Schulz endocrinology 2009

NEUROENDOCRINOLOGY

Testosterone Programs Adult Social Behavior before and during, But Not after, Adolescence Kalynn M. Schulz, Julia L. Zehr, Kaliris Y. Salas-Ramirez, and Cheryl L. Sisk Department of Psychology and Neuroscience Program, Michigan State University, East Lansing, Michigan 48824

Whereas the adolescent brain is a major target for gonadal hormones, our understanding of hormonal influences on adolescent neural and behavioral development remains limited. These experiments investigated how variations in the timing of testosterone (T) exposure, relative to adolescence, alters the strength of steroid-sensitive neural circuits underlying social behavior in male Syrian hamsters. Experiment 1 simulated early, on-time, and late pubertal development by gonadectomizing males on postnatal d 10 and treating with SILASTIC brand T implants for 19 d before, during, or after adolescence. T treatment before or during, but not after, adolescence facilitated mating behavior in adulthood. In addition, preadolescent T treatments most effectively increased mating behavior overall, indicating that the timing of exposure to pubertal hormones contributes to individual differences in adult behavior. Experiment 2 examined the effects of preadolescent T treatment on behavior and brain regional volumes within the mating neural circuit of juvenile males (i.e. still preadolescent). Although preadolescent T treatment did not induce reproductive behavior in juvenile males, it did increase volumes of the bed nucleus of the stria terminalis, sexually dimorphic nucleus, posterodorsal medial amygdala, and posteroventral medial amygdala to adult-typical size. In contrast, juvenile anterodorsal medial amygdala and ventromedial hypothalamus volumes were not changed by preadolescent T treatment yet differed significantly in volume from adult controls, suggesting that further maturation of these brain regions during adolescence is required for the expression of male reproductive behavior. Thus, adolescent maturation of social behavior may involve both steroid-independent and -dependent processes, and adolescence marks the end of a postnatal period of sensitivity to steroid-dependent organization of the brain. (Endocrinology 150: 3690 –3698, 2009)

he brain is a key target of gonadal hormones such as estradiol and testosterone (T), and numerous animal studies document the powerful effects of these hormones on a variety of behaviors ranging from sexual interest to cognitive abilities. We have known for almost 50 yr that exposure to gonadal hormones during early neural development permanently alters sex-specific behavioral responses to gonadal hormones in adulthood, a seminal finding that spawned the idea that hormones organize behavioral circuits during sensitive periods of development (1). Despite this understanding, only a handful of studies have investigated the contribution of pubertal hormones to adolescent brain development and whether hormone action in the brain during this postnatal developmental period results in enduring changes in adult behavior.

The scarcity of animal research investigating pubertal hormone influences on adolescent brain development is particularly

T

notable considering that deviations in pubertal timing are associated with adolescent-emerging psychopathologies such as depression, anxiety, disordered eating, and conduct disorder (2–10). The effects of variation in pubertal timing on psychopathology have largely been attributed to psychosocial factors that come into play with the intense changes in experience that accompany sexual maturation. However, gonadal hormones secreted at pubertal onset also have wide-ranging effects on the brain. Thus, mistimed, direct hormonal influences on the brain may also skew the course of adolescent brain development toward increased risk for psychopathology (8). If so, then the timely diagnosis and treatment of disorders of pubertal timing have repercussions that extend beyond normalization of reproductive function to more global influences on social behavior.

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-1708 Received December 9, 2008. Accepted April 28, 2009.

First Published Online May 7, 2009

Abbreviations: BSTpm, Posterior medial bed nucleus of the stria terminalis; GDX, gonadectomy; MeAD, medial anterodorsal; MeAV, medial anteroventral; MePD, medial posterodorsal; MePV, medial posteroventral; MPN, medial preoptic nucleus; P, postnatal day; SDN, sexually dimorphic nucleus; T, testosterone; VMH, ventromedial hypothalamus.

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Animal models have immense potential for elucidating the mechanisms by which gonadal hormones directly impact adolescent neural and behavioral development. For example, depriving Syrian hamsters of hormones during adolescence compromises social behaviors, even after hormones are replaced in adulthood (11). This suggests that exposure of the adolescent brain to gonadal hormones organizes behavioral circuits and programs long-lasting behavioral responses. Furthermore, these data imply that a window of sensitivity to organizational effects of gonadal steroid hormones may close after adolescence (12), a possibility we tested herein.

The current studies sought to define the temporal parameters spanning the pre- and postadolescent periods within which neural circuits mediating social behavior are sensitive to the organizational effects of gonadal steroid hormones. Although previous work has established that perinatal exposure to gonadal steroid hormones masculinizes and defeminizes behavioral neural circuits, more recent work suggests that a second window of sensitivity may open at adolescence. For example, steroid hormones fail to elicit maximal expression of male reproductive behavior immediately before adolescence (13, 14) but readily activate high levels of reproductive behavior after adolescence, raising the possibility that adolescence is a second sensitive period for the organizational effects of gonadal steroid hormones on male reproductive behavior (15). Experiment 1 tested the hypothesis that brain sensitivity to hormonal influences fluctuates over the course of postnatal development. After gonadectomy (GDX) on postnatal d 10, we simulated early, on-time, or delayed onset of pubertal hormone secretion by administering SILASTIC brand T capsules (Dow Corning Corp., Midland, MI) before, during, or after adolescent development and examined the potential for sexual behavior in adulthood.

