cm × 10 cm). Four 40 W reflector lamps suspended above the open-field illuminated the floor with a luminance of 250 lx and the start box with a luminance of 10 lx. A video camera was placed at 90 cm above the field in order to record the rats behavior. 2.1.2.2. Open-field test. This test used the open-field which was used in the emergence test without the start box. In this test, an inner wall (50 cm × 50 cm × 30 cm, l × w × h) separated the field into a peripheral area (a gallery with 20 cm width) and a center area (a 50 cm × 50 cm squares). Subjects could pass through these two areas freely by any of four openings (15 cm × 15 cm) located in each corner of the inner wall. The floor was illuminated with a luminance of 90 lx in the center area, and 15 lx in the peripheral area. 2.1.3. Procedure The tests were conducted during 20:00–24:00 in the dark phase of the daily light–dark cycle. On the first day of testing, the emergence test was conducted. Twenty minute before the start of the test, the subject with its home cage was moved from the colony room to the experiment room. At the beginning of the test, each animal was placed in the start box with the door closed. After 5 min passed, the door was opened and the rat was allowed to explore beyond the start box for 3 min. At the end of the test, the animal was returned to its home cage and the test apparatus was thoroughly cleaned with 70% ethanol solution. On the second day, the open-field test was conducted. Twenty minute before the start of the test, the subject with its home cage was transferred from the colony room to the experiment room. At the beginning of the 20 min test, the subject was placed in a corner of the peripheral area in the open-field. After the test trial was completed, the animal was returned to its home
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cage and the open-field was thoroughly cleaned with 70% ethanol solution. Behaviors of the subjects were videotaped and analyzed. An experimenter was blind to the condition of the animal videotaped in each test. In the emergence test, the latency that each animal first put its head outside the box and total duration of all bouts that each animal put its head outside the box added together were measured as behavioral indices. In the open-field test, the number of times that the subject’s entire body came into the center area was counted as entries. The number of times that the rat’s head, but not its entire body, entered the center area with stretching the body of the animal was also recorded as stretch-attend postures. The percentage of stretch-attend postures to the total number of exploratory behaviors was then calculated as the risk assessment ratio. 2.1.4. Statistics All the behavioral data were analyzed by two-way analyses of variance with rearing condition (IS or PS) and age (40, 65, or 130 days) as the between subjects factors. For multiple post hoc comparisons, the Fisher’s least significant difference test was used. A probability level of p < .05 was used as the statistical significance for all the tests. 2.2. Results 2.2.1. Emergence test Fig. 1a shows the mean latency of exploring for each group. A two-way ANOVA revealed significant main effects of rearing condition, F(1,42) = 5.33, p < .05, and age, F(1,42) = 7.08, p < .01. The interaction between rearing condition and age was significant, F(2,42) = 4.96, p < .05. Then, the latency was greater in Group IS-40 than in Group PS-40 (p < .01). No significant effects of rearing condition were found for animals tested at either 65 days or 130 days of age. The latency was longer in Group IS-40 than in either Groups IS-65 or IS-130 (p < .01). For the animals reared in pairs, no significant group difference was found. Fig. 1b represents the mean of total duration of exploring for each group. A two-way ANOVA revealed a significant main effects of age, F(1,42) = 27.19, p < .01, but not of rearing condition. There was a significant interaction between rearing condition and age, F(2,42) = 9.54, p < .01. Group IS-40 showed a
Fig. 1. Results of emergence test during 180 s in rats aged 40, 65, and 130 days, reared in isolation (IS) or pairs (PS) in Experiment 1. Data are expressed as mean ± S.E.M. Mean latency (a) and duration (b) that each animal put its head outside the start box during the test session. * Significant difference between rearing conditions, p < .05. # Significant difference between different age groups, p < .05.
lower total duration of exploration than Group PS-40 (p < .01), while no differences between rearing conditions were found for the animals tested at either 65 days or 130 days of age. The duration of exploration was less in Group IS-40 than in either Group IS-65 or Group IS-130, and the duration in Group IS-65 intermediated between Groups IS-40 and IS-130 (p < .01). For the animals reared in pairs, it was less in Group
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PS-40 than in Group PS-130 (p < .05). No other significant group differences were found. 2.2.2. Open-field test Fig. 2a reveals the mean number of entries in the center area for each group. A two-way ANOVA conducted on these scores found a significant main effect of age,
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F(2,42) = 6.43, p < .01, but not of rearing condition. The interaction between rearing condition and age was significant, F(2,42) = 8.67, p < .01. Group IS-40 had fewer entries than Group PS-40 (p < .05), whereas no differences were found between Groups IS-65 and PS-65. On the other hand, Group IS-130 had more entries than Group PS-130 (p < .01). The number of entries was less
Fig. 2. Results of open-field test during 20 min in rats aged 41, 66, and 131 days, reared in isolation (IS) or pairs (PS) in Experiment 1. Data are expressed as mean ± S.E.M. Mean number of entries (a) and stretch-attend postures (b) into the center area of an open-field, and percentage of risk assessment (c) that was calculated as the ratio of stretch-attend postures to total number of exploratory behaviors including entries and stretch-attend postures. * Significant difference between rearing conditions, p < .05. # Significant difference between different age groups, p < .05.
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in Group PS-130 than in either Group PS-40 or Group PS-65 (p < .01). Fig. 2b depicts the mean number of stretchattend postures for each group. A two-way ANOVA found a significant main effect of rearing condition, F(1,42) = 53.78, p < .01, but not of age. The interaction between rearing condition and age was significant, F(2,42) = 5.77, p < .01. Group IS-40 had fewer stretchattend postures than Group PS-40 (p < .01). Stretchattend postures was also less frequent in Group IS-130 compared to Group PS-130 (p < .01). On the other hand, no difference was found between Group IS-65 and Group PS-65. The number of stretch-attend posture events was greater in Group IS-65 than in either Group IS-40 or Group IS-130 (p < .01). The mean ratio of risk assessment for each group is shown in Fig. 2c. A two-way ANOVA revealed a significant main effect of rearing condition, F(1,42) = 19.66, p < .01, but not of age. The significant interaction between rearing condition and age was found, F(2,42) = 11.51, p < .01. The ratio was less in Group IS-130 than in Group PS-130 (p < .01), but no effects were found in the animals tested at either 40 days or 65 days of age. Group IS-130 had a lower ratio than Group IS-65 (p < .05), while the ratio was greater in Group PS-130 than in either Group PS-40 or Group PS-65 (p < .01).
