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Gonadal hormones during puberty organize environment-related social interaction in the male rat
HORMONES
AND
BEHAVIOR
24,
31 l-323 (1990)
Gonadal Hormones during Puberty Organize EnvironmentRelated Social Interaction in the Male Rat RENEE J. PRIMUS AND CAROL K. KELLOGG University
of Rochester,
Department Rochester,
of Psychology, Room New York 14627
186, Meiiora
Hall,
This study examined the role of gonadal androgens during puberty on the development of environment-related social interaction (SI) in male rats. SI in an unfamiliar environment versus SI in a familiar environment was evaluated in young adult rats as a function of sex and gonadal status. Intact male rats at 60 days of age exhibited a differential response to the two environments, whereas SI in intact female rats at 60 days was equivalent in the two environments. Furthermore, male rats castrated as juveniles and tested for SI at 60 days displayed a pattern of environment-related SI similar to SI in intact adult female rats. This effect of juvenile castration on SI in male rats was prevented by chronic exposure to testosterone propionate (TP) over Days 30 through 60. SI in male rats castrated in adulthood, on the other hand, was not altered either 2 or 4 weeks postcastration. The results from this study indicate that pubertal secretions of gonadal androgen(s) are necessary for the development of environment-related SI in male rats. In contrast, secretions of gonadal androgens in adulthood do not appear to be critical for the continued expression of environment-related SI, as suggested by the observation that environment-related SI in male rats remains unchanged by castration in adulthood. o IWO Academic press, 1~.
The observation that specific “stress-related” or “fear” responses in the rat emerge during pubertal development (Primus and Kellogg, 1989a; Candland and Campbell, 1962) suggests that hormonal events associated with puberty may contribute to the development of these responses. A role for gonadal secretions in the development of specific stress-related responses has been supported by the demonstration that several stressrelated responses are both sexually dimorphic and dependent on hormonal secretions during early development. For example, Brand and Slob (1988) and Slob, Huizer, and Van Der Werff Bosch (1986) have shown that peripubertal secretions of male gonadal androgens are necessary for the expression of the sex difference that has been reported in open-field ambulation in the adult rat (Beatty and Fessler, 1976; Blizard, Lippman, and Chen, 1975). Similarly, the sexual dimorphism in sensitivity to electric shock apparent at 50 days of age in the rat (Beatty 311 0018-506x/90 $1.50 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and Fessler, 1976) is also due to the influence of male gonadal androgens during peripubertal development (Beatty and Fessler, 1977). Evidence from this laboratory suggests that the secretion of gonadal androgens during peripubertal development may also be important for the expression of environment-related social behavior in the adult male rat (Primus and Kellogg, 1989a). Environment-related social behavior in the rat was examined by using the social interaction test (SI) of anxiety (File, 1980). In this test, the behavioral interaction between two rats (strangers to each other) placed in a neutral environment that is either familiar or unfamiliar to both rats is assessed. Previous work has shown that SI in adult male rats in an unfamiliar environment is decreased relative to SI in an environment that is familiar (File and Hyde, 1978). Decreased SI in an unfamiliar environment has been referred to as an adaptive response to an anxiogenic situation (File, 1988). This decrease in SI induced by an unfamiliar environment is accompanied by increases in both physiological (File and Peet, 1980) and behavioral (File and Hyde, 1978) indicators of stress. In addition, pretreatment with anxiolytic benzodiazepine compounds prevents environment-induced decreases in SI (File, 1980). Recently, Primus and Kellogg (1989a) have shown that the differential response to the two environments in male rats does not appear until the onset of puberty, at about 35 days of age. Furthermore, the appearance of environment-related SI with the onset of puberty is prevented by castration at 19 days of age. Therefore, the expression of this anxiety-related behavior in the male rat appears to be influenced by hormonal events associated with the onset of puberty. The purpose of the present study was to demonstrate that environmentrelated SI in the adult male rat is dependent on the presence of gonadal androgens during pubertal development. The sexually dimorphic nature of SI was first evaluated in both male and female adult rats. Then, the ability of testosterone replacement to reinstate environment-related SI in juvenile castrated male rats was examined. Finally, the requirement for intact gonadal function in the adult male rat for the maintenance of adult SI was also evaluated. METHODS Animals Male and female rats (Long-Evans, Harlan-Sprague-Dawley, Altamont, NY) were tested for SI as detailed below. All rats were group housed until 5 days prior to the SI test, at which time they were individually housed. Rats received food and water ad libitum and were maintained on a 12 hr light/dark cycle (lights on at 0600).
GONADAL
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SI
Apparatus SI was evaluated in a Plexiglas arena with a 41 x 25 cm floor and Plexiglas walls 30 cm high. Infrared photocells located 2.54 cm above the floor along the walls of the test arena were connected to a cumulative recorder and provided an automated measure of locomotor activity during the social interaction test. All social interactions were recorded on videotape by a video camera that was focused on the test arena. Both video camera and test arena were located in a sound-isolation chamber that was maintained at an illumination intensity of 13-15 scotopic lux. Procedure Adult rats were randomly assigned to either the familiar or unfamiliar test environment. Pairs of rats were assigned on the basis of body weight, so that rats within each pair did not differ by more than 10 g. Each pair was tested for SI only once. Individually housed rats were handled and weighed on each of the 5 days preceding the social interaction test. On each of the 2 days before the test, the rats to be tested in the familiar environment were placed singly in the arena for 7.5 min. The rats to be tested in the unfamiliar environment were transported to the test room but remained in their home cage for the duration of the 7.5 min. On the day of the social interaction test, each pair of rats was placed in the test arena and their behaviors were recorded on videotape for a total of 7.5 min. Both members of a pair received the same familiarization experience and prior treatment (castration, etc.). Rats were tested between 0800 and 1200 hr. The arena was cleaned with moistened paper towels between testing of each rat pair. The behaviors measured in the test were separated into two categories, curiosity and physical behavior. Curiosity behavior included sniffing and following, whereas physical behavior included pushing, jumping, wrestling, and grooming (of each other). Behaviors were scored from the videotapes and the amount of time spent in the various behaviors was added to give a total social interaction score. This score was multiplied by 2 in order to reflect the contribution of each rat in the pair. Additionally, both curiosity and physical behaviors were analyzed separately for each pair. Scoring of the videotapes was performed by individuals who had no knowledge of the particular condition being scored. Experiment
1: Environment-Related
SI as a Function
of Sex
Intact male and female rats were 60 days of age on the day of testing for SI. Seven pairs of male rats and five pairs of female rats were allocated to each environment. Locomotor activity was recorded for each pair in each group.