We also sought to extend the previous finding that 1 week of preadolescent T-treatment does not facilitate mating behavior in juveniles in Exp. 2 by 1) determining whether a longer duration of preadolescent T-treatment facilitates mating behavior in juveniles, 2) determining what behaviors are displayed by juveniles in the presence of estrous females, and 3) identifying which brain regions underlying social behaviors are capable of steroid-dependent organization before adolescence, and which regions undergo additional maturation during adolescence. Identifying the brain regions capable of steroid-dependent change before adolescence and those that require further maturation during adolescence is an essential first step in understanding the brain regions underlying the adolescent maturation of social behaviors.

Materials and Methods Animals Timed-pregnant female Syrian hamsters (Mesocricetus auratus) arrived from Harlan Sprague Dawley laboratories (Madison, WI) on gestational d 4. Pregnant females were housed in clear polycarbonate cages (37.5 ⫻ 33 ⫻ 17 cm) provided with nesting materials, food (Telkad Rodent diet no. 8640; Harlan, Madison, WI) and water. Females were exposed to a 14-h light, 10-h dark schedule, and the temperature was maintained at 21 ⫾ 2 C. Nests were checked twice daily for births beginning on gestational d 15, 1 d before the predicted birth. On postnatal

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d (P) 9, litters were sexed and culled to six to eight mixed-sex pups. Males were housed with mothers and littermates until weaning and single housing on P19 (cage dimensions 30.5 ⫻ 10.2 ⫻ 20.3 cm). Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and protocols were approved by the institutional animal care committee.

Experimental design Experiment 1: manipulation of the timing of periadolescent exposure to T Figure 1 depicts the design of experiment 1. Males were randomly assigned to treatment groups and GDX or sham castrated at P10. To

FIG. 1. Schematic depicting experiment 1 design. We define adolescence here as the time period encompassing not only reproductive maturation (puberty) but also cognitive, emotional, and social maturation (57), which occurs approximately between 28 and 56 d of age in male Syrian hamsters. Although puberty and adolescence are normally temporally coordinated, variations in the timing of puberty do occur and raise the question of whether brain development is similarly shaped by pubertal hormones when experienced outside the normal age range of adolescence. Here we attempted to examine the effects of temporal dissociations between puberty and adolescence on adult sexual behavior.

Experimental males were gonadectomized on P10. To simulate early, on-time, or delayed puberty, males were exposed to blank- or T-filled SILASTIC brand capsules before adolescence (P10 –29; row 1), during adolescence (P29 – 48; row 2), or after adolescence (P64 – 83; row 3). In adulthood, 4 wk after pellet removal, both blank and T-treated males were implanted with T-filled capsules and behavior tested 1 wk later (P64, 83, or 118). This design kept constant the time interval between the 19-d hormone treatment (blank or T pellet) and the time of adult T treatment and behavior testing (BEH). The 19- and 7-d T treatments served two different purposes. The 19-d T treatment period was intended to simulate early, on-time, or delayed onset of pubertal hormone secretion as a means of probing the ability of gonadal hormones to organize behavioral neural circuits at different ages relative to normal adolescence. The 1-wk treatment in adulthood was used to provide adult physiological levels of T as a means of probing the ability of gonadal hormones to activate sexual behavior after 1 wk of hormone priming in the groups that had experienced different histories of pubertal timing. In addition, every experimental group was raised with age-matched, sham-treated males that received all sham surgeries (sham gonadectomy and blank pellet implant) at the same time as experimental groups and were also behavior tested on the same day in adulthood. Behavioral comparisons between these groups controlled for the possible influence of age at the time of testing in young adulthood.

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simulate early, on-time, or delayed puberty, males were exposed to blank- or T-filled SILASTIC brand capsules before, during, or after adolescence. In adulthood, 4 wk after pellet removal, both blank and Ttreated males were implanted with T-filled capsules and behavior tested 1 wk later. This design kept constant the time interval between the 19-d hormone treatment (blank or T pellet) and the time of adult T treatment and behavior testing.

Because our experimental objectives required testing males at three different ages in young adulthood, sham males were included as age controls so that age-related differences in behavior could be detected independently of experimental manipulation (Fig. 1). These sham groups were chosen randomly from the same litters as GDX males and were sham GDX at P10 and tested for sexual behavior concurrently with experimental groups on P64, 83, or 118. The behaviors of these groups were statistically compared for detection of age-related changes in adult behavioral responses to T.

Experiment 2: juvenile vs. adult comparisons Males were GDX on P10 and administered 19 d of blank or T capsules. Males were tested for sexual behavior with a hormone-primed adult female on P29 (still juvenile), and brains were collected immediately after testing. The behavior and brain region volumes of juveniles were compared with a group of males that were GDX in adulthood, after experiencing endogenous T during perinatal and pubertal development.

