Journal of Integrative Neuroscience, Vol. 13, No. 1 (2014) 61–70 c Imperial College Press ° DOI: 10.1142/S0219635214500046
Brief neonatal handling alters sexually dimorphic behaviors in adult rats Tetsuya Fujimoto*,§, Kazuhiko Kubo†, Yasuo Nishikawa* and Shuji Aou‡ J. Integr. Neurosci. 2014.13:61-70. Downloaded from www.worldscientific.com by FLINDERS UNIVERSITY LIBRARY on 02/09/15. For personal use only.
*Department of Physiology, Osaka Dental University Hirakata 573-1121, Japan † Department of Otorhinolaryngology, Graduate School of Medical Sciences Kyushu University, Fukuoka 812-8633, Japan ‡ Department of Brain Science and Engineering Kyushu Institute of Technology, Kitakyushu 808-0196, Japan § [email protected]
[Received 21 December 2013; Accepted 3 February 2014; Published 11 March 2014] Several e®ects of neonatal handling on brain and behavior have been reported. We investigated the e®ects of neonatal handling on behaviors that have been shown to be sexually dimorphic in rats using an open-¯eld test. \Gender di®erences" were observed in locomotor activity, exploratory behavior and grooming in the handled group. However, clear gender di®erences in these behaviors were not observed in the non-handled group. Our ¯ndings show that brief daily handling sessions (1 min) in the ¯rst 2 weeks of postnatal life increased locomotor activity and exploratory behavior, and that these e®ects were more pronounced in females. Moreover, many rats in the non-handling group exhibited an increase in defecation relative to the handling group during the 10-min observation period. This suggests that the non-handling group experienced more stress in response to the novel open-¯eld arena, and that this resulted in the absence of gender di®erences. Notably, this anxiety-related response was attenuated by neonatal handling. Our study underscores the impact of brief neonatal handling on sexually dimorphic behaviors, and indicates that caution should be exercised in controlling for the e®ects of handling between experimental groups, particularly in neurotoxicological studies that evaluate gender di®erences. Keywords: Neonatal handling; sexually dimorphic behavior; gender di®erence.
1. Introduction Evaluation of sexually dimorphic behaviors is a common strategy used in the ¯eld of neurotoxicology (Alfonso-Loeches et al., 2013; Beronius et al., 2013; Cauli et al., 2013; Lafuente & Pereiro, 2013; Lilienthal et al., 2013). This is because these behaviors are thought to provide indices of di®erences in the central nervous systems
§
Corresponding author.
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(CNS) of males and females that are brought about by gender di®erentiation, a process in which the actions of sex steroid hormones during the perinatal period are deeply involved (MacLusky & Naftolin, 1981). Many environmental substances exert hormone-like actions, such as \environmental endocrine disruptors (EEDs)". In order to assess how the impact of EEDs on the CNS interact with gender, previous studies examined sexually dimorphic behaviors in an open-¯eld test (Beronius et al., 2013; Fujimoto et al., 2006, 2013; Kubo et al., 2001, 2003). However, in order to make this assessment, the behaviors examined must show clear gender di®erences at baseline. Generally, in behavioral tests, results tend to be a®ected by several factors, including elements in the environment, or the emotional state of the animal. Thus, the potential e®ects of several factors such as breeding conditions or laboratory environment need to be carefully examined (e.g., temperature, humidity, light, sound and smell). In this study, we focused on \neonatal handling" as a factor which could a®ect behavior in adulthood. Since the e®ects of neonatal handling were ¯rst reported by Levine (1956), many studies have explored the potential e®ects of handling on brain development. Handling has been reported to induced low emotionality, high activity and high performance in learning and memory tasks (Levine, 1956; Levine & Broadhurst, 1963; Levine et al., 1967; Meaney et al., 1988). In addition, handling has been shown to in°uence endocrine systems (Meaney et al., 1989; Panagiotaropoulos et al., 2004; Todeschin et al., 2009; Viau et al., 1993), monoamine systems (Madruga et al., 2006; Panagiotaropoulos et al., 2004; Papaioannou et al., 2002) and brain morphology (Bock et al., 2011; Lucion et al., 2003; Winkelmann-Duarte et al., 2007). These neurochemical and neuroanatomical studies suggested that neonatal handling may produce several changes in behavioral outcome. In addition, many studies reported that certain handling-induced alterations were gender-speci¯c. Handling in°uenced female rats more than males in inhibitory avoidance and less in object recognition (Kosten et al., 2007). Handling induced better learning performance in male rats (Stamatakis et al., 2008), but worse performance in the Y -maze (Noschang et al., 2012). Stress-induced plasma corticosterone level was increased by handling female rats (Park et al., 2003). Hippocampal dendritic arbor morphology was also a®ected by handling in a gender-dependent manner (Bock et al., 2011). Therefore, neonatal handling can produce dramatic e®ects on the evaluation of gender-di®erences. Some operations in neonatal rats, such as a bedding change, a health evaluation or a weight measurement are accompanied by handling. The impact of these manipulations has not been completely explored. In this study, we performed carefully controlled brief handling of neonatal rat pups and investigated whether this could a®ect sexually dimorphic behaviors in adulthood using an open¯eld test. Furthermore, we assessed whether clear and more dramatic gender differences in these behavioral parameters could be displayed. We used an open-¯eld test to assess sexually dimorphic behavioral parameters, including locomotor activity, rearing, grooming and defecation as an index of anxiety.