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Experiment 2: Testosterone Replacement in Juvenile Castrated Rats Castration of IPday-old male rats anesthetized with ether was performed by removing the testes via a midline perineal incision that was closed with sutures. All rats were returned to their mother following recovery from ether anesthesia. At 23 days of age, rats were weaned and placed in group housing. At 30 days of age, male rats castrated at 19 days were implanted with either empty or testosterone propionate (TP)-filled silastic tubing capsules under ether anesthesia. The TP-filled capsules (1.57 mm inner diameter, 3.18 mm outer diameter) were prepared as described by Sodersten, Damassa, and Smith (1977) and sealed with Silastic adhesive. A 30 mm length of TP-filled silastic tubing produces a T plasma level comparable to that measured in intact adult male rats (Damassa, Smith, Tennet, and Davidson, 1977). Verification of plasma T levels in TP-exposed rats was conducted at the conclusion of the study (see below). At 60 days of age, rats were tested for SI. Four to six pairs of castrated rats chronically exposed to TP and three to five pairs of castrated control rats were allocated to each environment. Locomotor activity was recorded in each group. Experiment
3: SI in Rats Castrated
in Adulthood
Male rats between 55 and 60 days of age were either castrated or shamcastrated and then tested for ST 2 or 4 weeks later. Rats were anesthetized with ketamine HCl(l2.5 mg/kg i.m.> and xylazine HCl(l5.0 mg/kg i.m.) and castrated as described under Experiment 2. Four to five pairs of rats to be tested 2 weeks postcastration and three pairs of rats to be tested 4 weeks postcastration were allocated to each environment. Two pairs of sham-castrated rats to be tested 2 weeks postsurgery were also allocated to each environment. Locomotor activity was recorded in each group. Measurement
of Testosterone
The effectiveness of both juvenile and adult castration and of chronic hormone administration was verified by analysis of serum testosterone (T) levels at the conclusion of the testing. Trunk blood was collected from randomly selected male rats (n = 8) in each treatment group. Total testosterone was measured using a Coat-A-kit supplied by Diagnostic Product Corporation. Aliquots of serum were placed in antibody-coated tubes. The antibody is highly specific for testosterone, having less than 10% cross-reactivity with dihydrotestosterone. Following the addition of I’*‘-labeled testosterone, the tubes were incubated for 3 hr at 37°C and then counted in a gamma counter after decantation. All samples were run in duplicate and read against a logit-log representation of the calibration curve prepared by the addition of known amounts of testosterone. The approximate sensitivity of the assay was 0.04 rig/ml.
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Statistics Data for social interaction and locomotor activity were analyzed by a commercial statistical package (SAS vol. 6.0) using a general linear model analysis of variance. This model was used to control for unequal sample size. Data were analyzed by an analysis of variance with environment and either sex or hormonal status (intact, castrated, or TP-exposed) as the independent variables. Posthoc analyses were performed using Duncan’s multiple range test. Statistical significance was attributed when the probability of a Type I error was less than 5%. RESULTS
Male versus Female SI The data in Fig. 1A show that environment-related SI in male adult rats differs significantly from SI in female adult rats, as indicated by an environment by sex interaction (F(1, 23) = 16.35, P = 0.0006). SI in male rats at 60 days was significantly decreased in the unfamiliar environment relative to SI in the familiar environment (P = 0.0001). In contrast, a differential response to the two environments was not evident in female rats at 60 days. Instead, SI was equivalent in both environments. Compared to SI in male rats, SI in female rats was significantly lower in the familiar environment (P = 0.014), but significantly greater in the unfamiliar environment (P = 0.007). Analysis of the behavioral composition of SI (Fig. 1B) indicated a significant interaction between sex and environment on both curiosity behavior (F(l, 14) = 7.10, P < 0.02) and physical behavior (F(1, 19) = 13.88, P < 0.002). Female rats showed a greater amount of both curiosity (P = 0.03) and physical (P = 0.05) behaviors in the unfamiliar environment compared to that shown by male rats in the unfamiliar environment. These increases correspond to the increase in total SI observed in female rats in the unfamiliar environment compared to total SI in male rats. On the other hand, male rats showed a greater amount of physical behavior in the familiar environment compared to that shown by female rats in the familiar environment (P < 0.006). Locomotor activity did not differ significantly as a function of sex or environment (data not shown). Effect of Testosterone Propionate on SI As previously reported (Primus and Kellogg, 1989a), castration at 19 days of age abolished the differential response to the familiar and unfamiliar environments in male rats tested at 60 days of age. Compared to SI in intact male rats at 60 days (Fig. IA), SI in prepubertal castrated rats at this age (Fig. 2A) was decreased in the familiar environment, but was increased in the unfamiliar environment, a difference similar to that observed between intact male and female rats (Fig. IA).
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n
Familiar Unfamiliar
FEMALE
MALE
SEX
C :, ‘5
MALE-U
B h
FEMALE-F
e
0
100
200
300
400
SECONDS FIG. 1. (A) Total SI as a function of sex at 60 days of age. * A statistically significant difference between SI in the familiar and unfamiliar environment. ’ A significant decrease in SI from male rats in the familiar environment. + A significant increase in SI from male rats in the unfamiliar environment. (B) Behavioral composition of SI as a function of sex at 60 days. See text for probability level of significance.