3. Experiment 2 In Experiment 1, rats that were isolated during their juvenile stage demonstrated reduced exploratory behavior compared to rats reared in pairs. Several studies have found that the effect of isolation during the juvenile stage remains for a long time, even when animals are thereafter returned to being reared in pairs (Arakawa, 2002; Ikemoto and Panksepp, 1992; Einon and Potegal, 1991; Van den Berg et al., 1999a). In Experiment 2, the consequences of isolating juveniles on their exploratory behavior after puberty were investigated, in order to determine if the effect of isolation persists after puberty. 3.1. Materials and methods The methods of Experiment 2 mirrored that of Experiment 1, except for the age of the subjects in
the isolated groups and their rearing conditions as described below. 3.1.1. Subjects and rearing conditions Thirty-two Wistar male rats were used as the subjects. After weaning at the age of 23 days, all the animals were housed in pairs with a male littermate (litter size = 5–12). At the age of 26 days, they were reared in either isolation or pairs for 14 days. After this period, all the animals were reared in pairs with a littermate again, and were either assigned to one of four groups (N = 8 for each group): isolated rats tested at the age of 65 days (Group JI-65), pair-reared rats tested at the age of 65 days (Group PS-65), isolated rats tested at the age of 130 days (Group JI-130), and pair-reared rats tested at the age of 130 days (Group PS-130). The same tests as those used in the previous experiment were used. All the behavioral indices were analyzed by a two-way ANOVA with rearing condition (JI or PS) and age at the test (65 or 130 days) as betweensubjects factors. 3.2. Results 3.2.1. Emergence test Fig. 3a depicts the mean latency of exploring for each group. A two-way ANOVA demonstrated a significant main effect of rearing condition, F(1,28) = 33.24, p < .01, while the main effect of age and the interaction between these two factors were not significant. Thus Group JI-65 showed longer latency than Group PS-65. Similarly, Group JI-130 revealed longer latency than Group PS-130. Fig. 3b shows the mean total duration of exploring for each group. A two-way ANOVA showed that there was a significant main effect of rearing condition, F(1,28) = 19.09, p < .01, however the main effect of age and the interaction between these two factors were not significant. Thus isolated rats (Groups JI-65 and JI-130) exhibited lower durations of exploring than pair-reared rats (Groups PS-65 and PS-130, respectively), regardless of their ages at testing. 3.2.2. Open-field test Fig. 4a represents the mean number of entries in the center area for each group. A two-way ANOVA conducted on these scores revealed no significant main effect of rearing condition, but a significant main
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JI-65 and PS-65) showed more entries than rats aged 130 days (Groups JI-130 and PS-130, respectively), regardless of their rearing conditions. Fig. 4b shows the mean number of stretch-attend posture events for each group. A two-way ANOVA exhibited a significant main effect of rearing condition, F(1,28) = 47.56, p < .01, but not of age. The interaction between rearing condition and age was significant, F(1,28) = 15.08, p < .01. Rats in Group JI-65 and Group JI-130 had less stretch-attend postures than rats in Group PS-65 (p < .05) and Group PS-130 (p < .01), respectively. In addition, rats in Group JI-65 showed more stretch-attend behavior than those in Group JI130 (p < .05), whereas rats in Group PS-65 exhibited significantly lower stretch-attend posture events than those in Group PS-130 (p < .01). The mean ratio of risk assessment for each group is depicted in Fig. 4c. A two-way ANOVA demonstrated significant main effects of rearing condition, F(1,28) = 6.94, p < .05, and age, F(1,28) = 7.20, p < .05. The interaction between rearing condition and age was significant, F(1,28) = 5.00, p < .05. Group JI-65 did not differ from Group PS-65, while Group JI-130 showed a lower ratio than Group PS-130 (p < .01). In addition, Group PS-130 exhibited greater ratio than Group PS65 (p < .01), while Group JI-130 did not differ from Group JI-65.
4. General discussion
Fig. 3. Results of emergence test during 180 s in rats aged 65 and 130 days, reared in isolation during the juvenile stage (JI) or pairs (PS) in Experiment 2. Data are expressed as mean ± S.E.M. Mean latency (a) and duration (b) that each animal put its head outside the start box during the test session. * Significant difference between rearing conditions, p < .05. # Indicates a significant difference between different age groups, p < .05.
effect of age, F(1,28) = 10.42, p < .01. The interaction between rearing condition and age was not significant. Isolated animals entered the center area to the same extent as pair-reared animals, at the ages of both 65 and 130 days. In addition, rats aged 65 days (Groups
In the present study, rats isolated during the juvenile stage displayed less exploratory behavior than non-isolated rats, whereas rats isolated during the post-pubertal stage demonstrated no such decrease in the emergence test (Experiment 1). Furthermore, the decrease in exploratory behavior of rats reared in isolation during the juvenile stage was also observed in adult rats, even when they were returned to pair-rearing after isolation (Experiment 2). Thus it appears that the development of exploratory behavior as a response to a novel environment has an irreversible critical period during the juvenile stage (Einon and Morgan, 1977). Several studies have reported that rats are remarkably sensitive to isolation rearing in the juvenile stage. For example, isolation during this stage has been associated with a decrease in the duration of staying in the open arms of an elevated plus maze (Wright et
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Fig. 4. Results of open-field test during 20 min in rats aged 41, 66, and 131 days, reared in isolation during the juvenile stage (JI) or pairs (PS) in Experiment 2. Data are expressed as mean ± S.E.M. Mean number of entries (a) and stretch-attend postures (b) into the center area of an open-field, and the percentage of risk assessment (c) that was calculated as the ratio of stretch-attend postures to total number of exploratory behaviors including entries and stretch-attend postures. *Significant difference between rearing conditions, p < .05. # Significant difference between different age groups, p < .05.
al., 1991), a lower frequency of social behavior with conspesifics (Van den Berg et al., 1999a), and a longer latency before entry into a phobic field from a narrow tube (Einon and Morgan, 1977). In contrast, isolation after puberty does not elicit these changes (Einon and Morgan, 1977; Van den Berg et al., 1999a; Wright et al., 1991). These findings also suggest that rats demon-
strate less exploratory behavior due to isolation during the juvenile stage. Rats reared in isolation during the juvenile stage displayed a reduced threshold for jumping responses to foot shocks (Arakawa, 2002) and showed enhanced defensiveness toward conspecifics (Einon and Potegal, 1991), suggesting that they became more sensitive to aversive stimuli than pair-reared rats.