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2. Material and Methods 2.1. Animals
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Pregnant Wistar rats (gestational day 10) were purchased at 13 weeks of age from Kyudo Corp. (Tosu, Japan). Five pregnant dams were used for the non-handling group, and ¯ve dams were used for handling group. The rats were kept in a closed colony room under a 12:12-h light–dark cycle (light period 08:00 AM to 08:00 PM) in a constant air-conditioned environment (23 C 1 C and 60 10% relative humidity). Animals had ad libitum access to laboratory chow (CE-2, CLEA Japan, Inc., Tokyo, Japan) and tap water. 2.2. Neonatal handling After birth, litters were balanced to contain four males and four females by removing any excess pups. The pups were selected according to body weight. First, runts, if any, were removed from the litter. Second, the average weights in both sexes were measured. The four male pups and four female ones that were selected for an experiment were closest to each average weight. In the handling group (H(þÞÞ, pups were separated from their mothers once a day by (1) putting them in our hands for 3–5 s, (2) measuring their weight, (3) placing them individually in another cage and (4) returning them to their home cage. These operations were completed in 1 min, and were repeated on a daily basis until postnatal day 14 (PND14). In the nonhandling group (H()), the pups were never handled between PND1 and PND14 except on the day of birth (PND0) during the culling operation. All PND21 o®spring were then weaned, classi¯ed and housed with same sex littermates (four males in one cage, four females in one cage) and were allowed ad libitum access to food and water. 2.3. Open-¯eld test At 6 weeks of age, the open-¯eld test was conducted. The open-¯eld apparatus was a square ¯eld (80 56 40 cm) in which an object (12 9 7 cm) was placed at the center. Novel object exploration was scored by measuring the number of entries into the center area. Each rat was placed along one inner side of the apparatus at the beginning of the test and was allowed to move freely for 10 min. The total distance traveled, the frequency of rearing and grooming, the number of entries into the center area and the number of defecations were recorded. The total distance travelled was used as an index of locomotor activity. The rearing and the number of entries into the center area were used as an index of exploratory behavior. The amount of defecation was used as an indicator of anxiety-level. 2.4. Statistics All data were analyzed using the Statview 5.0 software for Macintosh (SAS Inc.). Results of all behavioral tests, with the exception of the defecation test, were analyzed by two-way ANOVA followed by a post-hoc test (Fisher's PLSD test). The
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data for defecation were analyzed by Fisher's exact test. Statistical di®erences were considered signi¯cant when the p value was below 0.05. All results are presented as mean values standard errors (SEM).