The data in Fig. 2A show that chronic exposure of prepubertal castrated male rats to TP between Days 30 and 60 prevented the effect of early castration on SI, as revealed by a significant interaction between environment and gonadal status (F(2, 28) = 6.95, P = 0.004). Exposure to TP reinstated the differential response to the two environments, such that SI in the unfamiliar environment was significantly decreased relative to SI in the familiar environment (P = 0.03). This differential response to the two environments now evident in TP-exposed rats was primarily
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SI
A
n Familiar
CASTRATED
CASTRATED
+ TP
GONADAL STATUS
B
G 5
GDX-F
E z 2
GDX-U
5 2
2 z
TP-F
TP-U
0
100
200
300
400
SECONDS FIG. 2. (A) Total SI at 60 days of age in juvenile castrated male rats exposed to either empty or TP-filled silastic implants Days 30 through 60. * A significant difference between SI in the familiar and unfamiliar environment. (B) Behavioral composition of SI in juvenile castrated rats with or without testosterone replacement during puberty. See text for probability level of significance.
due to a significant decrease in SI in the unfamiliar environment compared to SI in this environment in juvenile castrated rats exposed to empty tubing (P = 0.003). Analysis of the behavioral composition of SI (Fig. 2B) revealed a significant interaction between hormonal status and environment on curiosity behavior (F(1, 14) = 4.91, P < 0.05) but no effect of either on
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physical behavior was indicated. The amount of time spent in curiosity behavior in the unfamiliar environment was significantly decreased in TP-exposed castrated rats compared to that of castrated control rats (P = 0.01). Serum levels of T were below detection at 60 days in male rats castrated as juveniles (co.04 ng/mg). In castrated male rats exposed to TP over Days 30 through 60, assay results indicated serum T levels of 21.1 + 1.26 rig/ml (compared to 12 f 2.5 rig/ml in intact male rats at 60 days). Locomotor activity was not significantly altered as a function of environment or hormonal status (data not shown). SZ in Adult Castrated Rats Analysis of SI in male rats sham-castrated or castrated between 55 and 60 days of age and tested either 2 or 4 weeks later revealed a significant effect of the environment on SI (F(1, 18) = 193.69, P = O.OOOl), such that SI in both castrated and sham-castrated rats was decreased in the unfamiliar environment relative to SI in the familiar environment (Fig. 3A). In addition, a significant effect of hormonal status on SI (F(1, 18) = 13.51, P = 0.0007) was also revealed. Collapsing across environments, SI in adult rats tested 4 weeks postcastration was significantly decreased compared to SI in sham-castrated rats (P = 0.001) and in rats tested 2 weeks postcastration (P = 0.0004). Considering the behavioral composition of SI, an effect of hormonal status on curiosity behavior was revealed (F(2, 18) = 10.53, P = 0.002), but physical behavior was not affected. Rats tested for SI 4 weeks postcastration spent less time in curiosity behavior (collapsed across environments) compared to sham-castrated rats (P = 0.0008) and rats tested 2 weeks postcastration (P > 0.002). Assay results indicated that serum T levels were diminished below detection (co.04 rig/ml) in male rats castrated in adulthood. Locomotor activity did not change as a function of environment or castration (data not shown). DISCUSSION A differential response to a familiar versus an unfamiliar environment has been shown to emerge with the onset of puberty (Primus and Kellogg, 1989a). Furthermore, prepubertal castration in male rats has been shown to prevent the appearance of the differential response to the familiar and unfamiliar environments during SI (Primus and Kellogg, 1989a). Thus, the purpose of the present study was to further determine the role of male gonadal androgens in the development and expression of environment-related SI. At 60 days of age, SI in male rats differed significantly from SI in female rats. A decrease in SI in the unfamiliar environment relative to SI in the familiar environment was evident in male rats, but not in female rats. This sexually dimorphic response to the unfamiliar environment is
GONADAL
HORMONES
ORGANIZE
"1
H
SHAMGDX
2 WEKS-GDX
319
SI
Famib
4 WEEKSGDX
GROUP
B
2 i E .L k 0 h 5
MALE-F MALE-U 2 WEEK-F 2 WEEK-U 4 WEEK-F
5 0 EL u
4 WEEK-U 0
100
200
300
400
SECONDS
FIG. 3. (A) Total SI at either 2 or 4 weeks following either sham-castration or castration between 55 and 60 days of age. * A statistically significant difference between SI in the familiar and unfamiliar environment. ’ Statistically different from sham-castrated rats and rats tested 2 weeks postcastration (familiar environment). (B) Behavioral composition of SI in rats either sham-castrated or castrated at 55-60 days and tested for SI either 2 or 4 weeks later. See text for probability level of significance.
reminiscent of other sex differences in anxiety/stress-related behavioral responses in that female rats appear to be less emotional than male rats in response to novelty. For example, Beatty and Fessler (1976) have shown that adult female rats ambulate more and defecate less than adult male rats when placed in an open field (i.e., a lesser emotional response). Interestingly, emotionality in the open field was increased by castration in adult female rats (Blizard et al., 1973, while castration in adult male rats had no effect on open-field behavior (Brand and Slob, 1988). This
320
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finding suggests that the hormonal environment of the adult female rat may exert an influence on anxiety-related responses. In fact, injections of ovarian hormones, such as progesterone and estrogen, have been shown to increase shock-punished responses in both females and neonatally castrated male adult rats (Rodriquez-Sierra, Hagley, and Hendricks, 1986). In the present study, therefore, the lack of a differential response to the two environments in adult female rats may be partially due to an anxiolytic/inhibitory effect of ovarian hormones on SI in the unfamiliar environment. In support of a role for ovarian hormones in the expression of SI in adult female rats is the finding that while both male and female rats at 35 days of age display a differential response to the two environments (Primus and Kellogg, 1989b), only male rats display the differential response at 60 days. This change in SI in female rats as a function of pubertal age implicates an influence of ovarian hormones on the development of SI. Exposure of juvenile castrated male rats to testosterone propionate over Days 30 through 60 prevented the effect of castration on SI in the unfamiliar environment. That is, SI was decreased in the unfamiliar environment relative to SI in the familiar environment in TP-exposed castrated male rats. Thus, the differential response to the two environments in intact adult male rats (Fig. 2A) was not present in TP-exposed juvenile castrated male rats (Fig. 2B). The presence of testosterone (or one of its metabolites) between 30 and 60 days of age, therefore, appears to be critical for the expression of environment-related changes in SI in adult male rats. The possibility exists, however, that TP exposure does not directly alter SI between rats, but rather alters behavioral responses of the rat to its surrounding environment. That is, decreased SI in the unfamiliar environment in TP-exposed male rats may have resulted from a concomitant increase in behavioral interactions with the surrounding unfamiliar environment. Previous work (File and Hyde, 1978) has shown, however, that displacement activity (rearing, grooming behavior), and not environmental exploration, increases in the unfamiliar environment during SI in intact adult male rats. In addition, intact adult male rats spend less time in exploration of novel stimuli than adult female rats (Russell, 1977), indicating that exploration of environmental novelty is not increased by the presence of testosterone. The reinstatement of environment-related SI by exposure to TP during puberty demonstrates, therefore, that secretion of gonadal androgens during puberty may indeed underlie the development of SI in male rats. Significant changes in the steroidogenic pathways of rat testes have been shown to occur between the onset of puberty (30-35 days of age) and early adulthood (55-60 days of age) (Ojeda and Urbanski, 1988). In addition, the induction of sex differences in central nervous system (CNS) function by gonadal steroids has been shown to occur during both peri-
