Dopaminergic Activation of Estrogen Receptors in Neonatal Brain Alters Progestin Receptor Expression and Juvenile Social Play Behavior
http://www.100md.com
《内分泌学杂志》
Departments of Psychology (K.M.O., H.M.J., A.P.A.) and Zoology (C.J.A.), University of Wisconsin, Madison, Wisconsin 53706
Abstract
Steroid receptor activation in developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. We report that estrogen receptors can be activated in a ligand-independent manner within developing brain by membrane dopamine receptors. Neonatal treatment with either estradiol or a dopamine D1 receptor agonist can increase the expression of an estrogen receptor-regulated gene (i.e. progestin receptors) and later juvenile social play. More importantly, increases in social play behavior induced by neonatal treatment with estradiol or a dopamine D1 receptor agonist can be prevented by prior treatment with an estrogen receptor antagonist. This suggests that changes in dopamine transmission in developing brain can activate estrogen receptors in a ligand-independent manner to influence gene expression and have lasting consequences on social behavior.
Introduction
STEROID RECEPTOR ACTIVATION in the developing brain results in a profound reorganization of synaptic connections, neuronal content, and differences in cell number between males and females. These changes last into adulthood and underlie many of the behavioral and physiological differences between the sexes. In humans, sex differences are observed in many cognitive functions as well as neurological and psychiatric disorders. In rodents, sex differences in brain and behavior are largely the result of early steroid hormone action in developing brain (1, 2). During development, male rats are exposed to high levels of testosterone, leading to masculinization and defeminization of the brain. The female brain, on the other hand, is exposed to significantly lower levels of testosterone (3) and estradiol (4). Altering the levels of steroid hormones during a critical period by castrating males or treating females with testosterone can sex reverse brain development (5, 6, 7). In addition to altering adult sexual behavior, exposure to steroid hormones also influences nonsexual social behavior, such as rough-and-tumble play (8, 9). Males engage in this form of social play behavior more frequently than females (10). The behavioral changes due to early steroid hormone exposure are likely due to testosterone’s effect on a variety of physiological factors, such as neuronal survival, neuronal migration, and the plasticity of both neurons and glia (i.e. dendritic spine density in neurons, and branching in glia) (1, 2, 11).
Steroid hormones produce transient and lasting changes within the developing brain mainly through their actions on intracellular steroid receptors. Upon steroid hormone binding, steroid receptors undergo a conformational change and form dimer complexes with other ligand-bound receptors. Steroid receptors then bind to DNA and recruit additional proteins, including coactivators, to form a transcriptional complex. This complex then initiates gene transcription and thereby changes in protein expression (12). Not only are steroid receptors themselves important in regulating brain differentiation, but also the additional factors recruited to the transcriptional complex, such as coactivators, are equally important (13, 14).
Recent evidence has shown that steroid receptors can be activated in the absence of steroid hormone ligand by a variety of neurochemical compounds. For example, progestin receptors (PRs) can be activated in vitro in the absence of progesterone by the neurotransmitter dopamine (15), 8-bromoadenosine-cAMP (16), and neuropeptides (17, 18). More importantly, ligand-independent activation of progestin receptors is reported to occur in adult brain to influence behavior. Mani et al. (19) found that a centrally administered dopamine receptor agonist activates PR in a ligand-independent manner to induce lordosis in estradiol-primed rats. Further research has shown that LHRH and cAMP (20) as well as exposure to reproductive stimuli (21) can also activate PRs to induce sexual behavior in the absence of progesterone. These data indicate that PRs in the adult brain can be activated by factors other than steroid hormones to influence brain physiology and behavior.
Whereas progestin receptors influence some aspects of brain development, the majority of differences between males and females are due to estrogen receptor (ER) activation. Recent data indicate that ERs can also be activated in the absence of ligand. For example, ER knockout mice fail to show normal uterine responses to epidermal growth factor (EGF) (22), suggesting that EGF may act through ER. In vitro evidence suggests that EGF (23), IGF-I (24), caveolin-1 (25), and activators of the protein kinase A and protein kinase C pathways (26) can also induce ER-dependent gene transcription by activating ER in the absence of estradiol. Uterine EGF (27) and IGF-I (28) also result in ER-dependent gene transcription that can be blocked by an ER antagonist in vivo. Ligand-independent activation of ER by both EGF and IGF-I can also induce female sexual behavior in the absence of estrogen (29), indicating that ligand-independent activation of ER occurs in the adult brain. Furthermore, ligand-independent activation of ER by IGF-I appears to induce adult neurogenesis (30).
Whereas evidence exists that ERs can be activated in vitro in the absence of estradiol by dopamine (15, 31, 32), it is unclear whether dopamine can activate ERs during brain development to influence sexual differentiation. Dopamine itself appears to play a role in sexual differentiation of the brain. During early postnatal development, males show increased hypothalamic dopamine content contrasted to females (33). Additionally, neonatally administered dopamine antagonists interfere with normal masculinization of sexual behavior in males (34, 35), and neonatal treatment with lisuride, a dopamine D1 receptor agonist, masculinizes rough-and-tumble social play and adult sex behavior in females (36, 37). Because masculinization of adult sex behavior is at least partially dependent on perinatal exposure to estradiol (1), this suggests that dopamine may activate ER to influence brain differentiation and subsequently social behavior.
Although there is evidence that ERs can be activated in a ligand-independent manner by dopamine in vitro, it is unclear whether dopamine can activate ERs in the developing brain. We now report that stimulation of dopamine D1-like receptors can lead to the activation of ERs within developing brain in a ligand-independent manner. Dopaminergic activation of ERs in developing brain increases the expression of an ER-responsive gene, PRs, and profoundly alters the later development of juvenile social play behavior.
Materials and Methods
Animals
Adult female Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were mated in our animal facility and allowed to deliver normally. Cages were checked regularly to determine the day of birth. Once born, neonatal rats were injected with India ink into a paw to mark treatment groups. Each experiment included animals from at least two litters. Animals from each litter were distributed evenly across treatment groups. This research was approved by the University of Wisconsin Animal Care and Use Committee.