This group was sham operated at P10, GDX at P64, treated with T 4 wk later at P92, and tested for sexual behavior on P99. Thus, the behavior of this group is representative of males that underwent normal pubertal development and whose sexual behavior was assessed in adulthood using a reinstatement paradigm (i.e. hormone replacement 4 wk after GDX).

Tissue collection, histology, and volume measurements Male hamsters were euthanized with an overdose of sodium pentobarbital (130 mg/kg ip) after behavior testing and perfused intracardially with 4% paraformaldehyde (13). Brains were sectioned at 40 ␮m on a cryostat, and every fourth section was thaw mounted onto glass slides, allowed to dry, and then thionin stained and coverslipped. Hypothalamic and medial amygdalar regions were traced bilaterally at ⫻ 40 magnification by an observer blind to experimental treatment using Neurolucida (version 6; Microbrightfield, Williston, VT), and volumes were calculated by summing the traced cross-sectional areas and multiplying by the distance between sections (160 ␮m). The subnuclei traced for volume measurements were selected because of their steroid hormone sensitivity and/or well documented involvement in both mating and agonistic behaviors in hamsters and other rodent species (e.g. Refs. 16 –25). The hypothalamic regions traced included the sexually dimorphic nucleus (SDN), posterior medial bed nucleus of the stria terminalis (BSTpm) medial preoptic nucleus (MPN), and ventromedial hypothalamus (VMH). We did not measure the MPN magnocellular in this study because our sampling interval of 160 ␮m did not permit accurate estimation of volume, given that the entire rostral-caudal extent of this nucleus is only approximately 150 ␮m (26). The medial amygdalar regions traced included the anterodorsal (MeAD), and anteroventral (MeAV) regions, and also the posterodorsal (MePD), and posteroventral (MePV) subregions. The hamster brain atlas was used as a reference for tracings (27).

Surgical procedures GDX and sham surgeries were performed under isoflurane anesthesia and aseptic conditions (28). Two sterile SILASTIC brand T pellets (one 7 mm and one 15 mm; inner diameter 1.98 mm; outer diameter 3.18 mm) or two blank SILASTIC brand capsules were inserted sc through a 3- to 4-mm incision made on the dorsal midline, and the incision was sutured closed. Animals were returned to their mothers and littermates after surgery, and their health was monitored closely for the following week.

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geles, CA). Samples from each group were counterbalanced across three different assays, and the intrassay coefficients of variation were 7.7, 4.0, and 8.2%. The interassay coefficient of variation was 14.1%, and the lower detection limit was 0.1 ng/ml for all assays.

Behavioral testing All reproductive behavior tests were conducted 1–5 h after lights out, and the timing of testing across this period was counterbalanced between groups. The male was placed in a 10-gal glass aquarium (51 ⫻ 26 ⫻ 31.5 cm) and allowed to acclimate for 5 min before the introduction of a receptive stimulus female. The behavior tests were 15 min. Ovariectomized stimulus females were brought into behavioral estrus with an injection of 10 ␮g estradiol benzoate (0.2 mg/ml) in sesame oil 48 h before testing, and an injection of 250 ␮g progesterone (5 mg/ml) in sesame oil 3 h before testing. All females were checked for behavioral receptivity before behavior tests by pairing them with colony breeder males for approximately 1 min. Females were paired with only one experimental male per test day.

The behavioral tests were videotaped under dim red light illumination and later scored to assess the number of vaginally oriented mounts, intromissions, and the latencies to mount and intromit females. The criteria for these behaviors have been described previously (13). In addition, the risk assessment behavior stretch-attend and submissive behaviors tail-up walking and escape dashes were quantified. We used the criterion established by Albers et al. (29) when scoring these behaviors.

All videotapes were scored blind to experimental condition by a single observer.

Sample sizes and experimental attrition Twelve males were assigned to each group on P9, 1 day before the first surgical procedures (GDX or sham). Some animals were lost from groups during surgeries and in the postoperative period. Videotape failures also precluded the analysis of some behavioral data. Finally, data were not analyzed if damaged or broken T pellets were found at the time of tissue collection. Therefore, final sample sizes in both experiments ranged from eight to 12 per group.

Statistics Experiment 1: manipulation of the timing of periadolescent exposure to testosterone Mounting behavior of males treated with blank- or T-filled capsules before, during, and after adolescence was analyzed using a 2 ⫻ 3 twoway ANOVA (⫾T treatment ⫻ before, during, or after adolescent age of treatment), and significant differences were probed using Fisher’s protected least significant difference test. This same analysis was used to evaluate the plasma T levels during both the organizational 19-d treatment and the 7-d adult/activational T treatment. Intromissive behavior did not pass the test for normality (skewness ⱕ1.96), and 2 ⫻ 2 ␹2 tests were conducted to determine whether the proportion of males intromitting the female differed between blank and T-treated males. ␹2 tests were conducted separately for males receiving T treatments before, during, or after adolescence.

Experiment 2: juvenile vs. adult comparisons Social behavior. The behaviors of blank-treated juveniles, T-treated juveniles, and adult T-treated controls were compared. Planned comparisons were conducted as opposed to a single-factor ANOVA because the three groups do not represent varying levels of a single factor.