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natal (MacLusky and Naftolin, 1981) and peripubertal (Brand and Slob, 1988; Clough and Rodriquez-Sierra, 1983) periods of development. Hence, the expression of sexually dimorphic SI in the rat may depend on an organizational effect of testicular secretions on the CNS during pubertal development. Not only do gonadal steroids exert important organizational influences on behavior during early development, the presence of such steroids in adulthood are often required for the activation of these behaviors (Arnold and Breedlove, 1985). That TP exposure over Days 30 through 60 exerted an organizational effect on environment-related SI and that its presence at 60 days did not simply activate appropriate circuitry was verified by testing male rats castrated in adulthood. In the present study, the differential response to the two environments was present in rats castrated in early adulthood and tested for SI either 2 or 4 weeks later. Thus, the presence of testicular secretions in adult male rats appeared not to be necessary for the maintenance of environment-related SI. The lack of effect of adult castration on SI in male rats also demonstrated that testosterone, per se, does not induce the decrease in SI in the unfamiliar environment. That testosterone does not induce anxiety-related responses in the rat is further supported by the observation that the amount of emotionality displayed in male rats in the open-field test is unaltered by castration in adulthood (Brand and Slob, 1988). The findings from this study indicate that environment-related SI in the adult rat is a sexually dimorphic behavior. Furthermore, the expression of the differential response to the two environments in adult male rats appears to depend on exposure to testicular secretions of testosterone (or one of its metabolites) during the pubertal period of development. In addition, the finding that SI in male rats is unaltered at both 2 and 4 weeks following adult castration provides additional support for puberty as a critical period for the development of environment-related SI. The 4-week postcastration interval was of comparable duration to the period of TP exposure following juvenile castration. That exposure to gonadal androgens during puberty is necessary for the continued development of environment-related SI is supported by the findings from this study. However, it is unknown whether testicular function, only during a restricted period of puberty, is required for the development of environment-related SI in male rats. In addition, since metabolites of testosterone are also produced during puberty, it is impossible to state at this time whether it is testosterone or one of its metabolites that is critical for the development of SI in the male rat. Nevertheless, the fact that both male and female rats demonstrate environment-related SI at 3.5 days, but only male rats show the effect of the unfamiliar environment at 60 days, suggests that the emergence of the different gonadal hormones in the two sexes during adolescence may differentially influence under-
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lying neural systems. The results of this study clearly reveal the importance of gonadal hormones during puberty in the expression of environment-related SI in the rat. ACKNOWLEDGMENTS This work was supported in part by Grant No. MH 31850 and by a Research Scientist Development Award (MHOO651) to C. Kellogg, both from the National Institute of Mental Health. We thank Dr. Richard Aslin for the loan of video taping equipment and Dr. Ernest Nordeen for his helpful guidance in the preparation of the manuscript.
REFERENCES Arnold, A. P., and Breedlove, S. M. (1985). Organizational and activational effect of sex steroids on brain and behavior: A reanalysis. Harm. Behav. 19, 469-499. Beatty, W. W., and Fessler, R. G. (1977). Gonadectomy and sensitivity to electric shock in the rat. Physiol. Behav. 19, l-6. Beatty, W. W., and Fessler, R. G. (1976). Ontogeny of sex differences in open-field behavior and sensitivity to electric shock in the rat. Physiol. Behav. 16, 413-417. Blizard, D. A., Lippman, R. H., and Chen, J. J. (1975). Sex differences in open-field behavior in the rat: The inductive and activational role of gonadal hormone. Physiol. Behav. 14, 601-608. Brand, T., and Slob, A. K. (1988). Peripubertal castration of male rats, adult open field ambulation and partner preference behavior. Behav. Brain Res. 30, 11 l-l 17. Candland, D. K., and Campbell, B. A. (1962). Development of fear in the rat as measured by behavior in the open field. J. Comp. Physiol. Psych. 55, 593-596. Clough, R. W., and Rodriquez-Sierra, J. F. (1983). Synaptic changes in the hypothalamus of the prepubertal female rat administered estrogen. Amer. J. Anat. 167, 205-214. Damassa, D. A., Smith, E. R., Tennet, B., and Davidson, J. M. (1977). The relationship between circulating testosterone levels and male sexual behavior in rats. Horm. and Behav.
8, 275-286.
File, S. E. (1980). The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J. Neurosci. Methods 2, 219-238. File, S. E. (1988). How good is social interaction as a test of anxiety? Anim. Mode/s Psych. Disord. 1, 151-166. File, S. E., and Hyde, J. R. G. (1978). Can social interaction be used to measure anxiety. Brit. J. Pharmacol. 64, 19-24. File, S. E., and Peet, L. A. (1980). The sensitivity of the rat corticosterone response to environmental manipulations and to chronic chlordiazepoxide treatment. Physiol. Behav.
25, 753-758.
MacLusky, N. J., and Naftolin, F. (1981). Sexual differentiation of the central nervous system. Science 221, 1294-1311. Ojeda, S. R., and Urbanski, H. F. (1988). Puberty in the rat. In E. Knobil and J. Neil1 (Eds.), The Physiology of Reproduction. Raven Press, New York. Primus, R., and Kellogg, C. K. (1989a). Pubertal-related changes influence the development of environment-related social interaction in male rats. Dev. Psychobiol. 22(6), 633643. Primus, R., and Kellogg, C. K. (1989b). Sexually dimorphic aspects of social interaction in adult rats are altered by prenatal exposure to diazepam. Sot. Neurosci. Abstr. 15(l), 415, 167.7. Rodriguez-Sierra, J. F., Hagley, M. T., and Hendricks, S. E. (1986). Anxiolytic effects of progesterone are sexually dimorphic. Life Sci. 38, 1841-1845.
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Russell, P. A. (1977). Sex differences in rats’ stationary exploration as a function of stimulus and environmental novelty. Anim. Learn Behav. S(3), 297-302. Slob, A. K., Huizer, T., and Van Der Werff Ten Bosch, J. J. (1986). Ontogeny of sex differences in open-field ambulation in the rat. Physiol. Behav. 37, 313-315. Sodersten, P., Damassa, D. A., and Smith, E. R. (1977). Sexual behavior in developing male rats. Horm. Behav. 8, 320-341.