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In addition, the phobic aspects of exploratory behavior in isolation-reared rats can be attenuated by the injection of anxiolytics (Da Silva et al., 1996). Thus, it is proposed that animals isolated during the juvenile stage did not become less reactive to stimuli, but rather displayed more phobic responses to a novel environment accompanied by their higher level of anxiety as compared to group-reared animals. In the current study, isolation during adulthood did not affect the level of exploratory behavior. Rather, adult rats reared in isolation (IS-130) demonstrated a shift in the exploratory behavior to a more active pattern, as displayed by more entries and less stretchattend postures into the center area of the openfield than pair-reared rats (PS-130). Furthermore, pair-reared rats were less likely to perform active exploratory behaviors in adulthood (PS-130), as they performed fewer entries into the center area of the open-field than those before puberty (PS-40 and PS65) (Experiment 1). After puberty, male rodents often establish a variety of social relationships involving social hierarchies and dominance relationships (Berdoy et al., 1995; Lore and Stipo-Flaherty, 1984). It has been found that differentiation of dominant and subordinate animals in these relationships associate with individual differences in patterns of exploratory behavior in both adult rats and mice (De Vries et al., 2003; Koolhaas et al., 1999; Von Holst, 1998). When exposed to a novel environment, subordinate male rats displayed less active exploratory behavior than did dominant males. When housed together before puberty, however, post-pubertal male rats and mice formed mild dominance relationships without overt victory and defeat (Lore and Stipo-Flaherty, 1984), and no differences were found in exploratory behavior between dominants and subordinates (Bartolomucci et al., 2001). Similarly in the present experiments, pair-reared adult rats were likely to form non-aggressive mild dominance relationships in adulthood. Group formation with adult males can also be considered as a factor affecting exploratory behavior (Arakawa, 2005; Bartolomucci et al., 2004). For example, both dominant and subordinate rats demonstrated less active exploratory behavior than adult rats reared with adult females, who do not develop male-like dominance relationships (Blanchard et al., 1998, 2001; Williams and Lierle, 1988). Furthermore, even rats reared in pairs with littermates showed
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a lower frequency of active exploratory behavior than adult rats housed with a female (Haller and Hal´asz, 2000). These studies suggest that adult males who have established social relationships by being reared with other adult males show a decrease in the active pattern of exploratory behavior accompanied by an emergence of social tension among them, regardless of whether they become dominants or subordinates (Arakawa, 2005; Palanza et al., 2001; Sachser and Lick, 1991). In other words, isolation during adulthood can be considered to be deprivation of such dominance relationships (Palanza, 2001; Von Frijtag et al., 2002), which is responsible for the increased active pattern of exploration in rats isolated during adulthood. In contrast, rats that were isolated during the juvenile stage did not demonstrate a change in exploratory pattern after puberty (Experiment 2). This suggests that the increase in passive exploratory behavior that rats typically display due to social contact with an adult cage mate was inhibited through deprivation of social contacts during the juvenile stage. It has been reported that rats isolated during the juvenile stage exhibit a decrease in social activity as demonstrated by less social contacts and anogenital sniffing than that shown by group-reared rats, even when they were returned to group-rearing after puberty (Hol et al., 1999; Ikemoto and Panksepp, 1992; Van den Berg et al., 1999b). In studies on colony formation, adult rats that were reared in isolation during the juvenile stage displayed less submissive postures and suffered from more serious physical injuries than rats reared in groups (e.g., Lore and Flannelly, 1977; Luciano and Lore, 1975; Van den Berg et al., 1999a). Based on the hypothesis that the change in adult rats’ exploratory patterns in a novel environment arises from an adaptive response to social situations in adulthood, the present findings, that rats isolated during the juvenile stage showed no change in exploratory patterns as adult, can probably be ascribed to an irreversible effect of the lack of social experience in this period (see also Von Frijtag et al., 2002). It has been demonstrated that rats display social play behavior most frequently in the juvenile stage (Pellis and Pellis, 1990, 1991; Takahashi and Lore, 1983; Thor and Holloway, 1984). Several researchers indicate that the experience of play behavior during the juvenile stage is important for the regulation of social stress responses and the generation of appropriate responses to changing social circumstances in adulthood. Furthermore, these
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adaptive behaviors are not acquirable through social contacts with conspecifics in the adult stage (Pellis and Pellis, 1998b; Van den Berg et al., 1999a). However, it should be noted that rats isolated during the juvenile stage showed less passive exploratory behavior even at the age of 65 days, in which pair-reared rats did not exhibit a shift in exploratory patterns. There are several explanations for this finding. It has been found that rats display changes in exploratory pattern depending on intensity of the stimuli to which they are exposed (Albonetti and Farabollini, 1994; Blanchard et al., 2001). That is, the exploratory pattern in rats shifts to a more passive pattern with increasing the intensity of aversive stimuli. To the presence of intensely threatening stimuli, rats show a marked decrease in exploratory behavior. Thus, it appears that rats isolated during the juvenile stage decreased exploration per se on account of their higher anxiety due to hypersensitivity to stimuli or lack of social experience. In addition, since the same size open-field was used for all ages in the present experiments, the magnitude of the threat for a juvenile compared to an adult may be different because of their different body sizes. Thus, it is possible that the open-field area is a relatively more intense stimulus for a juvenile than for an adult, and so this may affect them differently, leading to some of the age-related differences found in the present studies. However, another explanation is also plausible. Social interactions have been shown to be rewarding for male juvenile rats (Douglas et al., 2004), which is likely to facilitate the neural maturation of mesolimbic dopamine systems during puberty (Andersen et al., 1997; Vanderschuren et al., 1997). This may modulate the rewarding properties of novelty and drugs (Douglas et al., 2004; Spear, 2000). This neural maturation of dopamine systems may contribute to the emergence of the age-specific behavior in prepubertal male rats, who show marked propensities for risk-taking, noveltyseeking, and active exploration (Bardo et al., 1996; Palanza, 2001; Spear, 2000), which has been called a behavioral approach system (Palanza, 2001). Based on this hypothesis, juvenile isolation may inhibit the ontogenetic maturation of this system, which may be responsible for the inhibition of exploratory behavior in rats isolated during the juvenile stage. Indeed, basal dopamine synthesis in the nucleus accumbens is lower in juvenile rats than in adults (Andersen et al., 1997), and isolated male mice also exhibit reduced
mesoaccumbens dopamine responses to restraint stress compared to a group reared with conspecifics (Cabib et al., 2002). Taken together, the results of the present study demonstrate that isolation as juveniles reduced the exploratory tendency, while isolation in adulthood changed the exploratory patterns. These findings suggest that exploratory patterns in response to a novel environment emerge through the experience of social interaction with other rats during the juvenile stage and are modified by the social conditions in adulthood (see also De Vries et al., 2003; Sachser et al., 1998). Further experiments are needed to understand the determinants of the ontogenetic emergence of the exploratory pattern as well as the underlying neural mechanisms.