When behavioral data were not normally distributed, 2 ⫻ 2 ␹2 analyses were conducted to determine whether the proportions of males displaying a behavior differed between juvenile (blank or T treated) and adult males.

T RIA Plasma concentrations of T were measured in duplicate 50-␮l samples using the Coat-A-Count total T kit (Diagnostic Products, Los An-

Brain regional volumes. A repeated-measures ANOVA determined that right and left hemispheres did not significantly differ for any brain region.

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FIG. 2. Effects of periadolescent T exposure on adult reproductive behaviors. T treatments were designed to simulate early, on-time, and late pubertal development, and all behavior testing occurred in adulthood. Only pre- and midadolescent T treatments increased mounts (A) and decreased mount latencies (B) in response to T in adulthood. Adult intromissive behavior was increased only by preadolescent T treatments (C). These data suggest that early T treatments enhance behavioral responsiveness to T in adulthood. *, Significant difference (P ⬍ 0.05) between groups.

Thus, hemispheres were summed and planned comparisons were carried out using unpaired t tests to determine regional volume differences between untreated juveniles, T-treated juveniles, and T-treated adult control males.

Planned comparisons were conducted rather than a single-factor ANOVA because the three groups do not represent varying levels of a single factor.

Results Experiment 1: manipulation of the timing of periadolescent exposure to testosterone Mount number and latency, and proportion of males intromitting Treatment (T or blank capsule) and time (before, during, or after adolescence) interacted to influence adult mounting behavior in adulthood 关Fig. 2A; F(2,52) ⫽ 5.70, P ⬍ 0.006兴. T treatment before 关F(2,52) ⫽ 29.65, P ⬍ 0.0001兴 and during adolescence 关F(2,52) ⫽ 9.16, P ⬍ 0.01兴 significantly increased mount number relative to blank-treated males, whereas postadolescent T treatment did not 关F(2,52) ⫽ 0.190, P ⬎ 0.05兴.

T treatment significantly reduced overall mount latencies 关Fig. 2B; F(2,52) ⫽ 15.76, P ⬍ 0.0002兴, and a trend toward an interaction was also observed 关F(1,52) ⫽ 3.03, P ⫽ 0.057兴. T treatments reduced mount latencies when administered before 关F(2,52) ⫽ 15.70, P ⬍ 0.001兴 and during adolescence 关F(2,52) ⫽ 6.84, P ⬍ 0.02兴 but not after 关F(2,52) ⫽ 0.15, P ⬎ 0.05兴.

Preadolescent T treatment significantly increased the proportion of males intromitting in adulthood 关blank: two of 11; T: eight of 10; ␹2 ⫽ 8.03, P ⬍ 0.005兴, whereas mid- 关␹2 ⫽ 0.69, P ⬎ 0.05兴 and postadolescent 关␹2 ⫽ 0.11, P ⬎ 0.05兴 treatments did not (Fig. 2C).

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Behavior of sham-operated age-control groups and unmanipulated controls One-way ANOVA was used to compare the behaviors of adult males sham-operated before, during, or after adolescence with unmanipulated controls that were not exposed to any surgical procedure. These four groups did not differ significantly in any behavior, indicating that the behavioral differences observed between the castrate groups described above are not attributable to age at the time of testing or surgical procedures 关Table 1; mounts: F(3,38) ⫽ 0.890, P ⫽ 0.46; mount latency: F(3,38) ⫽ 1.9, P ⫽ 0.15; intromissions: F(3,38) ⫽ 1.91, P ⫽ 0.144; intromission latency: F(3,38) ⫽ 0.96, P ⫽ 0.423兴.

Plasma T concentrations Blood samples were taken at two experimental time points to determine: 1) the plasma concentrations of T produced by SILASTIC brand capsules implanted for 19 d before, during, or after adolescence and designed to simulate early, on-time, and delayed puberty; and 2) the plasma concentrations of T produced

by SILASTIC brand capsules implanted for 7 d in adulthood and designed to probe activation of mating behavior. At the end of the 19-d treatment period, males administered T-filled capsules had significantly higher concentrations of T than males given blank-filled capsules 关F(1,44)⫽19.52, P ⬍ 0.001兴. No significant differences in plasma T concentrations were found across the three 19-d treatment periods before, during, or after adolescence 关F(1,44)⫽1.66, P ⫽ 0.20兴, indicating that circulating levels of T were similar during the three organizational treatment times (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

The 7-d activational T treatment received by all animals in adulthood yielded small but significant differences in plasma T concentrations among groups 关F(2,52) ⫽ 8.62, P ⬍ 0.001兴.

Specifically, the 7-d capsule implanted in adulthood yielded higher T concentrations in the two groups that had received 19-d blank or T-filled capsules as juveniles, compared with the groups that had received blank or T-filled capsules during adolescence (P ⬍ 0.003) or in adulthood (P ⬍ 0.001; supplemental Table 1). However, for all six groups, mean plasma T concentrations were within the normal adult physiological range of 2–7 ng/ml (30, 31).