AND
BEHAVIOR
24,
31 l-323 (1990)
Gonadal Hormones during Puberty Organize EnvironmentRelated Social Interaction in the Male Rat RENEE J. PRIMUS AND CAROL K. KELLOGG University
of Rochester,
Department Rochester,
of Psychology, Room New York 14627
186, Meiiora
Hall,
This study examined the role of gonadal androgens during puberty on the development of environment-related social interaction (SI) in male rats. SI in an unfamiliar environment versus SI in a familiar environment was evaluated in young adult rats as a function of sex and gonadal status. Intact male rats at 60 days of age exhibited a differential response to the two environments, whereas SI in intact female rats at 60 days was equivalent in the two environments. Furthermore, male rats castrated as juveniles and tested for SI at 60 days displayed a pattern of environment-related SI similar to SI in intact adult female rats. This effect of juvenile castration on SI in male rats was prevented by chronic exposure to testosterone propionate (TP) over Days 30 through 60. SI in male rats castrated in adulthood, on the other hand, was not altered either 2 or 4 weeks postcastration. The results from this study indicate that pubertal secretions of gonadal androgen(s) are necessary for the development of environment-related SI in male rats. In contrast, secretions of gonadal androgens in adulthood do not appear to be critical for the continued expression of environment-related SI, as suggested by the observation that environment-related SI in male rats remains unchanged by castration in adulthood. o IWO Academic press, 1~.
The observation that specific “stress-related” or “fear” responses in the rat emerge during pubertal development (Primus and Kellogg, 1989a; Candland and Campbell, 1962) suggests that hormonal events associated with puberty may contribute to the development of these responses. A role for gonadal secretions in the development of specific stress-related responses has been supported by the demonstration that several stressrelated responses are both sexually dimorphic and dependent on hormonal secretions during early development. For example, Brand and Slob (1988) and Slob, Huizer, and Van Der Werff Bosch (1986) have shown that peripubertal secretions of male gonadal androgens are necessary for the expression of the sex difference that has been reported in open-field ambulation in the adult rat (Beatty and Fessler, 1976; Blizard, Lippman, and Chen, 1975). Similarly, the sexual dimorphism in sensitivity to electric shock apparent at 50 days of age in the rat (Beatty 311 0018-506x/90 $1.50 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PRIMUS AND KELLOGG
and Fessler, 1976) is also due to the influence of male gonadal androgens during peripubertal development (Beatty and Fessler, 1977). Evidence from this laboratory suggests that the secretion of gonadal androgens during peripubertal development may also be important for the expression of environment-related social behavior in the adult male rat (Primus and Kellogg, 1989a). Environment-related social behavior in the rat was examined by using the social interaction test (SI) of anxiety (File, 1980). In this test, the behavioral interaction between two rats (strangers to each other) placed in a neutral environment that is either familiar or unfamiliar to both rats is assessed. Previous work has shown that SI in adult male rats in an unfamiliar environment is decreased relative to SI in an environment that is familiar (File and Hyde, 1978). Decreased SI in an unfamiliar environment has been referred to as an adaptive response to an anxiogenic situation (File, 1988). This decrease in SI induced by an unfamiliar environment is accompanied by increases in both physiological (File and Peet, 1980) and behavioral (File and Hyde, 1978) indicators of stress. In addition, pretreatment with anxiolytic benzodiazepine compounds prevents environment-induced decreases in SI (File, 1980). Recently, Primus and Kellogg (1989a) have shown that the differential response to the two environments in male rats does not appear until the onset of puberty, at about 35 days of age. Furthermore, the appearance of environment-related SI with the onset of puberty is prevented by castration at 19 days of age. Therefore, the expression of this anxiety-related behavior in the male rat appears to be influenced by hormonal events associated with the onset of puberty. The purpose of the present study was to demonstrate that environmentrelated SI in the adult male rat is dependent on the presence of gonadal androgens during pubertal development. The sexually dimorphic nature of SI was first evaluated in both male and female adult rats. Then, the ability of testosterone replacement to reinstate environment-related SI in juvenile castrated male rats was examined. Finally, the requirement for intact gonadal function in the adult male rat for the maintenance of adult SI was also evaluated. METHODS Animals Male and female rats (Long-Evans, Harlan-Sprague-Dawley, Altamont, NY) were tested for SI as detailed below. All rats were group housed until 5 days prior to the SI test, at which time they were individually housed. Rats received food and water ad libitum and were maintained on a 12 hr light/dark cycle (lights on at 0600).
GONADAL
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SI
Apparatus SI was evaluated in a Plexiglas arena with a 41 x 25 cm floor and Plexiglas walls 30 cm high. Infrared photocells located 2.54 cm above the floor along the walls of the test arena were connected to a cumulative recorder and provided an automated measure of locomotor activity during the social interaction test. All social interactions were recorded on videotape by a video camera that was focused on the test arena. Both video camera and test arena were located in a sound-isolation chamber that was maintained at an illumination intensity of 13-15 scotopic lux. Procedure Adult rats were randomly assigned to either the familiar or unfamiliar test environment. Pairs of rats were assigned on the basis of body weight, so that rats within each pair did not differ by more than 10 g. Each pair was tested for SI only once. Individually housed rats were handled and weighed on each of the 5 days preceding the social interaction test. On each of the 2 days before the test, the rats to be tested in the familiar environment were placed singly in the arena for 7.5 min. The rats to be tested in the unfamiliar environment were transported to the test room but remained in their home cage for the duration of the 7.5 min. On the day of the social interaction test, each pair of rats was placed in the test arena and their behaviors were recorded on videotape for a total of 7.5 min. Both members of a pair received the same familiarization experience and prior treatment (castration, etc.). Rats were tested between 0800 and 1200 hr. The arena was cleaned with moistened paper towels between testing of each rat pair. The behaviors measured in the test were separated into two categories, curiosity and physical behavior. Curiosity behavior included sniffing and following, whereas physical behavior included pushing, jumping, wrestling, and grooming (of each other). Behaviors were scored from the videotapes and the amount of time spent in the various behaviors was added to give a total social interaction score. This score was multiplied by 2 in order to reflect the contribution of each rat in the pair. Additionally, both curiosity and physical behaviors were analyzed separately for each pair. Scoring of the videotapes was performed by individuals who had no knowledge of the particular condition being scored. Experiment
1: Environment-Related
SI as a Function
of Sex
Intact male and female rats were 60 days of age on the day of testing for SI. Seven pairs of male rats and five pairs of female rats were allocated to each environment. Locomotor activity was recorded for each pair in each group.