Acknowledgements The author would thank to Professor Kiyoshi Ishii for his critical reading to this article, and two anonymous reviewers for their helpful comments.
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Interaction between isolation rearing and social development on exploratory behavior in male rats Hiroyuki Arakawa ∗ Department of Psychology, Graduate School of Letters, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Received 13 January 2005; received in revised form 10 July 2005; accepted 10 July 2005
Abstract The effect of isolation on exploratory behavior has been shown to differ depending on the developmental stages of male rats. However, there has been little systematic comparison of the frequencies and the patterns of exploratory behavior across the developmental stages. The present study assessed the frequencies of exploration using the emergence test and exploratory patterns in the open-field test in three developmental stages of male rats: juvenile, post-puberty, and adult. A lower propensity for exploration was observed in rats isolated during the juvenile stage, as assessed by increased latency and decreased duration of exploratory behaviors compared to pair-reared rats, and this tendency was maintained in adulthood. Altered patterns of exploratory behavior were demonstrated both in rats isolated in adulthood, who showed an increased active pattern, and those pair-reared following puberty, who shifted to a more passive pattern. However, rats isolated during the juvenile stage did not change their exploratory patterns following puberty. These results suggest that the changes in the exploratory pattern, which can be observed in adulthood, are associated with the emergence of adult-like dominance relationships. Juvenile-isolated rats did not show these changes following puberty, suggesting the importance of social interaction as juveniles for the ontogenetic emergence of behavioral flexibility implicated in the regulation of exploratory patterns. © 2005 Elsevier B.V. All rights reserved. Keywords: Isolation rearing; Exploratory behavior; Social development; Anxiety; Rats
1. Introduction Isolation rearing of animals after weaning is considered to be one model of social stress (Blanchard et al., 1998; Palanza, 2001; Sutanto and de Kloet, 1994). Animals reared in isolation have been found ∗ Present address: School of Psychology, Chukyo University, 1012 Yagoto-Honmachi, Showa-ku, Nagoya 466-8666, Japan. Tel.: +81 52 789 2263; fax: +81 52 835 7144. E-mail address: [email protected].
0376-6357/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2005.07.002
to demonstrate a variety of behavioral and physiological changes in comparison to those reared in groups (Hall, 1998; Palanza, 2001). A previous study by the author (Arakawa, 2003) suggested that one particularly important factor is the age of the subjects during isolation. Specifically, that study demonstrated that rats isolated during the juvenile stage demonstrated diminished exploratory behavior, as indicated by a decrease in duration of stay at the center area of an open-field, compared to rats reared in pairs. In contrast, rats isolated during the pubertal stage showed no reduction in
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exploratory behavior, while rats isolated in adulthood demonstrated increased exploratory behavior. One possible explanation of these findings is that developmental changes in social behavior of rats are responsible for this age-dependent effect of isolation (Arakawa, 2002, 2003; Von Frijtag et al., 2002). For rats, the frequency of social contacts changes with age (Pellis and Pellis, 1998a). As such, the effect of isolation, in which rats are deprived of social contacts with conspecifics (Hall, 1998; Holloway and Suter, 2004), would also be expected to differ as a function of the age of the animals at the time of isolation. However, another possibility is plausible. Based on the analysis of individual differences in behavioral patterns associated with social stress, Blanchard et al. (1998, 2001) found that rats exhibited two patterns of exploratory behavior in a novel environment: an active pattern in which rats approach a novel stimulus, indicated by the amount of locomotion or the duration of stay at the center area of an open-field, and a passive pattern (risk assessment behavior) in which rats participate in behaviors that allow the gathering of information about potential threats, including behaviors such as sniffing or stretching towards the phobic area without ambulation (see also Arakawa, 2005; Haller and Hal´asz, 1999). Thus it is also possible to attribute the age-dependent effect of isolation not to the developmental changes in the frequency of social activity that affect exploratory behavior, but to the characteristics of exploratory behavior that are affected by isolation. However, a simple open-field composed of a dimly lit undifferentiated square floor is not a suitable apparatus for determining whether isolation-reared rats exhibit changes in the frequency or pattern of exploratory behavior. For example, it is often difficult to determine whether the responses of animals staying motionlessly in a corner of the field reflect a decrease in activity or an increase in anxiety. In other words, it is unavoidable that in such testing conditions a behavioral index might reflect a number of factors such as activity level, anxiety level, and exploratory patterns (De Passill´e et al., 1995; Ramos and Morm`ede, 1998; Rushen, 2000). In order to further elucidate the exploratory behavior of rats in a novel environment, it is necessary to use a test apparatus that includes a less phobic area into which animals can escape freely from a more phobic area, and to observe both the level