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3. Results In this study, an open-¯eld test was conducted in adult (6 weeks old) rat o®spring. Both neonatal handling and gender were found to a®ect the total distance traveled. (two-way ANOVA, main e®ect of handling: Fð1;72Þ ¼ 5:43, p < 0:05, main e®ect of gender, Fð1;72Þ ¼ 9:53; p < 0:01). However, no interaction was found between these factors (Fig. 1(a)). A gender di®erence (male < female) in locomotor activity was observed in the handling-group, with females exhibiting a signi¯cant increase in total distance traveled (Fig. 1(a)). Main e®ects of both handling and gender were also observed on the number of entries made into the center area (two-way ANOVA: main e®ect of handling: Fð1;72Þ ¼ 9:57; p < 0:01, main e®ect of gender: Fð1;72Þ ¼ 5:46; p < 0:05, respectively), but a signi¯cant interaction between these factors was not observed. A gender di®erence was observed in the handling group (male < female), with handled females exhibiting a signi¯cantly greater number of entries in comparison to handled males (Fig. 1(b)). A gender di®erence was observed in the frequency of rearing (two-way ANOVA, main e®ect of gender: Fð1;72Þ ¼ 17:81; p < 0:001, Fig. 1(c)). Handling did not a®ect this behavioral parameter in the
(a)
(b)
Fig. 1. E®ects of neonatal handling in the open-¯eld test. The parameters were (a) total distance travelled (b), number of entries into the center area (c), number of rearings (d), and number of grooming responses. The black and white columns represent mean values SEM for data from males vs. females, respectively (n ¼ 18–20 animals per group). H() and H(þ) represent the non-handling and handling groups, respectively. *p < 0:05, **p < 0:01, ***p < 0:001, by Fisher's PLSD test between the data of males vs. females in same group. #p < 0:05, ##p < 0:01, by Fisher's PLSD test between the data of H() vs. H(þ) of the female rats.
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BRIEF NEONATAL HANDLING ALTERS SEXUALLY DIMORPHIC BEHAVIORS
(c)
(d) Fig. 1.
Table 1.
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(Continued )
E®ects of neonatal handling on defecation. Male
H() (n ¼ 18)
H(þ) (n ¼ 20)
Female H() (n ¼ 18)
H(þ) (n ¼ 20)
D() D(þ) D(); number of rats that did not defecate, D(þÞ; number of rats that defecated at least one time. # p < 0:05, ### p < 0:001, H() vs. H(þÞ, Fisher's exact test.
open¯eld test (Fig. 1(c)). The frequency of grooming was also only a®ected by gender (two-way ANOVA, main e®ect of gender: Fð1;72Þ ¼ 5:03; p < 0:05, Fig. 1(d)). Anxiety was assessed by classifying animals into one of two groups: (1) rats that defecated at least one time (Dþ) or (2) rats that did not defecate during the 10 min observation period (D). In the handling group, all animals were classi¯ed into group 2 (D). In the non-handling group, 10 out of 18 males and 4 out of 18 females were classi¯ed into group 1 (Dþ) (Table 1). Thus, neonatal handling reduced anxiety assessed by defecation in the open-¯eld test in both male and female rats (Table 1). 4. Discussion In this study, we evaluated the e®ects of neonatal handling on sexually dimorphic behaviors in rats using an open-¯eld test. In the handling group, signi¯cant gender di®erences were observed in all the parameters (Fig. 1). Remarkably, daily handling
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sessions of 1 min during the ¯rst 2 weeks of postnatal life had an impact on an index of anxiety during the open ¯eld test in adulthood, as well as on locomotor activity and exploratory behavior. In contrast, in the non-handling group, gender di®erences were only observed in rearing frequency; gender di®erences in the other behavioral parameters did not reach signi¯cance (Fig. 1). Having a clear understanding of how environmental factors may di®erentially interact with gender in certain behaviors is important for neurotoxicology studies in which manipulations are suspected to have di®erential gender-speci¯c e®ects. Gender di®erences at baseline are presumed to re°ect di®erences in brain development that are caused by events during gender di®erentiation. Perinatal gonadal hormones, which di®er between males and females, are deeply involved in these events (MacLusky & Naftolin, 1981). Therefore, in order to evaluate the impact of various environment chemicals such as EEDs on gender di®erentiation of the developing brain, it will be a useful \endpoint" to determine how other environmental factors can a®ect brain gender di®erentiation. Neonatal handling had previously been reported to cause several alterations in brain development. In addition to producing e®ects on behavior, handling has been shown to a®ect endocrine systems (Meaney et al., 1989; Panagiotaropoulos et al., 2004; Todeschin et al., 2009; Viau et al., 1993), monoamine systems (Madruga et al., 2006; Panagiotaropoulos et al., 2004; Papaioannou et al., 2002) and brain morphology (Bock et al., 2011; Lucion et al., 2003; Winkelmann-Duarte et al., 2007). Routine events, such as bedding changes and weight measurements, require a minimal amount of handling. In addition, certain experimental manipulations may require varying degrees of handling, and may be di®erent across treatment and control groups. Thus, we sought to determine whether brief and gentle neonatal handling sessions would be su±cient to induce changes in sexually dimorphic behaviors, and whether the e®ects would di®er between the genders. Our