Experiment 1: can treatment with a D1 receptor agonist induce PR in the developing brain
On the day after birth [postnatal day (PN) 1], neonatal female rats (n = 6–7 per group) were given sc injections of 100 μg SKF 38393 (a dopamine D1-like receptor agonist), 100 μg cAMP, 100 μg estradiol benzoate, or vehicle. Drug dosages were based on published literature. One hundred micrograms estradiol benzoate (EB) is the dosage necessary to induce a male-typical level of estradiol in the brain of female rats during development (4). SKF 38393 and cAMP doses were adapted for neonates based on the dosages used to induce Fos expression during development and sexual behavior in adult rats (20, 38). Brains were collected 24 h after injection and immediately immersed in 5% acrolein overnight at 4 C and then placed in 0.1 M Tris-buffered saline (TBS) containing 30% sucrose at 4 C until sunk. Brains were sectioned at 40 μm using a cryostat at –20 C and stored in cryoprotectant at –20 C until processed for PR immunocytochemistry.
Experiment 2: can SKF 38393 induction of PR be blocked by an ER antagonist
Female rat pups (n = 6 per group) were injected sc on PN1 with either 100 μg tamoxifen or vehicle and then 2 h later with either 100 μg SKF 38393 or vehicle. The dose of tamoxifen chosen has been previously reported to reduce masculinization of the sexually dimorphic nucleus of the preoptic area in neonatal male rats (39). Brains were collected 24 h after the second injection and immediately immersed in 5% acrolein overnight at 4 C and then placed in 0.1 M TBS containing 30% sucrose at 4 C until sunk. Brains were sectioned at 40 μm using a cryostat at –20 C and stored in cryoprotectant at –20 C until processed for PR immunocytochemistry.
Experiment 3: does neonatal treatment with estradiol alter social play behavior
Newborn female rats (n = 4–7 per group) were injected sc once daily from PN0 to PN2 with either 100 μg tamoxifen or vehicle and then 3 h later with either 100 μg EB or vehicle. Newborn males (n = 5) were injected with vehicle according to the same paradigm. Animals were weaned on PN21 and housed in groups of six composed of mixed sexes and treatment groups. Each group contained at least one male and at least one female from each of the four treatment groups. Rough-and-tumble social play behavior was scored between PN25 and PN29 as described below. All animals were gonadally intact at the time of testing.
Experiment 4: does neonatal treatment with SKF 38393 alter social play behavior
Newborn female rats (n = 6 per group) were injected sc with either 100 μg SKF 38393 or vehicle once daily from PN0 to PN2. Females were weaned on PN21 and housed in groups of six, with each cage containing mixed treatment groups. Each cage contained three animals from each of the two treatment groups. Rough-and-tumble social play behavior was scored between PN25 and PN29 as described below. All animals were gonadally intact at the time of testing. Previous studies have reported that treatment of neonatal rats for 10 d with a dopamine agonist, lisuride, can increase social play behavior (36). Our pilot studies suggested that 3 d of treatment with the more specific dopamine D1 receptor agonist, SKF 38393, could be sufficient to increase play behavior.
Experiment 5: can the SKF 38393-induced increase in social play be blocked by tamoxifen
Newborn female rats (n = 7–8 per group) were injected sc once daily from PN0 to PN2 with either 100 μg tamoxifen or vehicle and then 2 h later with either 100 μg SKF 38393 or vehicle. Females were weaned on PN21 and housed in groups of six, with each group containing at least one animal from each of the four treatment groups. Rough-and-tumble social play behavior was scored between PN25 and PN29 as described below. All animals were gonadally intact at the time of testing.
Experiment 6: does SKF 38393 alter serum estradiol concentrations
Newborn female rats (n = 4 per group) were sc injected with 100 μg SKF 38393, 100 μg estradiol benzoate, or vehicle on PN1. Animals were killed 6 h after injection and trunk blood collected. Blood samples were centrifuged for 10 min at 10,000 rpm at 4 C to separate plasma. Plasma was pipetted off and stored at –80 C until processed by enzyme immunoassay for estradiol.
PR immunocytochemistry
Sections representing half of the brain from each animal were washed three times for 5 min each in 0.1 M TBS (pH 7.4) and then placed in TBS containing 1% H2O2 and 20% normal goat serum for 1 h to reduce endogenous peroxidase activity and nonspecific staining. Sections were then incubated in PR antibody (catalog no. A0098, DAKO USA, Carpinteria, CA; 1:1000 dilution) overnight at room temperature in TBS containing 0.3% Triton X-100 (TTBS), 2% normal goat serum, and 0.5% gelatin. After primary incubation, sections were washed three times for 5 min each in TTBS and then incubated in biotinylated goat antirabbit IgG (catalog no. BA-1000, 1:500 dilution; Vector Laboratories, Burlingame, CA) for 90 min at room temperature. Sections were then washed three times for 5 min each in TTBS and two times for 5 min each in TBS. After washes, sections were incubated in Vectastain ABC (catalog no. PK-6100, 1:400 dilution; Vector Laboratories) for 1 h. Sections were rinsed three times for 5 min each in TBS then treated with Vector SG (catalog no. SK-4700, diluted as directed; Vector Laboratories) for 30 min. Developed sections were mounted on gelatin-coated slides and coverslipped using Permount mounting medium. Omission of PR primary eliminated all immunoreactivity.
Computer-aided image analysis
Brain areas examined include the medial preoptic area (mPOA), dorsolateral bed nucleus of the stria terminalis (BST), ventromedial hypothalamus (VMH), central amygdala (CeA), and arcuate nucleus. One section per area was matched according to the rat brain atlas of Paxinos and Watson (40).
Bilateral counts of PR-immunoreactive cells were obtained using an BX61 microscope (Olympus, Melville, NY) fitted with an Olympus FV II digital camera, connected to a PC compatible computer. The software used for analysis was Olympus MicroSuite (Soft Imaging System Corp., Lakewood, CO). Data were analyzed with a one-way ANOVA and Tukey post hoc comparison tests using the SigmaStat Statistical Analysis System 2.03 software (Jandel Scientific, Corta Madera, CA).