and the pattern of approach behavior to the phobic area (Blanchard et al., 1998; Fujita et al., 1994). The present study, which investigated whether the age-dependent effect of isolation found in the previous study (Arakawa, 2003) was ascribed to changes in the frequency or in the pattern of exploratory behavior, addressed these issues by using two types of apparatus and an appropriate procedure designed to minimize such confounds. Specifically, an emergence test was conducted in order to measure the level of anxiety. In this test, rats were first placed in a small and dimly lit box for several minutes and then were allowed to explore a phobic area (i.e. a large and bright area) outside it. Rats show several exploratory behaviors including active and passive patterns for searching a phobic area. Then the indices of anxiety included latency and duration spent to search the phobic area (Crawley and Goodwin, 1980; Einon and Morgan, 1977; Pijlman et al., 2002). Patterns of exploratory behavior were measured using an open-field apparatus that was divided by an inner wall into a brightly lit center square and a dimly lit peripheral alley. Exploratory patterns of rats were identified according to the type of approach behavior to the center square (Ossenkopp et al., 1994; Roth and Katz, 1979; Seliger, 1977). Both the number of area entries (representing the active pattern of exploratory behavior) and the number of stretch-attend postures for the center area (representing the passive pattern of exploratory behavior) were counted. The ratio of the number of stretch-attend postures to the total number of exploratory behaviors was defined as the risk assessment ratio. The frequency of play fighting in rats peaks during juvenile stage, then declines with the onset of puberty (Pellis and Pellis, 1990, 1991; Takahashi and Lore, 1983; Thor and Holloway, 1984). Aggressive fighting with conspecifics increases gradually with puberty, and peaks in adult rats after the age of 100 days (Pellis and Pellis, 1987; Takahashi and Lore, 1982). Thus, male rats exhibit a transition between these social behaviors at puberty (Matuszcayk et al., 1994; Meaney and Stewart, 1981; S¨odersten et al., 1977). The animals in the present experiments were tested at 40, 65, or 130 days of age (Arakawa, 2002, 2003), which represent the juvenile, post-pubertal and adult stages in which they were isolated, respectively. The period of isolation for the different developmental stages tested was the 14
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days that immediately preceded the corresponding test age (Arakawa, 2002).
2. Experiment 1 Experiment 1 examined the effect of isolation rearing on exploratory behavior of rats in the three developmental stages. The emergence test and the open-field test were conducted by a two-way factorial design with rearing condition (isolation or pair) and age (40, 65, or 130 days) as the between-subjects factors. 2.1. Materials and methods 2.1.1. Subjects and rearing conditions Forty-eight male Wistar rats, bred and reared in the colony room of the Department of Psychology at Nagoya University, were used as subjects. They were obtained from 12 Wistar dams (litter size = 4–12), and were undisturbed and housed until weaning with the parent and siblings in standard opaque plastic cages (36 cm × 30 cm × 18 cm, l × w × h) with pine shavings as bedding material. Immediately after weaning at the age of 23 days, male rats were housed in pairs with same-sex littermates. At the age of 26 days, they were randomly divided into one of six groups (N = 8 for each group). Then each animal obtained from a litter of the Wistar dams were assigned into other groups as possible. Of these, three groups of rats were in the isolated rearing condition (Groups IS) and each group was tested after a 14-day isolation period. Thus, rats tested at 40 days old (IS-40) were isolated at 26 days, rats tested at 65 days old (IS-65) were isolated at 51 days, and rats tested at 130 days old (IS-130) were isolated at 116 days, respectively. The other three groups of animals were in the paired rearing condition (Groups PS-40, PS-65, and PS-130), and were reared in pairs with a littermate throughout the experiment. All subjects were reared in opaque plastic cages (36 cm × 30 cm × 18 cm, l × w × h) with lids of stainless steel wire grid. The floors of the cage were covered with pine shavings, which was changed every week. The colony room was illuminated on a 12:12 light–dark cycle with light from 07:00 to 19:00 h. The temperature and humidity were maintained at 23 ± 1 ◦ C and 60%, respectively. Subjects were allowed access to food and water freely throughout the experiment. All animal pro-
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tocols were in accordance with the Nagoya University guidelines for the use of animals. 2.1.2. Apparatus 2.1.2.1. Emergence test. This test apparatus was composed of a simple open-field (90 cm × 90 cm × 40 cm, l × w × h), in which a gray plastic start box (20 cm × 20 cm × 20 cm, l × w × h) with a door (10 cm × 10 cm), was placed in the corner. The open-field was made of opaque gray plastic with the floor divided by black lines into 81 equal squares (10 cm × 10 cm). Four 40 W reflector lamps suspended above the open-field illuminated the floor with a luminance of 250 lx and the start box with a luminance of 10 lx. A video camera was placed at 90 cm above the field in order to record the rats behavior. 2.1.2.2. Open-field test. This test used the open-field which was used in the emergence test without the start box. In this test, an inner wall (50 cm × 50 cm × 30 cm, l × w × h) separated the field into a peripheral area (a gallery with 20 cm width) and a center area (a 50 cm × 50 cm squares). Subjects could pass through these two areas freely by any of four openings (15 cm × 15 cm) located in each corner of the inner wall. The floor was illuminated with a luminance of 90 lx in the center area, and 15 lx in the peripheral area. 2.1.3. Procedure The tests were conducted during 20:00–24:00 in the dark phase of the daily light–dark cycle. On the first day of testing, the emergence test was conducted. Twenty minute before the start of the test, the subject with its home cage was moved from the colony room to the experiment room. At the beginning of the test, each animal was placed in the start box with the door closed. After 5 min passed, the door was opened and the rat was allowed to explore beyond the start box for 3 min. At the end of the test, the animal was returned to its home cage and the test apparatus was thoroughly cleaned with 70% ethanol solution. On the second day, the open-field test was conducted. Twenty minute before the start of the test, the subject with its home cage was transferred from the colony room to the experiment room. At the beginning of the 20 min test, the subject was placed in a corner of the peripheral area in the open-field. After the test trial was completed, the animal was returned to its home