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Experiment 2: Testosterone Replacement in Juvenile Castrated Rats Castration of IPday-old male rats anesthetized with ether was performed by removing the testes via a midline perineal incision that was closed with sutures. All rats were returned to their mother following recovery from ether anesthesia. At 23 days of age, rats were weaned and placed in group housing. At 30 days of age, male rats castrated at 19 days were implanted with either empty or testosterone propionate (TP)-filled silastic tubing capsules under ether anesthesia. The TP-filled capsules (1.57 mm inner diameter, 3.18 mm outer diameter) were prepared as described by Sodersten, Damassa, and Smith (1977) and sealed with Silastic adhesive. A 30 mm length of TP-filled silastic tubing produces a T plasma level comparable to that measured in intact adult male rats (Damassa, Smith, Tennet, and Davidson, 1977). Verification of plasma T levels in TP-exposed rats was conducted at the conclusion of the study (see below). At 60 days of age, rats were tested for SI. Four to six pairs of castrated rats chronically exposed to TP and three to five pairs of castrated control rats were allocated to each environment. Locomotor activity was recorded in each group. Experiment
3: SI in Rats Castrated
in Adulthood
Male rats between 55 and 60 days of age were either castrated or shamcastrated and then tested for ST 2 or 4 weeks later. Rats were anesthetized with ketamine HCl(l2.5 mg/kg i.m.> and xylazine HCl(l5.0 mg/kg i.m.) and castrated as described under Experiment 2. Four to five pairs of rats to be tested 2 weeks postcastration and three pairs of rats to be tested 4 weeks postcastration were allocated to each environment. Two pairs of sham-castrated rats to be tested 2 weeks postsurgery were also allocated to each environment. Locomotor activity was recorded in each group. Measurement
of Testosterone
The effectiveness of both juvenile and adult castration and of chronic hormone administration was verified by analysis of serum testosterone (T) levels at the conclusion of the testing. Trunk blood was collected from randomly selected male rats (n = 8) in each treatment group. Total testosterone was measured using a Coat-A-kit supplied by Diagnostic Product Corporation. Aliquots of serum were placed in antibody-coated tubes. The antibody is highly specific for testosterone, having less than 10% cross-reactivity with dihydrotestosterone. Following the addition of I’*‘-labeled testosterone, the tubes were incubated for 3 hr at 37°C and then counted in a gamma counter after decantation. All samples were run in duplicate and read against a logit-log representation of the calibration curve prepared by the addition of known amounts of testosterone. The approximate sensitivity of the assay was 0.04 rig/ml.
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Statistics Data for social interaction and locomotor activity were analyzed by a commercial statistical package (SAS vol. 6.0) using a general linear model analysis of variance. This model was used to control for unequal sample size. Data were analyzed by an analysis of variance with environment and either sex or hormonal status (intact, castrated, or TP-exposed) as the independent variables. Posthoc analyses were performed using Duncan’s multiple range test. Statistical significance was attributed when the probability of a Type I error was less than 5%. RESULTS
Male versus Female SI The data in Fig. 1A show that environment-related SI in male adult rats differs significantly from SI in female adult rats, as indicated by an environment by sex interaction (F(1, 23) = 16.35, P = 0.0006). SI in male rats at 60 days was significantly decreased in the unfamiliar environment relative to SI in the familiar environment (P = 0.0001). In contrast, a differential response to the two environments was not evident in female rats at 60 days. Instead, SI was equivalent in both environments. Compared to SI in male rats, SI in female rats was significantly lower in the familiar environment (P = 0.014), but significantly greater in the unfamiliar environment (P = 0.007). Analysis of the behavioral composition of SI (Fig. 1B) indicated a significant interaction between sex and environment on both curiosity behavior (F(l, 14) = 7.10, P < 0.02) and physical behavior (F(1, 19) = 13.88, P < 0.002). Female rats showed a greater amount of both curiosity (P = 0.03) and physical (P = 0.05) behaviors in the unfamiliar environment compared to that shown by male rats in the unfamiliar environment. These increases correspond to the increase in total SI observed in female rats in the unfamiliar environment compared to total SI in male rats. On the other hand, male rats showed a greater amount of physical behavior in the familiar environment compared to that shown by female rats in the familiar environment (P < 0.006). Locomotor activity did not differ significantly as a function of sex or environment (data not shown). Effect of Testosterone Propionate on SI As previously reported (Primus and Kellogg, 1989a), castration at 19 days of age abolished the differential response to the familiar and unfamiliar environments in male rats tested at 60 days of age. Compared to SI in intact male rats at 60 days (Fig. IA), SI in prepubertal castrated rats at this age (Fig. 2A) was decreased in the familiar environment, but was increased in the unfamiliar environment, a difference similar to that observed between intact male and female rats (Fig. IA).
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n
Familiar Unfamiliar
FEMALE
MALE
SEX
C :, ‘5
MALE-U
B h
FEMALE-F
e
0
100
200
300
400
SECONDS FIG. 1. (A) Total SI as a function of sex at 60 days of age. * A statistically significant difference between SI in the familiar and unfamiliar environment. ’ A significant decrease in SI from male rats in the familiar environment. + A significant increase in SI from male rats in the unfamiliar environment. (B) Behavioral composition of SI as a function of sex at 60 days. See text for probability level of significance.
The data in Fig. 2A show that chronic exposure of prepubertal castrated male rats to TP between Days 30 and 60 prevented the effect of early castration on SI, as revealed by a significant interaction between environment and gonadal status (F(2, 28) = 6.95, P = 0.004). Exposure to TP reinstated the differential response to the two environments, such that SI in the unfamiliar environment was significantly decreased relative to SI in the familiar environment (P = 0.03). This differential response to the two environments now evident in TP-exposed rats was primarily
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n Familiar
CASTRATED
CASTRATED
+ TP
GONADAL STATUS
B
G 5
GDX-F
E z 2
GDX-U
5 2
2 z
TP-F
TP-U
0
100
200
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400
SECONDS FIG. 2. (A) Total SI at 60 days of age in juvenile castrated male rats exposed to either empty or TP-filled silastic implants Days 30 through 60. * A significant difference between SI in the familiar and unfamiliar environment. (B) Behavioral composition of SI in juvenile castrated rats with or without testosterone replacement during puberty. See text for probability level of significance.