results show that daily 1-min handling sessions during the ¯rst 2 weeks of postnatal life is su±cient to induce changes in behavioral parameters in an open-¯eld test in both genders in adulthood. In the open-¯eld test, gender di®erences (female > male) were reported in locomotor activity, rearing and entries into the center area of the open ¯eld (Archer, 1975). The trend of gender di®erence had also been reported for grooming behavior (female < male) (Moore, 1986). Consistent with these previous studies, our results indicate that handled females exhibited increased locomotor activity and a greater number of entries into the center arena in the open-¯eld test (Fig. 1). Furthermore, anxiety assessed by frequency of defecation (Fernandez-Teruel et al., 1992; Levine & Broadhurst, 1963; Levine et al., 1967) in response to the stress in the open-¯eld was higher in both male and female rats in the non-handling group in comparison to the handling group (Table 1). Some studies using the \Tsukuba Emotional rats" reported that highly emotional rats displayed low activity and a high frequency of defecation (Fujita et al., 1994; Kitaoka & Fujita, 1991). The results from the present study, together with these previous ¯ndings, suggest that non-handled rats are more highly emotional. That is, non-handled rats exposed to a novel environment such as an
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open-¯eld arena, ¯nd the experience more stressful. Neonatal handling may therefore enhance stress coping ability in response to several environmental stressors. In the present study, we show that the well-known gender di®erence in locomotion (male < female) (Archer, 1975) can be eliminated by withholding neonatal-handling (Fig. 1). Furthermore, non-handling particularly compromised stress coping ability in females. This may be consistent with the fact that women appear to be more susceptible to stress-related mental illness (Kornstein, 1997). In a previous study, female rats exhibited decreased locomotion and increased corticosterone levels in response to stress compared to males (Dalla et al., 2005). Indeed, these ¯ndings support our present data; non-handled female rats in the present study were more vulnerable to stress. In addition, serotonergic activities in the hippocampus and the hypothalamus have been reported to decrease in female rats after giving stress (Drossopoulou et al., 2004; Dalla et al., 2005). The hippocampus and hypothalamus are brain structures rich in corticoid receptors (Karandrea et al., 2000; McEwen, 2000). After exposure to stress, female rats exhibited an increase in mineralcorticoid receptor mRNA levels in the hippocampus, and a decrease in glucocorticoid receptor levels in the hypothalamus (Karandrea et al., 2000). We propose that gender differences in brain serotonin and endocrine systems underlie our ¯nding that females are more vulnerable to the e®ects of withholding neonatal-handling. The \disruption of gender di®erence" is a sensitive endpoint for the study of EEDs (Fujimoto et al., 2006, 2013; Kubo et al., 2003). In the present study, the presence or absence of neonatal handling was a critical factor in determining the behavioral performance in several of the behavioral parameters examined. Moreover, a daily handling session of 1 min was su±cient to produce these e®ects. Thus, our results show that the amount of handling should be tightly controlled across experimental groups when performing studies on neonatal rats. While our statistical analyses did not reveal an interaction in the results for the total distance traveled, the number of entries into the center and the number of grooming, gender di®erences appeared to be ampli¯ed in the group that was handled. Thus, handling may be a strategy that could be used in experiments in which the targeted endpoint is \disruption of gender di®erence". A gender di®erence has also been reported in the incidence of degenerative diseases, such as Alzheimer's disease (AD) (www.alz.org/documents custom/ report alzfacts¯gures2010.pdf). An interaction between genetics and sex hormones has been proposed as a mechanism underlying this di®erence between the sexes. Consistent with this, gender di®erences in stress response have been reported in an animal model of AD (3xTg-AD mice) (Clinton et al., 2007). A higher level of stressinduced corticosterone release and impaired performance in cognition tasks have also been observed in female 3xTg-AD mice compared to males. It is possible that handling could be a signi¯cant factor in these ¯ndings as well. Therefore, we propose that neonatal handling should be carefully controlled in studies linking gender di®erences to the incidence or the stage of AD, and studies exploring gender di®erences in stressresponses. Our study suggests that brief neonatal handling will produce robust data
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on sexually dimorphic parameters, and may be useful for several ethopharmacological studies, which evaluate gender di®erences. Acknowledgments The study is supported by Grants-in-Aid for Scienti¯c Research (No. 16209006, S.A.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Brief neonatal handling alters sexually dimorphic behaviors in adult rats Tetsuya Fujimoto*,§, Kazuhiko Kubo†, Yasuo Nishikawa* and Shuji Aou‡ J. Integr. Neurosci. 2014.13:61-70. Downloaded from www.worldscientific.com by FLINDERS UNIVERSITY LIBRARY on 02/09/15. For personal use only.