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cage and the open-field was thoroughly cleaned with 70% ethanol solution. Behaviors of the subjects were videotaped and analyzed. An experimenter was blind to the condition of the animal videotaped in each test. In the emergence test, the latency that each animal first put its head outside the box and total duration of all bouts that each animal put its head outside the box added together were measured as behavioral indices. In the open-field test, the number of times that the subject’s entire body came into the center area was counted as entries. The number of times that the rat’s head, but not its entire body, entered the center area with stretching the body of the animal was also recorded as stretch-attend postures. The percentage of stretch-attend postures to the total number of exploratory behaviors was then calculated as the risk assessment ratio. 2.1.4. Statistics All the behavioral data were analyzed by two-way analyses of variance with rearing condition (IS or PS) and age (40, 65, or 130 days) as the between subjects factors. For multiple post hoc comparisons, the Fisher’s least significant difference test was used. A probability level of p < .05 was used as the statistical significance for all the tests. 2.2. Results 2.2.1. Emergence test Fig. 1a shows the mean latency of exploring for each group. A two-way ANOVA revealed significant main effects of rearing condition, F(1,42) = 5.33, p < .05, and age, F(1,42) = 7.08, p < .01. The interaction between rearing condition and age was significant, F(2,42) = 4.96, p < .05. Then, the latency was greater in Group IS-40 than in Group PS-40 (p < .01). No significant effects of rearing condition were found for animals tested at either 65 days or 130 days of age. The latency was longer in Group IS-40 than in either Groups IS-65 or IS-130 (p < .01). For the animals reared in pairs, no significant group difference was found. Fig. 1b represents the mean of total duration of exploring for each group. A two-way ANOVA revealed a significant main effects of age, F(1,42) = 27.19, p < .01, but not of rearing condition. There was a significant interaction between rearing condition and age, F(2,42) = 9.54, p < .01. Group IS-40 showed a
Fig. 1. Results of emergence test during 180 s in rats aged 40, 65, and 130 days, reared in isolation (IS) or pairs (PS) in Experiment 1. Data are expressed as mean ± S.E.M. Mean latency (a) and duration (b) that each animal put its head outside the start box during the test session. * Significant difference between rearing conditions, p < .05. # Significant difference between different age groups, p < .05.
lower total duration of exploration than Group PS-40 (p < .01), while no differences between rearing conditions were found for the animals tested at either 65 days or 130 days of age. The duration of exploration was less in Group IS-40 than in either Group IS-65 or Group IS-130, and the duration in Group IS-65 intermediated between Groups IS-40 and IS-130 (p < .01). For the animals reared in pairs, it was less in Group
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PS-40 than in Group PS-130 (p < .05). No other significant group differences were found. 2.2.2. Open-field test Fig. 2a reveals the mean number of entries in the center area for each group. A two-way ANOVA conducted on these scores found a significant main effect of age,
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F(2,42) = 6.43, p < .01, but not of rearing condition. The interaction between rearing condition and age was significant, F(2,42) = 8.67, p < .01. Group IS-40 had fewer entries than Group PS-40 (p < .05), whereas no differences were found between Groups IS-65 and PS-65. On the other hand, Group IS-130 had more entries than Group PS-130 (p < .01). The number of entries was less
Fig. 2. Results of open-field test during 20 min in rats aged 41, 66, and 131 days, reared in isolation (IS) or pairs (PS) in Experiment 1. Data are expressed as mean ± S.E.M. Mean number of entries (a) and stretch-attend postures (b) into the center area of an open-field, and percentage of risk assessment (c) that was calculated as the ratio of stretch-attend postures to total number of exploratory behaviors including entries and stretch-attend postures. * Significant difference between rearing conditions, p < .05. # Significant difference between different age groups, p < .05.
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in Group PS-130 than in either Group PS-40 or Group PS-65 (p < .01). Fig. 2b depicts the mean number of stretchattend postures for each group. A two-way ANOVA found a significant main effect of rearing condition, F(1,42) = 53.78, p < .01, but not of age. The interaction between rearing condition and age was significant, F(2,42) = 5.77, p < .01. Group IS-40 had fewer stretchattend postures than Group PS-40 (p < .01). Stretchattend postures was also less frequent in Group IS-130 compared to Group PS-130 (p < .01). On the other hand, no difference was found between Group IS-65 and Group PS-65. The number of stretch-attend posture events was greater in Group IS-65 than in either Group IS-40 or Group IS-130 (p < .01). The mean ratio of risk assessment for each group is shown in Fig. 2c. A two-way ANOVA revealed a significant main effect of rearing condition, F(1,42) = 19.66, p < .01, but not of age. The significant interaction between rearing condition and age was found, F(2,42) = 11.51, p < .01. The ratio was less in Group IS-130 than in Group PS-130 (p < .01), but no effects were found in the animals tested at either 40 days or 65 days of age. Group IS-130 had a lower ratio than Group IS-65 (p < .05), while the ratio was greater in Group PS-130 than in either Group PS-40 or Group PS-65 (p < .01).