Rough-and-tumble play behavioral testing
The social play behavior paradigm was similar to those published by Casto et al. (41) and Meaney and McEwen (42). Animals were weaned on PN21 and housed in groups of six containing animals from all treatment groups and maintained in these groups throughout the experiment. Animals were coded on the tails with a Sharpie marker to identify individuals. Rough-and-tumble social play behavior was scored on PN25–29, using a paradigm adapted from Casto et al. (41) and Meaney and McEwen (42). Animals were videorecorded for four 2-min trials per day over 5 d, with two trials occurring 3 h after lights-off and two trials occurring 6 h after lights-off, for a total observation time of 40 min per animal. The intertrial interval was 2 min. All animals were videotaped in their home cages. The tapes were scored by an observer blind to the treatment groups. All animals in a cage were observed and scored simultaneously. The play behavior scores were calculated by totaling the number of times each animal engaged in wrestling/boxing, biting, pinning, and pouncing over the entire observation time. Play behaviors were scored using the following criteria adapted from Casto et al. (41) and Meaney and McEwen (42): 1)wrestling/boxing: two animals engaged in rolling and tumbling over each other or making jabbing movements at each other with the forepaws; 2) biting: one rat biting another; 3) pouncing: one rat pounces or lunges at another; and 4) pinning: one rat standing over another, with its forepaws on the ventral surface of the opposing rat. Behavioral data were analyzed using a two-tailed Student t test (experiment 4) and a one-way ANOVA with Tukey post hoc comparison tests (experiments 3 and 5).
Estradiol enzyme immunoassay
An estradiol enzyme immunoassay kit (catalog no. 58251, Cayman Chemical, Ann Arbor, MI) was used. Estradiol standards were prepared according to the manufacturer’s instructions. The standard containing the highest concentration of estradiol was removed and another lower standard was created by diluting the lowest standard to allow the detection of lower levels of estradiol. After the preparation of estradiol standards, the standards, controls, and serum samples were loaded into a 96-well plate precoated with mouse antirabbit IgG. Next, estradiol acetylcholinesterase tracer was added, followed by estradiol antiserum. Tracer and antiserum were not added to specific control wells. The plate was then incubated for 1 h at room temperature on an orbital shaker. The plate was then rinsed five times with wash buffer, and then Ellman’s reagent was added to the empty wells. The plate was then developed in the dark on an orbital shaker until the absorbance of the maximum binding wells equaled 0.3 AU. After development, the plate was read at a wavelength of 415 nm with a plate reader. The results were calculated using a computer spreadsheet program provided by Cayman Chemicals (www.caymanchem.com/eiatools/promo/kit).
Results
Experiment 1: can treatment with a D1-like receptor agonist induce PR in the developing brain
Neonatal treatment with SKF 38393 or cAMP increased PR immunoreactivity in the BST and CeA contrasted to vehicle treatment (P < 0.05, Fig. 1). Interestingly, SKF 38393 and cAMP was without effect on PR cell number within the mPOA and the VMH. In contrast, estradiol induced PR immunoreactivity in the mPOA, and VMH as well as the CeA (P < 0.05). Estradiol treatment did not statistically alter PR cell number within the BST; however; this is consistent with findings by Greco et al. (44).
Experiment 2: can SKF 38393 induction of PR be blocked by an ER antagonist
As expected, neonatal treatment with SKF 38393 increased the number of cells expressing PR immunoreactivity only within the BST and CeA. More importantly, prior treatment with the ER antagonist, tamoxifen, completely blocked SKF 38393-induced PR immunoreactivity within the BST and CeA (P < 0.05, Fig. 2). Interestingly, whereas SKF 38393 was without effect on PR immunoreactivity within the mPOA, neonatal treatment with tamoxifen lead to a small but statistically significant increase in PR immunoreactivity contrasted to vehicle-only-treated animals within the mPOA (P < 0.05). This indicates that tamoxifen acts as a weak agonist within this particular brain region. No significant effects were present in the VMH.
Experiment 3: does neonatal treatment with estradiol alter social play behavior
As expected, control males exhibited higher levels of social play than control females (P < 0.001, Fig. 3 and Table 1). Treatment of females with estradiol increased the frequency of social play behavior contrasted to control females (P < 0.0001). Estradiol-treated females engaged in social play at levels statistically similar to those of control males. The estradiol-induced increase in social play was completely blocked by prior treatment with the ER antagonist, tamoxifen, indicating that ERs are required for estrogen regulation of social play behavior.
Experiment 4: does neonatal treatment with SKF 38393 alter social play behavior
Neonatal treatment with SKF 38393 dramatically increased juvenile social play behavior contrasted to control animals (P = 0.004, Fig. 3).
Experiment 5: can the SKF 38393-induced increase in social play be blocked by tamoxifen
As expected, neonatal treatment with SKF 38393 increased the instances of juvenile social play behavior on PN25-PN29 contrasted to controls (P < 0.001, Fig. 3). More importantly, prior treatment with the ER antagonist, tamoxifen, completely blocked SKF 38393-induced social play behavior, indicating ERs are required for SKF 38393 regulation of social play behavior. Tamoxifen treatment alone was without effect on social play behavior.
Experiment 6: does SKF 38393 alter serum estradiol concentrations
Although estradiol treatment dramatically increased serum estradiol concentrations above control levels (P = 0.002, Fig. 4), SKF 38393 treatment did not alter serum estradiol concentrations.
Discussion
We report that ERs in developing brain are not only sensitive to changes in serum estradiol levels but also can be activated by increased dopaminergic transmission. To determine whether dopamine activates ERs in developing brain, we used immunocytochemistry to examine whether stimulation of dopamine D1-like receptors increased the expression of an ER-regulated gene, PR. Neonatal female rats were treated with the dopamine D1-like receptor agonist SKF 38393, cAMP, estradiol benzoate, or vehicle and examined for PR protein expression 24 h later. As expected, estradiol increased PR expression in the CeA, mPOA, and VMH, areas that have all been shown to contain both ER and ER during development (45). Estradiol treatment did not alter PR cell number within the BST, even though this region contains high levels of both ER and ER during development (45); however, this finding is consistent with those published previously by Greco et al. (44). Interestingly, treatment with SKF 38393 and cAMP increased PR only within the BST and CeA contrasted to control females. SKF 38393 had no effect on PR expression within the mPOA or VMH (Fig. 1). These data suggest the possibility that ligand-independent and ligand activation of ER are functionally distinct at least with respect to PR expression in some areas. This idea is supported by recent in vitro studies indicating that ligand-independent activation of ER by protein kinase A occurs through a different pathway and has a different functional consequence on ER expression than does ligand activation (46).
Because estradiol is known to regulate PR expression during development (47) and the expression of PR in developing brain is dependent on ER (48), the up-regulation of PR by cAMP and SKF 38393 suggests that dopamine can induce ER-regulated gene expression during brain development. To confirm that SKF 38393-induced PR expression was a result of ER activation, we treated female rats with the ER antagonist, tamoxifen, 2 h before injection of SKF 38393 or estradiol. We found that the SKF 38393 induction of PR in the BST and CeA was completely blocked by prior treatment with tamoxifen, indicating that ER is a critical component for this induction (Fig. 2). This suggests that stimulation of dopamine D1 receptors leads to the activation of ER in a ligand-independent manner in restricted regions of developing brain. These results are consistent with previous studies showing ligand-independent activation of ER by dopamine in vitro (15, 31, 32) and by IGF-I in the adult brain (30).