*Department of Physiology, Osaka Dental University Hirakata 573-1121, Japan † Department of Otorhinolaryngology, Graduate School of Medical Sciences Kyushu University, Fukuoka 812-8633, Japan ‡ Department of Brain Science and Engineering Kyushu Institute of Technology, Kitakyushu 808-0196, Japan § [email protected]
[Received 21 December 2013; Accepted 3 February 2014; Published 11 March 2014] Several e®ects of neonatal handling on brain and behavior have been reported. We investigated the e®ects of neonatal handling on behaviors that have been shown to be sexually dimorphic in rats using an open-¯eld test. \Gender di®erences" were observed in locomotor activity, exploratory behavior and grooming in the handled group. However, clear gender di®erences in these behaviors were not observed in the non-handled group. Our ¯ndings show that brief daily handling sessions (1 min) in the ¯rst 2 weeks of postnatal life increased locomotor activity and exploratory behavior, and that these e®ects were more pronounced in females. Moreover, many rats in the non-handling group exhibited an increase in defecation relative to the handling group during the 10-min observation period. This suggests that the non-handling group experienced more stress in response to the novel open-¯eld arena, and that this resulted in the absence of gender di®erences. Notably, this anxiety-related response was attenuated by neonatal handling. Our study underscores the impact of brief neonatal handling on sexually dimorphic behaviors, and indicates that caution should be exercised in controlling for the e®ects of handling between experimental groups, particularly in neurotoxicological studies that evaluate gender di®erences. Keywords: Neonatal handling; sexually dimorphic behavior; gender di®erence.
1. Introduction Evaluation of sexually dimorphic behaviors is a common strategy used in the ¯eld of neurotoxicology (Alfonso-Loeches et al., 2013; Beronius et al., 2013; Cauli et al., 2013; Lafuente & Pereiro, 2013; Lilienthal et al., 2013). This is because these behaviors are thought to provide indices of di®erences in the central nervous systems
§
Corresponding author.
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(CNS) of males and females that are brought about by gender di®erentiation, a process in which the actions of sex steroid hormones during the perinatal period are deeply involved (MacLusky & Naftolin, 1981). Many environmental substances exert hormone-like actions, such as \environmental endocrine disruptors (EEDs)". In order to assess how the impact of EEDs on the CNS interact with gender, previous studies examined sexually dimorphic behaviors in an open-¯eld test (Beronius et al., 2013; Fujimoto et al., 2006, 2013; Kubo et al., 2001, 2003). However, in order to make this assessment, the behaviors examined must show clear gender di®erences at baseline. Generally, in behavioral tests, results tend to be a®ected by several factors, including elements in the environment, or the emotional state of the animal. Thus, the potential e®ects of several factors such as breeding conditions or laboratory environment need to be carefully examined (e.g., temperature, humidity, light, sound and smell). In this study, we focused on \neonatal handling" as a factor which could a®ect behavior in adulthood. Since the e®ects of neonatal handling were ¯rst reported by Levine (1956), many studies have explored the potential e®ects of handling on brain development. Handling has been reported to induced low emotionality, high activity and high performance in learning and memory tasks (Levine, 1956; Levine & Broadhurst, 1963; Levine et al., 1967; Meaney et al., 1988). In addition, handling has been shown to in°uence endocrine systems (Meaney et al., 1989; Panagiotaropoulos et al., 2004; Todeschin et al., 2009; Viau et al., 1993), monoamine systems (Madruga et al., 2006; Panagiotaropoulos et al., 2004; Papaioannou et al., 2002) and brain morphology (Bock et al., 2011; Lucion et al., 2003; Winkelmann-Duarte et al., 2007). These neurochemical and neuroanatomical studies suggested that neonatal handling may produce several changes in behavioral outcome. In addition, many studies reported that certain handling-induced alterations were gender-speci¯c. Handling in°uenced female rats more than males in inhibitory avoidance and less in object recognition (Kosten et al., 2007). Handling induced better learning performance in male rats (Stamatakis et al., 2008), but worse performance in the Y -maze (Noschang et al., 2012). Stress-induced plasma corticosterone level was increased by handling female rats (Park et al., 2003). Hippocampal dendritic arbor morphology was also a®ected by handling in a gender-dependent manner (Bock et al., 2011). Therefore, neonatal handling can produce dramatic e®ects on the evaluation of gender-di®erences. Some operations in neonatal rats, such as a bedding change, a health evaluation or a weight measurement are accompanied by handling. The impact of these manipulations has not been completely explored. In this study, we performed carefully controlled brief handling of neonatal rat pups and investigated whether this could a®ect sexually dimorphic behaviors in adulthood using an open¯eld test. Furthermore, we assessed whether clear and more dramatic gender differences in these behavioral parameters could be displayed. We used an open-¯eld test to assess sexually dimorphic behavioral parameters, including locomotor activity, rearing, grooming and defecation as an index of anxiety.