3. Experiment 2 In Experiment 1, rats that were isolated during their juvenile stage demonstrated reduced exploratory behavior compared to rats reared in pairs. Several studies have found that the effect of isolation during the juvenile stage remains for a long time, even when animals are thereafter returned to being reared in pairs (Arakawa, 2002; Ikemoto and Panksepp, 1992; Einon and Potegal, 1991; Van den Berg et al., 1999a). In Experiment 2, the consequences of isolating juveniles on their exploratory behavior after puberty were investigated, in order to determine if the effect of isolation persists after puberty. 3.1. Materials and methods The methods of Experiment 2 mirrored that of Experiment 1, except for the age of the subjects in
the isolated groups and their rearing conditions as described below. 3.1.1. Subjects and rearing conditions Thirty-two Wistar male rats were used as the subjects. After weaning at the age of 23 days, all the animals were housed in pairs with a male littermate (litter size = 5–12). At the age of 26 days, they were reared in either isolation or pairs for 14 days. After this period, all the animals were reared in pairs with a littermate again, and were either assigned to one of four groups (N = 8 for each group): isolated rats tested at the age of 65 days (Group JI-65), pair-reared rats tested at the age of 65 days (Group PS-65), isolated rats tested at the age of 130 days (Group JI-130), and pair-reared rats tested at the age of 130 days (Group PS-130). The same tests as those used in the previous experiment were used. All the behavioral indices were analyzed by a two-way ANOVA with rearing condition (JI or PS) and age at the test (65 or 130 days) as betweensubjects factors. 3.2. Results 3.2.1. Emergence test Fig. 3a depicts the mean latency of exploring for each group. A two-way ANOVA demonstrated a significant main effect of rearing condition, F(1,28) = 33.24, p < .01, while the main effect of age and the interaction between these two factors were not significant. Thus Group JI-65 showed longer latency than Group PS-65. Similarly, Group JI-130 revealed longer latency than Group PS-130. Fig. 3b shows the mean total duration of exploring for each group. A two-way ANOVA showed that there was a significant main effect of rearing condition, F(1,28) = 19.09, p < .01, however the main effect of age and the interaction between these two factors were not significant. Thus isolated rats (Groups JI-65 and JI-130) exhibited lower durations of exploring than pair-reared rats (Groups PS-65 and PS-130, respectively), regardless of their ages at testing. 3.2.2. Open-field test Fig. 4a represents the mean number of entries in the center area for each group. A two-way ANOVA conducted on these scores revealed no significant main effect of rearing condition, but a significant main
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JI-65 and PS-65) showed more entries than rats aged 130 days (Groups JI-130 and PS-130, respectively), regardless of their rearing conditions. Fig. 4b shows the mean number of stretch-attend posture events for each group. A two-way ANOVA exhibited a significant main effect of rearing condition, F(1,28) = 47.56, p < .01, but not of age. The interaction between rearing condition and age was significant, F(1,28) = 15.08, p < .01. Rats in Group JI-65 and Group JI-130 had less stretch-attend postures than rats in Group PS-65 (p < .05) and Group PS-130 (p < .01), respectively. In addition, rats in Group JI-65 showed more stretch-attend behavior than those in Group JI130 (p < .05), whereas rats in Group PS-65 exhibited significantly lower stretch-attend posture events than those in Group PS-130 (p < .01). The mean ratio of risk assessment for each group is depicted in Fig. 4c. A two-way ANOVA demonstrated significant main effects of rearing condition, F(1,28) = 6.94, p < .05, and age, F(1,28) = 7.20, p < .05. The interaction between rearing condition and age was significant, F(1,28) = 5.00, p < .05. Group JI-65 did not differ from Group PS-65, while Group JI-130 showed a lower ratio than Group PS-130 (p < .01). In addition, Group PS-130 exhibited greater ratio than Group PS65 (p < .01), while Group JI-130 did not differ from Group JI-65.
4. General discussion
Fig. 3. Results of emergence test during 180 s in rats aged 65 and 130 days, reared in isolation during the juvenile stage (JI) or pairs (PS) in Experiment 2. Data are expressed as mean ± S.E.M. Mean latency (a) and duration (b) that each animal put its head outside the start box during the test session. * Significant difference between rearing conditions, p < .05. # Indicates a significant difference between different age groups, p < .05.
effect of age, F(1,28) = 10.42, p < .01. The interaction between rearing condition and age was not significant. Isolated animals entered the center area to the same extent as pair-reared animals, at the ages of both 65 and 130 days. In addition, rats aged 65 days (Groups
In the present study, rats isolated during the juvenile stage displayed less exploratory behavior than non-isolated rats, whereas rats isolated during the post-pubertal stage demonstrated no such decrease in the emergence test (Experiment 1). Furthermore, the decrease in exploratory behavior of rats reared in isolation during the juvenile stage was also observed in adult rats, even when they were returned to pair-rearing after isolation (Experiment 2). Thus it appears that the development of exploratory behavior as a response to a novel environment has an irreversible critical period during the juvenile stage (Einon and Morgan, 1977). Several studies have reported that rats are remarkably sensitive to isolation rearing in the juvenile stage. For example, isolation during this stage has been associated with a decrease in the duration of staying in the open arms of an elevated plus maze (Wright et
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Fig. 4. Results of open-field test during 20 min in rats aged 41, 66, and 131 days, reared in isolation during the juvenile stage (JI) or pairs (PS) in Experiment 2. Data are expressed as mean ± S.E.M. Mean number of entries (a) and stretch-attend postures (b) into the center area of an open-field, and the percentage of risk assessment (c) that was calculated as the ratio of stretch-attend postures to total number of exploratory behaviors including entries and stretch-attend postures. *Significant difference between rearing conditions, p < .05. # Significant difference between different age groups, p < .05.
al., 1991), a lower frequency of social behavior with conspesifics (Van den Berg et al., 1999a), and a longer latency before entry into a phobic field from a narrow tube (Einon and Morgan, 1977). In contrast, isolation after puberty does not elicit these changes (Einon and Morgan, 1977; Van den Berg et al., 1999a; Wright et al., 1991). These findings also suggest that rats demon-
strate less exploratory behavior due to isolation during the juvenile stage. Rats reared in isolation during the juvenile stage displayed a reduced threshold for jumping responses to foot shocks (Arakawa, 2002) and showed enhanced defensiveness toward conspecifics (Einon and Potegal, 1991), suggesting that they became more sensitive to aversive stimuli than pair-reared rats.