The region-specific induction of PR by SKF 38393 in developing brain appears to correlate with the distribution of dopaminergic innervation. SKF 38393 increases PR within the BST and CeA but not the mPOA or VMH. This pattern correlates with the expression of the dopamine D1 receptor marker, DARPP-32. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, is found primarily within cells containing dopamine D1 receptors (49) and is expressed at high levels within the BST and CeA. In contrast, low levels of DARPP-32 immunoreactivity are found within the mPOA and VMH (50). This suggests that the lack of SKF 38393-induced PR in the mPOA and VMH may be due to lower levels of dopamine D1 receptors within these regions. The BST and CeA also express more dopamine D1 receptors and tyrosine hydroxylase immunoreactivity than the mPOA or VMH (51, 52). Because tyrosine hydroxylase is involved in the synthesis of dopamine, these data indicate that the BST and CeA are more heavily innervated by dopamine than the mPOA and VMH.
Hormonal activation of steroid receptors during development is known to organize sex differences in the BST (2) and CeA (53). Both of these regions are involved in development of social behavior (54, 55, 56), with the CeA being critical for sexual differentiation of juvenile social play (54). The region-specific induction of PR by SKF 38393 in the CeA and BST suggests that ligand-independent activation of ER by dopamine during development may alter behaviors associated with these brain regions later in life. For example, the BST and CeA modulate numerous nonsexual social behaviors (e.g. social play behavior), whereas the mPOA and VMH modulate sexual behavior. Because the BST and CeA are targets of dopaminergic-induced ligand-independent activation of ER, we examined the ability of the dopamine D1-like receptor agonist, SKF 38393, to alter the development of prepubertal social play behavior by treating neonatal female rats with SKF 38393 or vehicle. As expected, animals treated with SKF 38393 showed higher levels of social play behavior, contrasted to vehicle-treated animals. These results are consistent with previous studies showing masculinization of rough-and-tumble play in response to neonatal treatment with the dopamine agonist lisuride (36, 37), indicating dopamine modifies the development of social play behavior. To determine whether the increase in social play by SKF 38393 was due to ER activation, we attempted to block the effect with tamoxifen. We found that prior treatment with the ER antagonist, tamoxifen, completely blocked the SKF 38393-induced increase in social play, indicating that ER is a critical component in mediating dopaminergic regulation of social play behavior (Fig. 3). These data suggest that stimulation of dopamine D1 receptors can activate ER in a ligand-independent manner within developing brain and have lasting consequences on social behavior.
The development of social play behavior is known to be sexually dimorphic, with males engaging in more social play behavior contrasted to females (9). The sexually dimorphic patterns of social play are organized by exposure to testosterone during the perinatal period. Castration of neonatal males before PN6 reduces the frequency of social play to female-typical levels (9, 57). Additionally, peripheral testosterone (58) treatment and implants of testosterone into the amygdala (43, 59) during the early neonatal period both masculinize the social play behavior of females. The effects of testosterone on the sexual differentiation of social play have been attributed to activation of androgen receptors. Meaney and Stewart (9) found that although peripheral treatment with testosterone and its androgenic metabolite, dihydrotestosterone, masculinized social play behavior, 5 μg EB had no effect. It is possible that the lack of effect of EB was due to the dosage. Recent data indicate that it takes 100 μg peripheral EB to reach male-typical levels of estradiol in the brain (4), leaving open the possibility that estradiol may influence social play behavior. Furthermore, males rendered insensitive to androgens by the testicular feminization mutation showed decreased levels of play behavior (60). Although testicular feminization mutation males showed lower frequencies of play behavior contrasted to normal males, they did tend to engage in play more frequently than females, providing support for the idea that ERs may play a role in differentiating social play behavior. We examined the ability of a male-like dose of estradiol to influence the development of social play behavior by treating neonatal female rats with 100 μg estradiol or vehicle. Control males engaged in significantly more social play contrasted to control females. This is consistent with previously reported sex differences in social play behavior (9). Estradiol-treated females engaged in significantly more social play behavior contrasted to vehicle-treated females. The levels of social play exhibited by estradiol-treated females were statistically similar to those exhibited by control males. More importantly, the estradiol-induced increase in social play behavior was completely blocked by prior treatment with tamoxifen, indicating that ER is required for estradiol regulation of social play.
Although the neonatal ovary does not begin producing steroid hormones until PN8 (61), we needed to confirm that treatment with SKF 38393 did not result in early maturation of the ovary. We tested this by measuring serum estradiol concentrations 6 h after treatment with SKF 38393, estradiol, or saline vehicle. We found that SKF 38393 did not alter serum levels of estradiol, indicating that the changes in gene expression and behavior by SKF 38393 occurred independently of changes in serum estradiol concentrations. This is consistent with previous work demonstrating that cAMP did not alter neonatal ovarian synthesis of estrogens before PN14 (43). Furthermore, if SKF 38393 was causing peripheral release of estradiol, then this released estradiol would have increased PR expression within the mPOA and VMH; however, SKF 38393 treatment altered only PR expression within the BST and the CeA. Alternatively, a localized increase in estradiol levels may have occurred within the BST and CeA but not the mPOA and VMH. Recent data suggest that synthesis of estradiol can occur in newborn rat brain (4). Therefore, we cannot rule out the possibility that SKF 38393 stimulated local synthesis of estradiol.
In summary, our data indicate that the dopamine receptor agonist, SKF 38393, activates ER in a ligand-independent manner within developing brain to induce gene expression and subsequently alters social play behavior. Because there are no clear data on the role of PRs in the development of social play behavior, it is likely that the induction of PR expression and the regulation of social play behavior by ER are separate phenomena. However, these data do suggest that the activity of ER in developing brain can be regulated by both steroid hormones and dopamine. Our data illustrate a potential pathway by which endogenous or exogenous signals from the environment, leading to changes in dopamine transmission, can activate ERs and have lasting consequences on the development of social behavior.
Acknowledgments
We thank Elizabeth Zao for her assistance.
Footnotes
This work was supported by funding from National Institutes of Health Grant K01MH002035 (to A.P.A.).