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2. Material and Methods 2.1. Animals
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Pregnant Wistar rats (gestational day 10) were purchased at 13 weeks of age from Kyudo Corp. (Tosu, Japan). Five pregnant dams were used for the non-handling group, and ¯ve dams were used for handling group. The rats were kept in a closed colony room under a 12:12-h light–dark cycle (light period 08:00 AM to 08:00 PM) in a constant air-conditioned environment (23 C 1 C and 60 10% relative humidity). Animals had ad libitum access to laboratory chow (CE-2, CLEA Japan, Inc., Tokyo, Japan) and tap water. 2.2. Neonatal handling After birth, litters were balanced to contain four males and four females by removing any excess pups. The pups were selected according to body weight. First, runts, if any, were removed from the litter. Second, the average weights in both sexes were measured. The four male pups and four female ones that were selected for an experiment were closest to each average weight. In the handling group (H(þÞÞ, pups were separated from their mothers once a day by (1) putting them in our hands for 3–5 s, (2) measuring their weight, (3) placing them individually in another cage and (4) returning them to their home cage. These operations were completed in 1 min, and were repeated on a daily basis until postnatal day 14 (PND14). In the nonhandling group (H()), the pups were never handled between PND1 and PND14 except on the day of birth (PND0) during the culling operation. All PND21 o®spring were then weaned, classi¯ed and housed with same sex littermates (four males in one cage, four females in one cage) and were allowed ad libitum access to food and water. 2.3. Open-¯eld test At 6 weeks of age, the open-¯eld test was conducted. The open-¯eld apparatus was a square ¯eld (80 56 40 cm) in which an object (12 9 7 cm) was placed at the center. Novel object exploration was scored by measuring the number of entries into the center area. Each rat was placed along one inner side of the apparatus at the beginning of the test and was allowed to move freely for 10 min. The total distance traveled, the frequency of rearing and grooming, the number of entries into the center area and the number of defecations were recorded. The total distance travelled was used as an index of locomotor activity. The rearing and the number of entries into the center area were used as an index of exploratory behavior. The amount of defecation was used as an indicator of anxiety-level. 2.4. Statistics All data were analyzed using the Statview 5.0 software for Macintosh (SAS Inc.). Results of all behavioral tests, with the exception of the defecation test, were analyzed by two-way ANOVA followed by a post-hoc test (Fisher's PLSD test). The
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data for defecation were analyzed by Fisher's exact test. Statistical di®erences were considered signi¯cant when the p value was below 0.05. All results are presented as mean values standard errors (SEM).