due to a significant decrease in SI in the unfamiliar environment compared to SI in this environment in juvenile castrated rats exposed to empty tubing (P = 0.003). Analysis of the behavioral composition of SI (Fig. 2B) revealed a significant interaction between hormonal status and environment on curiosity behavior (F(1, 14) = 4.91, P < 0.05) but no effect of either on
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physical behavior was indicated. The amount of time spent in curiosity behavior in the unfamiliar environment was significantly decreased in TP-exposed castrated rats compared to that of castrated control rats (P = 0.01). Serum levels of T were below detection at 60 days in male rats castrated as juveniles (co.04 ng/mg). In castrated male rats exposed to TP over Days 30 through 60, assay results indicated serum T levels of 21.1 + 1.26 rig/ml (compared to 12 f 2.5 rig/ml in intact male rats at 60 days). Locomotor activity was not significantly altered as a function of environment or hormonal status (data not shown). SZ in Adult Castrated Rats Analysis of SI in male rats sham-castrated or castrated between 55 and 60 days of age and tested either 2 or 4 weeks later revealed a significant effect of the environment on SI (F(1, 18) = 193.69, P = O.OOOl), such that SI in both castrated and sham-castrated rats was decreased in the unfamiliar environment relative to SI in the familiar environment (Fig. 3A). In addition, a significant effect of hormonal status on SI (F(1, 18) = 13.51, P = 0.0007) was also revealed. Collapsing across environments, SI in adult rats tested 4 weeks postcastration was significantly decreased compared to SI in sham-castrated rats (P = 0.001) and in rats tested 2 weeks postcastration (P = 0.0004). Considering the behavioral composition of SI, an effect of hormonal status on curiosity behavior was revealed (F(2, 18) = 10.53, P = 0.002), but physical behavior was not affected. Rats tested for SI 4 weeks postcastration spent less time in curiosity behavior (collapsed across environments) compared to sham-castrated rats (P = 0.0008) and rats tested 2 weeks postcastration (P > 0.002). Assay results indicated that serum T levels were diminished below detection (co.04 rig/ml) in male rats castrated in adulthood. Locomotor activity did not change as a function of environment or castration (data not shown). DISCUSSION A differential response to a familiar versus an unfamiliar environment has been shown to emerge with the onset of puberty (Primus and Kellogg, 1989a). Furthermore, prepubertal castration in male rats has been shown to prevent the appearance of the differential response to the familiar and unfamiliar environments during SI (Primus and Kellogg, 1989a). Thus, the purpose of the present study was to further determine the role of male gonadal androgens in the development and expression of environment-related SI. At 60 days of age, SI in male rats differed significantly from SI in female rats. A decrease in SI in the unfamiliar environment relative to SI in the familiar environment was evident in male rats, but not in female rats. This sexually dimorphic response to the unfamiliar environment is
GONADAL
HORMONES
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"1
H
SHAMGDX
2 WEKS-GDX
319
SI
Famib
4 WEEKSGDX
GROUP
B
2 i E .L k 0 h 5
MALE-F MALE-U 2 WEEK-F 2 WEEK-U 4 WEEK-F
5 0 EL u
4 WEEK-U 0
100
200
300
400
SECONDS
FIG. 3. (A) Total SI at either 2 or 4 weeks following either sham-castration or castration between 55 and 60 days of age. * A statistically significant difference between SI in the familiar and unfamiliar environment. ’ Statistically different from sham-castrated rats and rats tested 2 weeks postcastration (familiar environment). (B) Behavioral composition of SI in rats either sham-castrated or castrated at 55-60 days and tested for SI either 2 or 4 weeks later. See text for probability level of significance.
reminiscent of other sex differences in anxiety/stress-related behavioral responses in that female rats appear to be less emotional than male rats in response to novelty. For example, Beatty and Fessler (1976) have shown that adult female rats ambulate more and defecate less than adult male rats when placed in an open field (i.e., a lesser emotional response). Interestingly, emotionality in the open field was increased by castration in adult female rats (Blizard et al., 1973, while castration in adult male rats had no effect on open-field behavior (Brand and Slob, 1988). This
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finding suggests that the hormonal environment of the adult female rat may exert an influence on anxiety-related responses. In fact, injections of ovarian hormones, such as progesterone and estrogen, have been shown to increase shock-punished responses in both females and neonatally castrated male adult rats (Rodriquez-Sierra, Hagley, and Hendricks, 1986). In the present study, therefore, the lack of a differential response to the two environments in adult female rats may be partially due to an anxiolytic/inhibitory effect of ovarian hormones on SI in the unfamiliar environment. In support of a role for ovarian hormones in the expression of SI in adult female rats is the finding that while both male and female rats at 35 days of age display a differential response to the two environments (Primus and Kellogg, 1989b), only male rats display the differential response at 60 days. This change in SI in female rats as a function of pubertal age implicates an influence of ovarian hormones on the development of SI. Exposure of juvenile castrated male rats to testosterone propionate over Days 30 through 60 prevented the effect of castration on SI in the unfamiliar environment. That is, SI was decreased in the unfamiliar environment relative to SI in the familiar environment in TP-exposed castrated male rats. Thus, the differential response to the two environments in intact adult male rats (Fig. 2A) was not present in TP-exposed juvenile castrated male rats (Fig. 2B). The presence of testosterone (or one of its metabolites) between 30 and 60 days of age, therefore, appears to be critical for the expression of environment-related changes in SI in adult male rats. The possibility exists, however, that TP exposure does not directly alter SI between rats, but rather alters behavioral responses of the rat to its surrounding environment. That is, decreased SI in the unfamiliar environment in TP-exposed male rats may have resulted from a concomitant increase in behavioral interactions with the surrounding unfamiliar environment. Previous work (File and Hyde, 1978) has shown, however, that displacement activity (rearing, grooming behavior), and not environmental exploration, increases in the unfamiliar environment during SI in intact adult male rats. In addition, intact adult male rats spend less time in exploration of novel stimuli than adult female rats (Russell, 1977), indicating that exploration of environmental novelty is not increased by the presence of testosterone. The reinstatement of environment-related SI by exposure to TP during puberty demonstrates, therefore, that secretion of gonadal androgens during puberty may indeed underlie the development of SI in male rats. Significant changes in the steroidogenic pathways of rat testes have been shown to occur between the onset of puberty (30-35 days of age) and early adulthood (55-60 days of age) (Ojeda and Urbanski, 1988). In addition, the induction of sex differences in central nervous system (CNS) function by gonadal steroids has been shown to occur during both peri-