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In addition, the phobic aspects of exploratory behavior in isolation-reared rats can be attenuated by the injection of anxiolytics (Da Silva et al., 1996). Thus, it is proposed that animals isolated during the juvenile stage did not become less reactive to stimuli, but rather displayed more phobic responses to a novel environment accompanied by their higher level of anxiety as compared to group-reared animals. In the current study, isolation during adulthood did not affect the level of exploratory behavior. Rather, adult rats reared in isolation (IS-130) demonstrated a shift in the exploratory behavior to a more active pattern, as displayed by more entries and less stretchattend postures into the center area of the openfield than pair-reared rats (PS-130). Furthermore, pair-reared rats were less likely to perform active exploratory behaviors in adulthood (PS-130), as they performed fewer entries into the center area of the open-field than those before puberty (PS-40 and PS65) (Experiment 1). After puberty, male rodents often establish a variety of social relationships involving social hierarchies and dominance relationships (Berdoy et al., 1995; Lore and Stipo-Flaherty, 1984). It has been found that differentiation of dominant and subordinate animals in these relationships associate with individual differences in patterns of exploratory behavior in both adult rats and mice (De Vries et al., 2003; Koolhaas et al., 1999; Von Holst, 1998). When exposed to a novel environment, subordinate male rats displayed less active exploratory behavior than did dominant males. When housed together before puberty, however, post-pubertal male rats and mice formed mild dominance relationships without overt victory and defeat (Lore and Stipo-Flaherty, 1984), and no differences were found in exploratory behavior between dominants and subordinates (Bartolomucci et al., 2001). Similarly in the present experiments, pair-reared adult rats were likely to form non-aggressive mild dominance relationships in adulthood. Group formation with adult males can also be considered as a factor affecting exploratory behavior (Arakawa, 2005; Bartolomucci et al., 2004). For example, both dominant and subordinate rats demonstrated less active exploratory behavior than adult rats reared with adult females, who do not develop male-like dominance relationships (Blanchard et al., 1998, 2001; Williams and Lierle, 1988). Furthermore, even rats reared in pairs with littermates showed
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a lower frequency of active exploratory behavior than adult rats housed with a female (Haller and Hal´asz, 2000). These studies suggest that adult males who have established social relationships by being reared with other adult males show a decrease in the active pattern of exploratory behavior accompanied by an emergence of social tension among them, regardless of whether they become dominants or subordinates (Arakawa, 2005; Palanza et al., 2001; Sachser and Lick, 1991). In other words, isolation during adulthood can be considered to be deprivation of such dominance relationships (Palanza, 2001; Von Frijtag et al., 2002), which is responsible for the increased active pattern of exploration in rats isolated during adulthood. In contrast, rats that were isolated during the juvenile stage did not demonstrate a change in exploratory pattern after puberty (Experiment 2). This suggests that the increase in passive exploratory behavior that rats typically display due to social contact with an adult cage mate was inhibited through deprivation of social contacts during the juvenile stage. It has been reported that rats isolated during the juvenile stage exhibit a decrease in social activity as demonstrated by less social contacts and anogenital sniffing than that shown by group-reared rats, even when they were returned to group-rearing after puberty (Hol et al., 1999; Ikemoto and Panksepp, 1992; Van den Berg et al., 1999b). In studies on colony formation, adult rats that were reared in isolation during the juvenile stage displayed less submissive postures and suffered from more serious physical injuries than rats reared in groups (e.g., Lore and Flannelly, 1977; Luciano and Lore, 1975; Van den Berg et al., 1999a). Based on the hypothesis that the change in adult rats’ exploratory patterns in a novel environment arises from an adaptive response to social situations in adulthood, the present findings, that rats isolated during the juvenile stage showed no change in exploratory patterns as adult, can probably be ascribed to an irreversible effect of the lack of social experience in this period (see also Von Frijtag et al., 2002). It has been demonstrated that rats display social play behavior most frequently in the juvenile stage (Pellis and Pellis, 1990, 1991; Takahashi and Lore, 1983; Thor and Holloway, 1984). Several researchers indicate that the experience of play behavior during the juvenile stage is important for the regulation of social stress responses and the generation of appropriate responses to changing social circumstances in adulthood. Furthermore, these
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adaptive behaviors are not acquirable through social contacts with conspecifics in the adult stage (Pellis and Pellis, 1998b; Van den Berg et al., 1999a). However, it should be noted that rats isolated during the juvenile stage showed less passive exploratory behavior even at the age of 65 days, in which pair-reared rats did not exhibit a shift in exploratory patterns. There are several explanations for this finding. It has been found that rats display changes in exploratory pattern depending on intensity of the stimuli to which they are exposed (Albonetti and Farabollini, 1994; Blanchard et al., 2001). That is, the exploratory pattern in rats shifts to a more passive pattern with increasing the intensity of aversive stimuli. To the presence of intensely threatening stimuli, rats show a marked decrease in exploratory behavior. Thus, it appears that rats isolated during the juvenile stage decreased exploration per se on account of their higher anxiety due to hypersensitivity to stimuli or lack of social experience. In addition, since the same size open-field was used for all ages in the present experiments, the magnitude of the threat for a juvenile compared to an adult may be different because of their different body sizes. Thus, it is possible that the open-field area is a relatively more intense stimulus for a juvenile than for an adult, and so this may affect them differently, leading to some of the age-related differences found in the present studies. However, another explanation is also plausible. Social interactions have been shown to be rewarding for male juvenile rats (Douglas et al., 2004), which is likely to facilitate the neural maturation of mesolimbic dopamine systems during puberty (Andersen et al., 1997; Vanderschuren et al., 1997). This may modulate the rewarding properties of novelty and drugs (Douglas et al., 2004; Spear, 2000). This neural maturation of dopamine systems may contribute to the emergence of the age-specific behavior in prepubertal male rats, who show marked propensities for risk-taking, noveltyseeking, and active exploration (Bardo et al., 1996; Palanza, 2001; Spear, 2000), which has been called a behavioral approach system (Palanza, 2001). Based on this hypothesis, juvenile isolation may inhibit the ontogenetic maturation of this system, which may be responsible for the inhibition of exploratory behavior in rats isolated during the juvenile stage. Indeed, basal dopamine synthesis in the nucleus accumbens is lower in juvenile rats than in adults (Andersen et al., 1997), and isolated male mice also exhibit reduced
mesoaccumbens dopamine responses to restraint stress compared to a group reared with conspecifics (Cabib et al., 2002). Taken together, the results of the present study demonstrate that isolation as juveniles reduced the exploratory tendency, while isolation in adulthood changed the exploratory patterns. These findings suggest that exploratory patterns in response to a novel environment emerge through the experience of social interaction with other rats during the juvenile stage and are modified by the social conditions in adulthood (see also De Vries et al., 2003; Sachser et al., 1998). Further experiments are needed to understand the determinants of the ontogenetic emergence of the exploratory pattern as well as the underlying neural mechanisms.
Acknowledgements The author would thank to Professor Kiyoshi Ishii for his critical reading to this article, and two anonymous reviewers for their helpful comments.
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