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3. Results In this study, an open-¯eld test was conducted in adult (6 weeks old) rat o®spring. Both neonatal handling and gender were found to a®ect the total distance traveled. (two-way ANOVA, main e®ect of handling: Fð1;72Þ ¼ 5:43, p < 0:05, main e®ect of gender, Fð1;72Þ ¼ 9:53; p < 0:01). However, no interaction was found between these factors (Fig. 1(a)). A gender di®erence (male < female) in locomotor activity was observed in the handling-group, with females exhibiting a signi¯cant increase in total distance traveled (Fig. 1(a)). Main e®ects of both handling and gender were also observed on the number of entries made into the center area (two-way ANOVA: main e®ect of handling: Fð1;72Þ ¼ 9:57; p < 0:01, main e®ect of gender: Fð1;72Þ ¼ 5:46; p < 0:05, respectively), but a signi¯cant interaction between these factors was not observed. A gender di®erence was observed in the handling group (male < female), with handled females exhibiting a signi¯cantly greater number of entries in comparison to handled males (Fig. 1(b)). A gender di®erence was observed in the frequency of rearing (two-way ANOVA, main e®ect of gender: Fð1;72Þ ¼ 17:81; p < 0:001, Fig. 1(c)). Handling did not a®ect this behavioral parameter in the
(a)
(b)
Fig. 1. E®ects of neonatal handling in the open-¯eld test. The parameters were (a) total distance travelled (b), number of entries into the center area (c), number of rearings (d), and number of grooming responses. The black and white columns represent mean values SEM for data from males vs. females, respectively (n ¼ 18–20 animals per group). H() and H(þ) represent the non-handling and handling groups, respectively. *p < 0:05, **p < 0:01, ***p < 0:001, by Fisher's PLSD test between the data of males vs. females in same group. #p < 0:05, ##p < 0:01, by Fisher's PLSD test between the data of H() vs. H(þ) of the female rats.
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BRIEF NEONATAL HANDLING ALTERS SEXUALLY DIMORPHIC BEHAVIORS
(c)
(d) Fig. 1.
Table 1.
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(Continued )
E®ects of neonatal handling on defecation. Male
H() (n ¼ 18)
H(þ) (n ¼ 20)
Female H() (n ¼ 18)
H(þ) (n ¼ 20)
D() D(þ) D(); number of rats that did not defecate, D(þÞ; number of rats that defecated at least one time. # p < 0:05, ### p < 0:001, H() vs. H(þÞ, Fisher's exact test.
open¯eld test (Fig. 1(c)). The frequency of grooming was also only a®ected by gender (two-way ANOVA, main e®ect of gender: Fð1;72Þ ¼ 5:03; p < 0:05, Fig. 1(d)). Anxiety was assessed by classifying animals into one of two groups: (1) rats that defecated at least one time (Dþ) or (2) rats that did not defecate during the 10 min observation period (D). In the handling group, all animals were classi¯ed into group 2 (D). In the non-handling group, 10 out of 18 males and 4 out of 18 females were classi¯ed into group 1 (Dþ) (Table 1). Thus, neonatal handling reduced anxiety assessed by defecation in the open-¯eld test in both male and female rats (Table 1). 4. Discussion In this study, we evaluated the e®ects of neonatal handling on sexually dimorphic behaviors in rats using an open-¯eld test. In the handling group, signi¯cant gender di®erences were observed in all the parameters (Fig. 1). Remarkably, daily handling
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sessions of 1 min during the ¯rst 2 weeks of postnatal life had an impact on an index of anxiety during the open ¯eld test in adulthood, as well as on locomotor activity and exploratory behavior. In contrast, in the non-handling group, gender di®erences were only observed in rearing frequency; gender di®erences in the other behavioral parameters did not reach signi¯cance (Fig. 1). Having a clear understanding of how environmental factors may di®erentially interact with gender in certain behaviors is important for neurotoxicology studies in which manipulations are suspected to have di®erential gender-speci¯c e®ects. Gender di®erences at baseline are presumed to re°ect di®erences in brain development that are caused by events during gender di®erentiation. Perinatal gonadal hormones, which di®er between males and females, are deeply involved in these events (MacLusky & Naftolin, 1981). Therefore, in order to evaluate the impact of various environment chemicals such as EEDs on gender di®erentiation of the developing brain, it will be a useful \endpoint" to determine how other environmental factors can a®ect brain gender di®erentiation. Neonatal handling had previously been reported to cause several alterations in brain development. In addition to producing e®ects on behavior, handling has been shown to a®ect endocrine systems (Meaney et al., 1989; Panagiotaropoulos et al., 2004; Todeschin et al., 2009; Viau et al., 1993), monoamine systems (Madruga et al., 2006; Panagiotaropoulos et al., 2004; Papaioannou et al., 2002) and brain morphology (Bock et al., 2011; Lucion et al., 2003; Winkelmann-Duarte et al., 2007). Routine events, such as bedding changes and weight measurements, require a minimal amount of handling. In addition, certain experimental manipulations may require varying degrees of handling, and may be di®erent across treatment and control groups. Thus, we sought to determine whether brief and gentle neonatal handling sessions would be su±cient