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HORMONES
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SI
321
natal (MacLusky and Naftolin, 1981) and peripubertal (Brand and Slob, 1988; Clough and Rodriquez-Sierra, 1983) periods of development. Hence, the expression of sexually dimorphic SI in the rat may depend on an organizational effect of testicular secretions on the CNS during pubertal development. Not only do gonadal steroids exert important organizational influences on behavior during early development, the presence of such steroids in adulthood are often required for the activation of these behaviors (Arnold and Breedlove, 1985). That TP exposure over Days 30 through 60 exerted an organizational effect on environment-related SI and that its presence at 60 days did not simply activate appropriate circuitry was verified by testing male rats castrated in adulthood. In the present study, the differential response to the two environments was present in rats castrated in early adulthood and tested for SI either 2 or 4 weeks later. Thus, the presence of testicular secretions in adult male rats appeared not to be necessary for the maintenance of environment-related SI. The lack of effect of adult castration on SI in male rats also demonstrated that testosterone, per se, does not induce the decrease in SI in the unfamiliar environment. That testosterone does not induce anxiety-related responses in the rat is further supported by the observation that the amount of emotionality displayed in male rats in the open-field test is unaltered by castration in adulthood (Brand and Slob, 1988). The findings from this study indicate that environment-related SI in the adult rat is a sexually dimorphic behavior. Furthermore, the expression of the differential response to the two environments in adult male rats appears to depend on exposure to testicular secretions of testosterone (or one of its metabolites) during the pubertal period of development. In addition, the finding that SI in male rats is unaltered at both 2 and 4 weeks following adult castration provides additional support for puberty as a critical period for the development of environment-related SI. The 4-week postcastration interval was of comparable duration to the period of TP exposure following juvenile castration. That exposure to gonadal androgens during puberty is necessary for the continued development of environment-related SI is supported by the findings from this study. However, it is unknown whether testicular function, only during a restricted period of puberty, is required for the development of environment-related SI in male rats. In addition, since metabolites of testosterone are also produced during puberty, it is impossible to state at this time whether it is testosterone or one of its metabolites that is critical for the development of SI in the male rat. Nevertheless, the fact that both male and female rats demonstrate environment-related SI at 3.5 days, but only male rats show the effect of the unfamiliar environment at 60 days, suggests that the emergence of the different gonadal hormones in the two sexes during adolescence may differentially influence under-
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lying neural systems. The results of this study clearly reveal the importance of gonadal hormones during puberty in the expression of environment-related SI in the rat. ACKNOWLEDGMENTS This work was supported in part by Grant No. MH 31850 and by a Research Scientist Development Award (MHOO651) to C. Kellogg, both from the National Institute of Mental Health. We thank Dr. Richard Aslin for the loan of video taping equipment and Dr. Ernest Nordeen for his helpful guidance in the preparation of the manuscript.
REFERENCES Arnold, A. P., and Breedlove, S. M. (1985). Organizational and activational effect of sex steroids on brain and behavior: A reanalysis. Harm. Behav. 19, 469-499. Beatty, W. W., and Fessler, R. G. (1977). Gonadectomy and sensitivity to electric shock in the rat. Physiol. Behav. 19, l-6. Beatty, W. W., and Fessler, R. G. (1976). Ontogeny of sex differences in open-field behavior and sensitivity to electric shock in the rat. Physiol. Behav. 16, 413-417. Blizard, D. A., Lippman, R. H., and Chen, J. J. (1975). Sex differences in open-field behavior in the rat: The inductive and activational role of gonadal hormone. Physiol. Behav. 14, 601-608. Brand, T., and Slob, A. K. (1988). Peripubertal castration of male rats, adult open field ambulation and partner preference behavior. Behav. Brain Res. 30, 11 l-l 17. Candland, D. K., and Campbell, B. A. (1962). Development of fear in the rat as measured by behavior in the open field. J. Comp. Physiol. Psych. 55, 593-596. Clough, R. W., and Rodriquez-Sierra, J. F. (1983). Synaptic changes in the hypothalamus of the prepubertal female rat administered estrogen. Amer. J. Anat. 167, 205-214. Damassa, D. A., Smith, E. R., Tennet, B., and Davidson, J. M. (1977). The relationship between circulating testosterone levels and male sexual behavior in rats. Horm. and Behav.
8, 275-286.
File, S. E. (1980). The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J. Neurosci. Methods 2, 219-238. File, S. E. (1988). How good is social interaction as a test of anxiety? Anim. Mode/s Psych. Disord. 1, 151-166. File, S. E., and Hyde, J. R. G. (1978). Can social interaction be used to measure anxiety. Brit. J. Pharmacol. 64, 19-24. File, S. E., and Peet, L. A. (1980). The sensitivity of the rat corticosterone response to environmental manipulations and to chronic chlordiazepoxide treatment. Physiol. Behav.
25, 753-758.
MacLusky, N. J., and Naftolin, F. (1981). Sexual differentiation of the central nervous system. Science 221, 1294-1311. Ojeda, S. R., and Urbanski, H. F. (1988). Puberty in the rat. In E. Knobil and J. Neil1 (Eds.), The Physiology of Reproduction. Raven Press, New York. Primus, R., and Kellogg, C. K. (1989a). Pubertal-related changes influence the development of environment-related social interaction in male rats. Dev. Psychobiol. 22(6), 633643. Primus, R., and Kellogg, C. K. (1989b). Sexually dimorphic aspects of social interaction in adult rats are altered by prenatal exposure to diazepam. Sot. Neurosci. Abstr. 15(l), 415, 167.7. Rodriguez-Sierra, J. F., Hagley, M. T., and Hendricks, S. E. (1986). Anxiolytic effects of progesterone are sexually dimorphic. Life Sci. 38, 1841-1845.
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Russell, P. A. (1977). Sex differences in rats’ stationary exploration as a function of stimulus and environmental novelty. Anim. Learn Behav. S(3), 297-302. Slob, A. K., Huizer, T., and Van Der Werff Ten Bosch, J. J. (1986). Ontogeny of sex differences in open-field ambulation in the rat. Physiol. Behav. 37, 313-315. Sodersten, P., Damassa, D. A., and Smith, E. R. (1977). Sexual behavior in developing male rats. Horm. Behav. 8, 320-341.