TABLE 1. The behaviors of sham-operated age controls and unmanipulated control males Mounts

lntromissions

Age at testing controls (d)

n Mean ⴞ SEM

Latency (sec) Mean ⴞ SEM

n Mean ⴞ SEM

Latency (sec) Mean ⴞ SEM

Prepubertal sham Adolescent sham Adult sham Unmanipulated

7.22 ⫾1.56 7.92 ⫾ 1.31 8.36 ⫾ 0.92 10.20 ⫾ 1.44

101.98 ⫾ 42.39 67.70 ⫾ 15.65 144.93 ⫾ 35.47 66.30 ⫾ 9.36

17.67 ⫾ 3.30 24.92 ⫾ 3.03 20.00 ⫾ 1.68 24.70 ⫾ 1.75

131.91 ⫾ 39.54 157.30 ⫾ 26.02 218.10 ⫾ 45.72 179.30 ⫾ 34.82

No significant differences were found between these control groups.

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Experiment 2: juvenile vs. adult comparisons Reproductive behavior Blank and T-treated juveniles were significantly less likely to mount the female than adult controls 关Fig. 3; Juv⫹0 (one of nine) vs. adult control (12 of 12): ␹2 ⫽ 17.23, P ⬍ 0.0001; Juv⫹T (five of 11) vs. adult control (12 of 12): ␹2 ⫽ 8.86, P ⬍ 0.003兴. The proportion of males mounting the female did not significantly differ between blank- and T-treated juvenile groups 关Fig. 3; one of nine vs. five of 11, ␹2 ⫽ 2.780, P ⫽ 0.10兴.

FIG. 3. Juvenile (Juv) vs. adult behavioral responses to estrous females.

Behavior testing occurred at 29 (juvenile) and 99 (adult) d of age, respectively. Preadolescent T treatment did not increase juvenile copulatory behavior to adult control levels. However, a significantly higher proportion of juvenile males displayed risk assessment (stretch attend) and submissive behaviors (tail up and escape dashes) than did adult males. Preadolescent T treatment also reduced the proportion of juvenile males displaying escape dashes. These data indicate that juveniles and adults display very different behavioral strategies during interactions with estrous females: adults engage in mating behavior, whereas juveniles engage in risk assessment and submissive behaviors. Bars sharing a letter do not differ significantly from one another.

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Blank and T-treated juveniles were significantly less likely than adults to display intromissive behavior 关Fig. 3; Juv⫹0 (none of nine) vs. adult control (nine of 12): ␹2 ⫽ 11.81, P ⬍ 0.0006; Juv⫹T (one of 11) vs. adult control (nine of 12): ␹2 ⫽ 10.15, P ⬍ 0.002兴, and the proportion intromitting did not differ between blank- and T-treated juvenile groups 关Fig. 3; none of nine vs. one of 11; ␹2 ⫽ 0.86, P ⬎ 0.05兴.

Risk assessment and submissive behaviors Given the relative lack of sexual behaviors observed in juveniles, we quantified three additional behaviors displayed by these young animals during encounters with the receptive female. The proportion of males displaying stretch-attend, a behavior associated with risk assessment, was significantly higher in both blank- and T-treated juvenile groups than the adult control group 关Fig. 3; Juv⫹0 (seven of nine) vs. adult control (0 of 12): ␹2 ⫽ 14.00, P ⬍ 0.0002; Juv⫹T (seven of 11) vs. adult control (none of 12): ␹2 ⫽ 11.00, P ⬍ 0.001兴. However, the proportion of blank- and T-treated juveniles displaying stretch-attend did not significantly differ 关Fig. 3; seven of nine vs. seven of 11, ␹2 ⫽ 0.50, P ⬎ 0.05兴.

Both blank- and T-treated juvenile groups had a significantly higher proportion of males displaying tail-up walking behavior than the adult control group 关Fig. 3; Juv⫹0 (nine of nine) vs. adult control (one of 12): ␹2 ⫽ 17.33, P ⬍ 0.0001; Juv⫹T (10 of 11) vs. adult control (one of 12): ␹2 ⫽ 15.7, P ⬍ 0.0001兴. A similar proportion of blank- and T-treated juveniles displayed tail-up walking 关Fig. 3; nine of nine vs. 10 of 11, ␹2 ⫽ 0.86, P ⬎ 0.05兴.

Both blank and T-treated juvenile groups had a significantly higher proportion of males displaying escape dashes than the adult control group 关Fig. 3; Juv⫹0 (eight of nine) vs. adult control (none of 12): ␹2 ⫽ 17.23, P ⬍ 0.0001; Juv⫹T (five of 11) vs. adult control (none of 12): ␹2 ⫽ 7.0, P ⬍ 0.01兴. A significantly higher proportion of blank-treated juveniles displayed escape dashes than the T-treated juveniles 关Fig. 3; eight of nine vs. five of 11, ␹2 ⫽ 4.10, P ⬍ 0.05兴.