Abbreviations: BST, Bed nucleus of the stria terminalis; CeA, central amygdala; EB, estradiol benzoate; EGF, epidermal growth factor; ER, estrogen receptor; mPOA, medial preoptic area; PN, postnatal day; PR, progestin receptor; TBS, Tris-buffered saline; TTBS, TBS containing Triton X-100; VMH, ventromedial hypothalamus.
References
Cooke B, Hegstrom CD, Villeneuve LS, Breedlove SM 1998 Sexual differentiation of the vertebrate brain: principles and mechanisms. Front Neuroendocrinol 19:323–362
Simerly RB 2002 Wired for reproduction: organization and development of sexually dimorphic circuits in the mammalian forebrain. Annu Rev Neurosci 25:507–536
Weisz J, Ward IL 1980 Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106:306–316
Amateau SK, Alt JJ, Stamps CL, McCarthy MM 2004 Brain estradiol content in newborn rats: sex differences, regional heterogeneity, and possible de novo synthesis by the female telencephalon. Endocrinology 145:2906–2917
Beach F, Holz AM 1946 Mating behavior in male rats castrated at various ages and injected with androgen. J Exp Zool 101:91–142
Whalen RE, Edwards DA 1967 Hormonal determinants of the development of masculine and feminine behavior in male and female rats. Anat Rec 157:173–180
Feder HH, Phoenix CH, Young WC 1966 Suppression of feminine behaviour by administration of testosterone propionate to neonatal rats. J Endocrinol 34:131–132
Pellis SM 2002 Sex differences in play fighting revisited: traditional and nontraditional mechanisms of sexual differentiation in rats. Arch Sex Behav 31:17–26
Meaney MJ, Stewart J 1981 Neonatal-androgens influence the social play of prepubescent rats. Horm Behav 15:197–213
Olioff M, Stewart J 1978 Sex differences in the play behavior of prepubescent rats. Physiol Behav 20:113–115
Mong JA, McCarthy MM 1999 Steroid-induced developmental plasticity in hypothalamic astrocytes: implications for synaptic patterning. J Neurobiol 40:602–619
Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486
Auger AP, Tetel MJ, McCarthy MM 2000 Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior. Proc Natl Acad Sci USA 97:7551–7555
Auger AP, Perrot-Sinal TS, Auger CJ, Ekas LA, Tetel MJ, McCarthy MM 2002 Expression of the nuclear receptor coactivator, cAMP response element-binding protein, is sexually dimorphic and modulates sexual differentiation of neonatal rat brain. Endocrinology 143:3009–3016
Power RF, Mani SK, Codina J, Conneely OM, O’Malley BW 1991 Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 254:1636–1639
Denner LA, Weigel NL, Maxwell BL, Schrader WT, O’Malley BW 1990 Regulation of progesterone receptor-mediated transcription by phosphorylation. Science 250:1740–1743
Waring DW, Turgeon JL 1992 A pathway for luteinizing hormone releasing-hormone self-potentiation: cross-talk with the progesterone receptor. Endocrinology 130:3275–3282
Turgeon JL, Waring DW 1994 Activation of the progesterone receptor by the gonadotropin-releasing hormone self-priming signaling pathway. Mol Endocrinol 8:860–869
Mani SK, Allen JMC, Clark JH, Blaustein JD, O’Malley BW 1994 Convergent pathways for steroid hormone-and neurotransmitter-induced rat sexual behavior. Science 265:1246–1249
Beyer C, Gonzalez-Flores O, Gonzalez-Mariscal G 1997 Progesterone receptor participates in the stimulatory effect of LHRH, prostaglandin E2, and cyclic AMP on lordosis and proceptive behaviors in rats. J Neuroendocrinol 9:609–614
Auger AP, Moffatt CA, Blaustein JD 1997 Progesterone-independent activation of rat brain progestin receptors by reproductive stimuli. Endocrinology 138:511–514
Curtis SW, Washburn T, Sewall C, DiAugustine R, Lindzey J, Couse JF, Korach KS 1996 Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. Proc Natl Acad Sci USA 93:12626–12630
El-Tanani MK, Green CD 1997 Two separate mechanisms for ligand-independent activation of the estrogen receptor. Mol Endocrinol 11:928–937
Aronica SM, Katzenellenbogen BS 1993 Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen. Mol Endocrinol 7:743–752
Schlegel A, Wang C, Katzenellenbogen BS, Pestell RG, Lisanti MP 1999 Caveolin-1 potentiates estrogen receptor (ER) signaling. caveolin-1 drives ligand-independent nuclear translocation and activation of ER. J Biol Chem 274:33551–33556
Cho H, Katzenellenbogen BS 1993 Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. Mol Endocrinol 7:441–452
Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658–4662
Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP, Korach KS 2002 Requirement of estrogen receptor- in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. J Biol Chem 277:8531–8537
Apostolakis EM, Garai J, Lohmann JE, Clark JH, O’Malley BW 2000 Epidermal growth factor activates reproductive behavior independent of ovarian steroids in female rodents. Mol Endocrinol 14:1086–1098
Perez-Martin M, Azcoitia I, Trejo JL, Sierra A, Garcia-Segura LM 2003 An antagonist of estrogen receptors blocks the induction of adult neurogenesis by insulin-like growth factor-I in the dentate gyrus of adult female rat. Eur J Neurosci 18:923–930
Smith CL, Conneely OM, O’Malley BW 1993 Modulation of the ligand-independent activation of the human estrogen receptor by hormone and antihormone. Proc Natl Acad Sci USA 90:6120–6124
Gangolli EA, Conneely OM, O’Malley BW 1997 Neurotransmitters activate the human estrogen receptor in a neuroblastoma cell line. J Steroid Biochem Mol Biol 61:1–9
Lesage J, Bernet F, Montel V, Dupouy JP 1996 Hypothalamic metabolism of neurotransmitters (serotonin, norepinephrine, dopamine) and NPY, and gonadal and adrenal activities, during the early postnatal period in the rat. Neurochem Res 21:87–96
Hull EM, Nishita JK, Bitran D, Dalterio S 1984 Perinatal dopamine-related drugs demasculinize rats. Science 224:1011–1013
Gonzales FG, Ortega JG, Salazar M 2000 Effect of neonatal administration of an antidopaminergic drug (metoclopramide) on sexual behavior of male rats. Arch Androl 45:137–142
Tonjes R, Gotz F, Maywald J, Dorner G 1989 Influence of a dopamine agonist (lisuride) on sex-specific behavioural patterns in rats. II. Long-term effects. Exp Clin Endocrinol 94:48–54
Gotz F, Tonjes R, Maywald J, Dorner G 1991 Short- and long-term effects of a dopamine agonist (lisuride) on sex-specific behavioural patterns in rats. Exp Clin Endocrinol 98:111–121
Weaver DR, Reppert SM 1995 Definition of the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the rat suprachiasmatic nucleus. Mol Brain Res 33:136–148