to induce changes in sexually dimorphic behaviors, and whether the e®ects would di®er between the genders. Our results show that daily 1-min handling sessions during the ¯rst 2 weeks of postnatal life is su±cient to induce changes in behavioral parameters in an open-¯eld test in both genders in adulthood. In the open-¯eld test, gender di®erences (female > male) were reported in locomotor activity, rearing and entries into the center area of the open ¯eld (Archer, 1975). The trend of gender di®erence had also been reported for grooming behavior (female < male) (Moore, 1986). Consistent with these previous studies, our results indicate that handled females exhibited increased locomotor activity and a greater number of entries into the center arena in the open-¯eld test (Fig. 1). Furthermore, anxiety assessed by frequency of defecation (Fernandez-Teruel et al., 1992; Levine & Broadhurst, 1963; Levine et al., 1967) in response to the stress in the open-¯eld was higher in both male and female rats in the non-handling group in comparison to the handling group (Table 1). Some studies using the \Tsukuba Emotional rats" reported that highly emotional rats displayed low activity and a high frequency of defecation (Fujita et al., 1994; Kitaoka & Fujita, 1991). The results from the present study, together with these previous ¯ndings, suggest that non-handled rats are more highly emotional. That is, non-handled rats exposed to a novel environment such as an
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open-¯eld arena, ¯nd the experience more stressful. Neonatal handling may therefore enhance stress coping ability in response to several environmental stressors. In the present study, we show that the well-known gender di®erence in locomotion (male < female) (Archer, 1975) can be eliminated by withholding neonatal-handling (Fig. 1). Furthermore, non-handling particularly compromised stress coping ability in females. This may be consistent with the fact that women appear to be more susceptible to stress-related mental illness (Kornstein, 1997). In a previous study, female rats exhibited decreased locomotion and increased corticosterone levels in response to stress compared to males (Dalla et al., 2005). Indeed, these ¯ndings support our present data; non-handled female rats in the present study were more vulnerable to stress. In addition, serotonergic activities in the hippocampus and the hypothalamus have been reported to decrease in female rats after giving stress (Drossopoulou et al., 2004; Dalla et al., 2005). The hippocampus and hypothalamus are brain structures rich in corticoid receptors (Karandrea et al., 2000; McEwen, 2000). After exposure to stress, female rats exhibited an increase in mineralcorticoid receptor mRNA levels in the hippocampus, and a decrease in glucocorticoid receptor levels in the hypothalamus (Karandrea et al., 2000). We propose that gender differences in brain serotonin and endocrine systems underlie our ¯nding that females are more vulnerable to the e®ects of withholding neonatal-handling. The \disruption of gender di®erence" is a sensitive endpoint for the study of EEDs (Fujimoto et al., 2006, 2013; Kubo et al., 2003). In the present study, the presence or absence of neonatal handling was a critical factor in determining the behavioral performance in several of the behavioral parameters examined. Moreover, a daily handling session of 1 min was su±cient to produce these e®ects. Thus, our results show that the amount of handling should be tightly controlled across experimental groups when performing studies on neonatal rats. While our statistical analyses did not reveal an interaction in the results for the total distance traveled, the number of entries into the center and the number of grooming, gender di®erences appeared to be ampli¯ed in the group that was handled. Thus, handling may be a strategy that could be used in experiments in which the targeted endpoint is \disruption of gender di®erence". A gender di®erence has also been reported in the incidence of degenerative diseases, such as Alzheimer's disease (AD) (www.alz.org/documents custom/ report alzfacts¯gures2010.pdf). An interaction between genetics and sex hormones has been proposed as a mechanism underlying this di®erence between the sexes. Consistent with this, gender di®erences in stress response have been reported in an animal model of AD (3xTg-AD mice) (Clinton et al., 2007). A higher level of stressinduced corticosterone release and impaired performance in cognition tasks have also been observed in female 3xTg-AD mice compared to males. It is possible that handling could be a signi¯cant factor in these ¯ndings as well. Therefore, we propose that neonatal handling should be carefully controlled in studies linking gender di®erences to the incidence or the stage of AD, and studies exploring gender di®erences in stressresponses. Our study suggests that brief neonatal handling will produce robust data
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on sexually dimorphic parameters, and may be useful for several ethopharmacological studies, which evaluate gender di®erences. Acknowledgments The study is supported by Grants-in-Aid for Scienti¯c Research (No. 16209006, S.A.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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