Brain regional volumes Preadolescent T treatment significantly increased the volume of the SDN in juveniles 关Fig. 4A; t(1,19) ⫽ 2.25, P ⫽ 0.04兴 relative to blank-treated juveniles. SDN volumes of adult controls were intermediate to that of T-treated and blank-treated juveniles but not significantly different from either group 关Juv⫹0 vs. adult: t(1,15) ⫽ 1.56, P ⬎ 0.05; Juv⫹T vs. adult: t(1,16) ⫽ 1.11, P ⬎ 0.05兴.

Preadolescent T treatment also significantly increased the volume of the BSTpm in juveniles relative to blank-treated juveniles 关Fig. 4B; t(1,19) ⫽ 4.81, P ⫽ 0.0001兴, such that T-treated juveniles and adult controls did not differ in volume 关t(1,16) ⫽ 0.89 P ⬎ 0.05兴, but volumes of both these groups were significantly larger than blank-treated juveniles 关Juv⫹0 vs. adults: t(1,15) ⫽ 4.04, P ⫽ 0.001兴.

MPN volumes did not differ among the three groups 关Fig. 4C; Juv⫹0 vs. Juv⫹T, t(1,19) ⫽ 0.29, P ⬎ 0.05; Juv⫹0 vs. adults, t(1,15) ⫽ 0.12, P ⬎ 0.05; Juv⫹T vs. adults, t(1,16) ⫽ 0.38, P ⬎ 0.05兴.

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controls had similar plasma T concentrations 关P ⫽ 0.97兴, and both displayed significantly higher T concentrations than blanktreated juveniles (Juv⫹0 vs. Juv⫹T, P ⬍ 0.0001; Juv⫹0 vs. adult controls, P ⬍ 0.0001; supplemental Table 1).

Discussion These data support the hypothesis that adolescence marks the end of a single, protracted postnatal period of nervous system sensitivity to the organizing actions of T, which is likely an extension of the perinatal FIG. 4. Juvenile (Juv) vs. adult regional volumes within the hypothalamus. Brains were collected at 29 period of hormone-dependent sexual differen(juvenile) and 99 (adult) d of age, respectively. Preadolescent T treatment significantly increased the tiation. When T exposure after P10 was revolumes of both the SDN (A) and BSTpm (B) in juvenile males but not the MPN (C). Preadolescent T treatment did not significantly influence VMH volume in juvenile males (D). However, adult controls had stricted to a discrete 19-d treatment period eisignificantly greater volumes than juvenile males, suggesting that the VMH does not reach adult size until ther before, during, or after adolescence, both after adolescence. Bars sharing a letter do not differ significantly from one another. Middle panel, pre- and midadolescent T treatment, but not Photomicrographs of representative Nissl-stained coronal sections containing the hypothalamic brain regions traced. 3V, Third ventricle; OT, optic tract. Scale bar, 250 ␮m. postadolescent T treatment, enhanced T-activated reproductive behavior later in adulthood, demonstrating that adolescence is not an isolated sensitive In the VMH, no significant differences between blank- and period for the organizing actions of T on adult reproductive behavT-treated juveniles were observed 关Fig. 4D; t (1,19) ⫽ 0.48, P ⬎ ior. Moreover, the potential for T to organize behavioral circuits 0.05兴. In contrast, the VMH of adult males was significantly appears to decrease across postnatal development because activalarger than both blank- 关t (1,15) ⫽ 2.77, P ⫽ 0.01兴 and T-treated tion of behavior by T in adulthood was enhanced when exposure to juveniles 关Fig. 4D; t (1,16) ⫽ 2.15, P ⫽ 0.046兴.

T occurred earlier rather than later. Thus, the classical view of orPreadolescent T treatment significantly increased the volume ganizational and activational mechanisms of steroid action should of both the MePD 关Fig. 5A; t(1,19) ⫽ 4.20, P ⬍ 0.0006兴 and be revised to incorporate an extended window of decreasing postMePV of juveniles 关Fig. 5B; t(1,19) ⫽ 2.14, P ⬍ 0.05兴, relative to natal sensitivity to the organization of adult mating behavior by blank-treated juveniles. T treatment increased volumes in the steroid hormones. Furthermore, these results have implications for MePD and MePV such that no differences were observed bethe treatment of delayed puberty because they indicate that the later tween T-treated juveniles and adult controls 关MePD: t(1,15) ⫽ hormone therapy is begun, the less organizational influences hor1.64, P ⬎ 0.05; MePV: t(1,15) ⫽ 1.62, P ⬎ 0.05兴, yet these mone replacement will have on behavioral circuits. groups had significantly greater volumes than blank-treated juPerinatal hormone secretions masculinize and defeminize veniles 关MePD: Juv⫹0 vs. adult control, t(1,16) ⫽ 2.70, P ⬍ neural circuits, but relatively few have investigated whether sim0.02; MePV: Juv⫹0 vs. adult control, t(1,16) ⫽ 3.78, P ⬍ 0.002兴. ilar effects are possible before (32) or during adolescence (11, In contrast, preadolescent T treatment had no effect on vol33–35). Furthermore, studies have not directly compared whether umes of the MeAD and MeAV subregions of the anterior medial T exposure across the pre-, mid-, and postadolescent periods has amygdala in juveniles (Fig. 5, C and D; MeAD: t(1,19) ⫽ 0.21, differential effects on neural circuits underlying social behavior.