Vancutsem PM, Roessler ML 1997 Neonatal treatment with tamoxifen causes immediate alterations of the sexually dimorphic nucleus of the preoptic area and medial preoptic area in male rats. Teratology 56:220–228
Paxinos G, Watson C 1997 The rat brain in stereotaxic coordinates. Compact ed. 3rd ed. Sydney, Australia: Academic Press
Casto JM, Ward OB, Bartke A 2003 Play, copulation, anatomy, and testosterone in gonadally intact male rats prenatally exposed to flutamide. Physiol Behav 79:633–641
Meaney MJ, McEwen BS 1986 Testosterone implants into the amygdala during the neonatal period masculinize the social play of juvenile female rats. Brain Res 398:324–328
Weniger JP, Zeis A, Chouraqui J 1993 Estrogen production by fetal and infantile rat ovaries. Reprod Nutr Dev 33:129–136
Greco B, Allegretto EA, Tetel MJ, Blaustein JD 2001 Coexpression of ER with ER and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment. Endocrinology 142:5172–5181
Perez SE, Chen EY, Mufson EJ 2003 Distribution of estrogen receptor and immunoreactive profiles in the postnatal rat brain. Brain Res Dev Brain Res 145:117–139
Tsai HW, Katzenellenbogen JA, Katzenellenbogen BS, Shupnik MA 2004 Protein kinase A activation of estrogen receptor transcription does not require proteasome activity and protects the receptor from ligand-mediated degradation. Endocrinology 145:2730–2738
Quadros PS, Pfau JL, Goldstein AY, De Vries GJ, Wagner CK 2002 Sex differences in progesterone receptor expression: a potential mechanism for estradiol-mediated sexual differentiation. Endocrinology 143:3727–3739
Wagner CK, Pfau JL, De Vries GJ, Merchenthaler IJ 2001 Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor expression. J Neurobiol 47:176–182
Schalling M, Djurfeldt M, Hokfelt T, Ehrlich M, Kurihara T, Greengard P 1990 Distribution and cellular localization of DARPP-32 mRNA in rat brain. Brain Res Mol Brain Res 7:139–149
Ouimet CC 1991 DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, is present in corticothalamic neurons of the rat cingulate cortex. Brain Res 562:85–92
Weiner DM, Levey AI, Sunahara RK, Niznik HB, O’Dowd BF, Seeman P, Brann MR 1991 D1 and D2 dopamine receptor mRNA in rat brain. Proc Natl Acad Sci USA 88:1859–1863
Lazarov NE, Schmidt U, Wanner I, Pilgrim C 1998 Mapping of D1 dopamine receptor mRNA by non-radioactive in situ hybridization. Histochem Cell Biol 109:271–279
Staudt J, Dorner G 1976 Structural changes in the medial and central amygdala of the male rat, following neonatal castration and androgen treatment. Endokrinologie 67:296–300
Meaney MJ, Dodge AM, Beatty WW 1981 Sex-dependent effects of amygdaloid lesions on the social play of prepubertal rats. Physiol Behav 26:467–472
Daenen EW, Wolterink G, Gerrits MA, Van Ree JM 2002 The effects of neonatal lesions in the amygdala or ventral hippocampus on social behaviour later in life. Behav Brain Res 136:571–582
De Vries GJ, Simerly RB 2002 Anatomy, development, and function of sexually dimorphic neural circuits in the mammalian brain. In: Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, eds. Hormones, brain and behavior. San Diego: Academic Press; 137–191
Beatty WW, Dodge AM, Traylor KL, Meaney MJ 1981 Temporal boundary of the sensitive period for hormonal organization of social play in juvenile rats. Physiol Behav 26:241–243
Thor DH, Holloway Jr WR 1986 Social play soliciting by male and female juvenile rats: effects of neonatal androgenization and sex of cagemates. Behav Neurosci 100:275–279
Tonjes R, Docke F, Dorner G 1987 Effects of neonatal intracerebral implantation of sex steroids on sexual behaviour, social play behaviour and gonadotrophin secretion. Exp Clin Endocrinol 90:257–263
Meaney MJ, Stewart J, Poulin P, McEwen BS 1983 Sexual differentiation of social play in rat pups is mediated by the neonatal androgen-receptor system. Neuroendocrinology 37:85–90
Sokka TA, Huhtaniemi IT 1995 Functional maturation of the pituitary-gonadal axis in the neonatal female rat. Biol Reprod 52:1404–1409(Kristin M. Olesen, Heathe)
Abstract
Steroid receptor activation in developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. We report that estrogen receptors can be activated in a ligand-independent manner within developing brain by membrane dopamine receptors. Neonatal treatment with either estradiol or a dopamine D1 receptor agonist can increase the expression of an estrogen receptor-regulated gene (i.e. progestin receptors) and later juvenile social play. More importantly, increases in social play behavior induced by neonatal treatment with estradiol or a dopamine D1 receptor agonist can be prevented by prior treatment with an estrogen receptor antagonist. This suggests that changes in dopamine transmission in developing brain can activate estrogen receptors in a ligand-independent manner to influence gene expression and have lasting consequences on social behavior.
Introduction
STEROID RECEPTOR ACTIVATION in the developing brain results in a profound reorganization of synaptic connections, neuronal content, and differences in cell number between males and females. These changes last into adulthood and underlie many of the behavioral and physiological differences between the sexes. In humans, sex differences are observed in many cognitive functions as well as neurological and psychiatric disorders. In rodents, sex differences in brain and behavior are largely the result of early steroid hormone action in developing brain (1, 2). During development, male rats are exposed to high levels of testosterone, leading to masculinization and defeminization of the brain. The female brain, on the other hand, is exposed to significantly lower levels of testosterone (3) and estradiol (4). Altering the levels of steroid hormones during a critical period by castrating males or treating females with testosterone can sex reverse brain development (5, 6, 7). In addition to altering adult sexual behavior, exposure to steroid hormones also influences nonsexual social behavior, such as rough-and-tumble play (8, 9). Males engage in this form of social play behavior more frequently than females (10). The behavioral changes due to early steroid hormone exposure are likely due to testosterone’s effect on a variety of physiological factors, such as neuronal survival, neuronal migration, and the plasticity of both neurons and glia (i.e. dendritic spine density in neurons, and branching in glia) (1, 2, 11).