P ⬎ 0.05; MeAV: t(1,19) ⫽ 1.16, P ⬎ 0.05兴. However, age Bloch and Mills (32) demonstrated that 15 d of juvenile T treatinfluenced regional volumes within the MeAD because volumes ment masculinizes and defeminizes adult reproductive behavior were significantly larger in both blank- 关t(1,16) ⫽ 3.52, P ⬍ in neonatally GDX male rats. The same hormone treatment also 0.003兴 and T-treated 关t(1,15) ⫽ 4.42, P ⬍ 0.0005兴 juvenile defeminizes adult lordosis behavior in females (36). Our data groups than adult controls. support their conclusion that organizing actions of T are possible To summarize, preadolescent T treatment increased the size well beyond the neonatal period and extend their findings by of the SDN, BSTpm, MePD, and MePV in juveniles relative to demonstrating that the window for organization by T closes in blank-treated juveniles. In contrast, preadolescent T treatment early adulthood. We note, however, that under normal develdid not affect VMH or MeAD volume of juveniles, yet volumes opmental circumstances, nervous system organization by T is of VMH and MeAD were, respectively, larger and smaller in driven by marked elevations in endogenous testicular secretions adults relative to both groups of juveniles, suggesting that adoduring two distinct phases of life: the perinatal and adolescent lescent changes in these two structures may be independent of puperiods. bertal hormones.

Many other social behaviors have organizational links to the adolescent period (for review see Ref. 37), but whether these Plasma T concentrations behaviors also exhibit an overall postnatal decline in sensitivity Plasma T levels differed across the experimental groups to the organizing actions of gonadal steroid hormones is un关F(2,29) ⫽ 25.77, P ⬍ 0.0001兴. T-treated juveniles and adult Downloaded from endo.endojournals.org at Univ of CO Denver Med Campus Hlth Sci Lib on October 10, 2009

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VMH volumes and larger MeAD volumes than adults. Thus, age-dependent and possibly steroid-independent maturation of these regions occurs during adolescence. In addition, the MeAD and VMH are key regions within a neural circuit responsible for suppressing mating behavior in the face of threatening stimuli in mice (38). Thus, the differences in VMH and MeAD volumes found between juvenile and adult males coupled with the highly defensive behaviors only juveniles display toward estrous females suggest that development of the medial amygdala and VMH during adolescence may facilitate the appropriate assessment of estrous females as nonthreatening potential mates, but this possibility FIG. 5. Juvenile (Juv) vs. adult regional volumes within medial amygdalar brain regions. Brains were collected at 29 (juvenile) and 99 (adult) d of age, respectively. Preadolescent T treatment significantly awaits further experimental verification. increased the volume of subdivisions within the posterior medial amygdala (A and B) of juvenile males but In contrast to the MeA and VMH, preadnot the anterior medial amygdala (C and D). However, age influenced regional volumes within the MeAD olescent T treatment significantly increased (C). Specifically, MeAD volumes were significantly larger in both blank- and T-treated juvenile groups than adult controls, suggesting that the MeAD does not reach adult size until after adolescence (C). Bars volumes of the SDN, BST, MePD, and MePV sharing a letter do not differ significantly from one another. Middle panel, Photomicrographs of in juveniles relative to blank-treated juverepresentative Nissl-stained coronal sections containing the medial amygdalar brain regions traced. 3V, niles. These regions may be the neural targets Third ventricle; OT, optic tract. Scale bar, 250 �m. for steroid-dependent organization of mating behavior across the pre- and midadolescent periods, as inknown and likely depends on both the sex of the subject and the dicated by the behavioral results of experiment 1. Although we particular behavior (37). Thus, additional studies are needed that cannot exclude the possibility that the observed volume increases carefully control for the timing of steroid hormone exposure reflect transient activational rather than long-term organizabefore, during, and after adolescence before adult behavioral tional effects of T on regional volumes, these results clearly inassessment. dicate that regional volumes of several brain areas important for An intriguing quandary is why preadolescent T treatments mating are responsive to T before adolescence, even in the abfacilitate the adult display of mating behavior, yet preadolescent sence of behavioral responses to T at this age. Although the T treatments fail to elicit mating behavior in juvenile males. The MePD and SDN are known for their plasticity in response to the primary objective of experiment 2 was to address this question. presence or absence of gonadal hormones in adulthood (16, 23, Extending the duration of preadolescent T treatment to 19 d was 39, 40), volumes of the BST and MePV are not as sensitive to no more successful in activating juvenile mating behavior than fluctuations in adult gonadal hormones (16). Thus, the T-inwere previously used 7-d T treatments (13), suggesting that reduced increases in regional volumes in juveniles likely reflect gardless of the duration of T treatment, juveniles are not capable both long-term organizational and transient activational effects of displaying adult-like mating behavior. In contrast, juveniles of T. Future studies will be aimed at determining whether the displayed very high levels of defensive behavior during testing, observed volume increases are due to increases in cell number, even though hormone-primed female Syrian hamsters assume a cell soma size, or changes in dendritic morphology. stationary lordosis posture during much of behavior testing The mechanism by which the postnatal sensitive period to (even in the absence of tactile stimuli) and therefore pose very organizational effects of T closes at the end of adolescence is little threat to males. Adult males, however, displayed little or no unknown. However, if the sensitive period for exposure to T defensive behavior in the presence of the receptive female, even shares features common to other well-known sensitive periods, when their mating behavior was negligible. These data raise the it may close as a consequence of circuit reorganization or conpossibility that female-elicited defensive behavioral displays solidation during adolescence (41– 44). Specifically, particular may, at least in part, inhibit the ability of T to activate mating experiences occurring during adolescence (e.g. the presence or behavior in juveniles. absence of T) may organize circuits in a fashion that render them Adolescent changes in volume of brain regions mediating soresistant to further modification or change. For example, patcial behaviors may help to explain the transition from defensive terns of synaptic connectivity change across adolescence within to mating interactions with estrous females. Our aim was to the hamster MePD, a key region within the neural circuit undistinguish brain regions capable of steroid-dependent organiderlying male reproductive behavior (17). Dendrites, spine denzation before adolescence from those that continue to develop sities, and spinophilin protein all decrease substantially across during adolescence. The MeAD and VMH differed significantly adolescence, concomitant with dramatic rises in gonadal steroid between juvenile and adult males, irrespective of T treatment, hormones (45). These changes in dendritic morphology may resuggesting these regions may require additional maturation during flect organizational changes induced by T during the adolescent adolescence to facilitate behavioral activation by T in adulthood. sensitive period. Indeed, decreased forebrain spine densities are Specifically, both blank- and T-treated juveniles exhibited smaller Downloaded from endo.endojournals.org at Univ of CO Denver Med Campus Hlth Sci Lib on October 10, 2009