Steroid hormones produce transient and lasting changes within the developing brain mainly through their actions on intracellular steroid receptors. Upon steroid hormone binding, steroid receptors undergo a conformational change and form dimer complexes with other ligand-bound receptors. Steroid receptors then bind to DNA and recruit additional proteins, including coactivators, to form a transcriptional complex. This complex then initiates gene transcription and thereby changes in protein expression (12). Not only are steroid receptors themselves important in regulating brain differentiation, but also the additional factors recruited to the transcriptional complex, such as coactivators, are equally important (13, 14).
Recent evidence has shown that steroid receptors can be activated in the absence of steroid hormone ligand by a variety of neurochemical compounds. For example, progestin receptors (PRs) can be activated in vitro in the absence of progesterone by the neurotransmitter dopamine (15), 8-bromoadenosine-cAMP (16), and neuropeptides (17, 18). More importantly, ligand-independent activation of progestin receptors is reported to occur in adult brain to influence behavior. Mani et al. (19) found that a centrally administered dopamine receptor agonist activates PR in a ligand-independent manner to induce lordosis in estradiol-primed rats. Further research has shown that LHRH and cAMP (20) as well as exposure to reproductive stimuli (21) can also activate PRs to induce sexual behavior in the absence of progesterone. These data indicate that PRs in the adult brain can be activated by factors other than steroid hormones to influence brain physiology and behavior.
Whereas progestin receptors influence some aspects of brain development, the majority of differences between males and females are due to estrogen receptor (ER) activation. Recent data indicate that ERs can also be activated in the absence of ligand. For example, ER knockout mice fail to show normal uterine responses to epidermal growth factor (EGF) (22), suggesting that EGF may act through ER. In vitro evidence suggests that EGF (23), IGF-I (24), caveolin-1 (25), and activators of the protein kinase A and protein kinase C pathways (26) can also induce ER-dependent gene transcription by activating ER in the absence of estradiol. Uterine EGF (27) and IGF-I (28) also result in ER-dependent gene transcription that can be blocked by an ER antagonist in vivo. Ligand-independent activation of ER by both EGF and IGF-I can also induce female sexual behavior in the absence of estrogen (29), indicating that ligand-independent activation of ER occurs in the adult brain. Furthermore, ligand-independent activation of ER by IGF-I appears to induce adult neurogenesis (30).
Whereas evidence exists that ERs can be activated in vitro in the absence of estradiol by dopamine (15, 31, 32), it is unclear whether dopamine can activate ERs during brain development to influence sexual differentiation. Dopamine itself appears to play a role in sexual differentiation of the brain. During early postnatal development, males show increased hypothalamic dopamine content contrasted to females (33). Additionally, neonatally administered dopamine antagonists interfere with normal masculinization of sexual behavior in males (34, 35), and neonatal treatment with lisuride, a dopamine D1 receptor agonist, masculinizes rough-and-tumble social play and adult sex behavior in females (36, 37). Because masculinization of adult sex behavior is at least partially dependent on perinatal exposure to estradiol (1), this suggests that dopamine may activate ER to influence brain differentiation and subsequently social behavior.
Although there is evidence that ERs can be activated in a ligand-independent manner by dopamine in vitro, it is unclear whether dopamine can activate ERs in the developing brain. We now report that stimulation of dopamine D1-like receptors can lead to the activation of ERs within developing brain in a ligand-independent manner. Dopaminergic activation of ERs in developing brain increases the expression of an ER-responsive gene, PRs, and profoundly alters the later development of juvenile social play behavior.
Materials and Methods
Animals
Adult female Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were mated in our animal facility and allowed to deliver normally. Cages were checked regularly to determine the day of birth. Once born, neonatal rats were injected with India ink into a paw to mark treatment groups. Each experiment included animals from at least two litters. Animals from each litter were distributed evenly across treatment groups. This research was approved by the University of Wisconsin Animal Care and Use Committee.
Experiment 1: can treatment with a D1 receptor agonist induce PR in the developing brain
On the day after birth [postnatal day (PN) 1], neonatal female rats (n = 6–7 per group) were given sc injections of 100 μg SKF 38393 (a dopamine D1-like receptor agonist), 100 μg cAMP, 100 μg estradiol benzoate, or vehicle. Drug dosages were based on published literature. One hundred micrograms estradiol benzoate (EB) is the dosage necessary to induce a male-typical level of estradiol in the brain of female rats during development (4). SKF 38393 and cAMP doses were adapted for neonates based on the dosages used to induce Fos expression during development and sexual behavior in adult rats (20, 38). Brains were collected 24 h after injection and immediately immersed in 5% acrolein overnight at 4 C and then placed in 0.1 M Tris-buffered saline (TBS) containing 30% sucrose at 4 C until sunk. Brains were sectioned at 40 μm using a cryostat at –20 C and stored in cryoprotectant at –20 C until processed for PR immunocytochemistry.
Experiment 2: can SKF 38393 induction of PR be blocked by an ER antagonist
Female rat pups (n = 6 per group) were injected sc on PN1 with either 100 μg tamoxifen or vehicle and then 2 h later with either 100 μg SKF 38393 or vehicle. The dose of tamoxifen chosen has been previously reported to reduce masculinization of the sexually dimorphic nucleus of the preoptic area in neonatal male rats (39). Brains were collected 24 h after the second injection and immediately immersed in 5% acrolein overnight at 4 C and then placed in 0.1 M TBS containing 30% sucrose at 4 C until sunk. Brains were sectioned at 40 μm using a cryostat at –20 C and stored in cryoprotectant at –20 C until processed for PR immunocytochemistry.
Experiment 3: does neonatal treatment with estradiol alter social play behavior
Newborn female rats (n = 4–7 per group) were injected sc once daily from PN0 to PN2 with either 100 μg tamoxifen or vehicle and then 3 h later with either 100 μg EB or vehicle. Newborn males (n = 5) were injected with vehicle according to the same paradigm. Animals were weaned on PN21 and housed in groups of six composed of mixed sexes and treatment groups. Each group contained at least one male and at least one female from each of the four treatment groups. Rough-and-tumble social play behavior was scored between PN25 and PN29 as described below. All animals were gonadally intact at the time of testing.