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the physiological manifestation of sexual imprinting in zebra finches (reviewed in Ref. 46). Furthermore, in the absence of imprinting stimuli, spine densities eventually decrease to the point at which subsequent imprinting stimuli can no longer influence spine densities, and the timing is contemporaneous with diminished behavioral sensitivity to imprinting stimuli (42, 47).

Whether time-dependent reductions in MePD dendritic branches and spine densities reflect both the organizational influence of T and the closing of the sensitive period remains to be determined, and experiments testing these possibilities are underway.

Human adolescents exhibit substantial individual variability in pubertal onset (48, 49), and extremes in pubertal timing (early or late) have been associated with a range of human psychopathologies such as depression (4, 10, 50, 51), anxiety (5, 8), conduct disorder (52, 53), and increased alcohol and tobacco use (54, 55). Although these studies demonstrate that pubertal timing influences adolescent and adult psychopathology, many questions remain regarding the underlying mechanisms. For example, it is not clear whether the perception of undergoing puberty earlier or later than peers is more important than an individual’s actual pubertal status in predicting later psychopathology or whether mistimed direct hormonal influences on the brain also influence behavioral psychopathology. To our knowledge, this is the first demonstration in an animal model of how variations in pubertal timing during adolescence (i.e. when hormone exposure occurs relative to adolescent brain development) program variations in adult brain morphology and behavior. Although this research did not directly model a psychiatric disease state, the finding that adult behaviors vary, depending on the timing of hormone exposure across the adolescent period, speaks to the possibility that direct hormone-brain interactions moderate the effects of pubertal timing on many behaviors, even in humans. However, the effects of gonadal hormones in humans on the developing brain may also be modulated by social and environmental factors that change dramatically across the pre-, mid-, and postadolescent periods (10, 56).

Additional research using animal models is needed to elucidate how gonadal hormones and an individual’s unique experience impact the developing adolescent brain to drive individual differences in behavior.

Acknowledgments We thank Sara Hunter, Pamela Montalto, Andrew Poole, and Elizabeth Hingst for their valuable assistance with surgeries, behavior testing, and general animal care. We also thank Lisa Rogers for tissue sectioning and Jane Venier and Lisa Rogers for tissue histology. Thanks also go to Jane Venier for assistance with RIAs. A special thank you goes to Heather Molenda-Figueira for helpful feedback on early manuscript drafts. A portion of manuscript editing was done as part of Julia Zehr’s official duties as a federal government employee.

Address all correspondence and requests for reprints to: Kalynn M. Schulz, Department of Psychiatry and Developmental Psychobiology Research Group, University of Colorado, Denver, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, Colorado 80045. E-mail: kalynn.schulz@ucdenver.edu.

This work was supported by National Institutes of Health Grants R01-MH068764 (to C.L.S.), F31-MH070125 (to K.M.S.), and F32-

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MH068975 (to J.L.Z.) and the American Psychological Association Diversity in Neuroscience Program (to K.Y.S.-R.).

Disclosure summary: The authors have nothing to disclose. The views expressed in this article do not necessarily represent the views of the National Institute of Mental Health, National Institutes of Health, Department of Health and Human Services, or the United States government.

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