Experiment 4: does neonatal treatment with SKF 38393 alter social play behavior
Newborn female rats (n = 6 per group) were injected sc with either 100 μg SKF 38393 or vehicle once daily from PN0 to PN2. Females were weaned on PN21 and housed in groups of six, with each cage containing mixed treatment groups. Each cage contained three animals from each of the two treatment groups. Rough-and-tumble social play behavior was scored between PN25 and PN29 as described below. All animals were gonadally intact at the time of testing. Previous studies have reported that treatment of neonatal rats for 10 d with a dopamine agonist, lisuride, can increase social play behavior (36). Our pilot studies suggested that 3 d of treatment with the more specific dopamine D1 receptor agonist, SKF 38393, could be sufficient to increase play behavior.
Experiment 5: can the SKF 38393-induced increase in social play be blocked by tamoxifen
Newborn female rats (n = 7–8 per group) were injected sc once daily from PN0 to PN2 with either 100 μg tamoxifen or vehicle and then 2 h later with either 100 μg SKF 38393 or vehicle. Females were weaned on PN21 and housed in groups of six, with each group containing at least one animal from each of the four treatment groups. Rough-and-tumble social play behavior was scored between PN25 and PN29 as described below. All animals were gonadally intact at the time of testing.
Experiment 6: does SKF 38393 alter serum estradiol concentrations
Newborn female rats (n = 4 per group) were sc injected with 100 μg SKF 38393, 100 μg estradiol benzoate, or vehicle on PN1. Animals were killed 6 h after injection and trunk blood collected. Blood samples were centrifuged for 10 min at 10,000 rpm at 4 C to separate plasma. Plasma was pipetted off and stored at –80 C until processed by enzyme immunoassay for estradiol.
PR immunocytochemistry
Sections representing half of the brain from each animal were washed three times for 5 min each in 0.1 M TBS (pH 7.4) and then placed in TBS containing 1% H2O2 and 20% normal goat serum for 1 h to reduce endogenous peroxidase activity and nonspecific staining. Sections were then incubated in PR antibody (catalog no. A0098, DAKO USA, Carpinteria, CA; 1:1000 dilution) overnight at room temperature in TBS containing 0.3% Triton X-100 (TTBS), 2% normal goat serum, and 0.5% gelatin. After primary incubation, sections were washed three times for 5 min each in TTBS and then incubated in biotinylated goat antirabbit IgG (catalog no. BA-1000, 1:500 dilution; Vector Laboratories, Burlingame, CA) for 90 min at room temperature. Sections were then washed three times for 5 min each in TTBS and two times for 5 min each in TBS. After washes, sections were incubated in Vectastain ABC (catalog no. PK-6100, 1:400 dilution; Vector Laboratories) for 1 h. Sections were rinsed three times for 5 min each in TBS then treated with Vector SG (catalog no. SK-4700, diluted as directed; Vector Laboratories) for 30 min. Developed sections were mounted on gelatin-coated slides and coverslipped using Permount mounting medium. Omission of PR primary eliminated all immunoreactivity.
Computer-aided image analysis
Brain areas examined include the medial preoptic area (mPOA), dorsolateral bed nucleus of the stria terminalis (BST), ventromedial hypothalamus (VMH), central amygdala (CeA), and arcuate nucleus. One section per area was matched according to the rat brain atlas of Paxinos and Watson (40).
Bilateral counts of PR-immunoreactive cells were obtained using an BX61 microscope (Olympus, Melville, NY) fitted with an Olympus FV II digital camera, connected to a PC compatible computer. The software used for analysis was Olympus MicroSuite (Soft Imaging System Corp., Lakewood, CO). Data were analyzed with a one-way ANOVA and Tukey post hoc comparison tests using the SigmaStat Statistical Analysis System 2.03 software (Jandel Scientific, Corta Madera, CA).
Rough-and-tumble play behavioral testing
The social play behavior paradigm was similar to those published by Casto et al. (41) and Meaney and McEwen (42). Animals were weaned on PN21 and housed in groups of six containing animals from all treatment groups and maintained in these groups throughout the experiment. Animals were coded on the tails with a Sharpie marker to identify individuals. Rough-and-tumble social play behavior was scored on PN25–29, using a paradigm adapted from Casto et al. (41) and Meaney and McEwen (42). Animals were videorecorded for four 2-min trials per day over 5 d, with two trials occurring 3 h after lights-off and two trials occurring 6 h after lights-off, for a total observation time of 40 min per animal. The intertrial interval was 2 min. All animals were videotaped in their home cages. The tapes were scored by an observer blind to the treatment groups. All animals in a cage were observed and scored simultaneously. The play behavior scores were calculated by totaling the number of times each animal engaged in wrestling/boxing, biting, pinning, and pouncing over the entire observation time. Play behaviors were scored using the following criteria adapted from Casto et al. (41) and Meaney and McEwen (42): 1)wrestling/boxing: two animals engaged in rolling and tumbling over each other or making jabbing movements at each other with the forepaws; 2) biting: one rat biting another; 3) pouncing: one rat pounces or lunges at another; and 4) pinning: one rat standing over another, with its forepaws on the ventral surface of the opposing rat. Behavioral data were analyzed using a two-tailed Student t test (experiment 4) and a one-way ANOVA with Tukey post hoc comparison tests (experiments 3 and 5).
Estradiol enzyme immunoassay
An estradiol enzyme immunoassay kit (catalog no. 58251, Cayman Chemical, Ann Arbor, MI) was used. Estradiol standards were prepared according to the manufacturer’s instructions. The standard containing the highest concentration of estradiol was removed and another lower standard was created by diluting the lowest standard to allow the detection of lower levels of estradiol. After the preparation of estradiol standards, the standards, controls, and serum samples were loaded into a 96-well plate precoated with mouse antirabbit IgG. Next, estradiol acetylcholinesterase tracer was added, followed by estradiol antiserum. Tracer and antiserum were not added to specific control wells. The plate was then incubated for 1 h at room temperature on an orbital shaker. The plate was then rinsed five times with wash buffer, and then Ellman’s reagent was added to the empty wells. The plate was then developed in the dark on an orbital shaker until the absorbance of the maximum binding wells equaled 0.3 AU. After development, the plate was read at a wavelength of 415 nm with a plate reader. The results were calculated using a computer spreadsheet program provided by Cayman Chemicals (www.caymanchem.com/eiatools/promo/kit).
Results
Experiment 1: can treatment with a D1-like receptor agonist induce PR in the developing brain
Neonatal treatment with SKF 38393 or cAMP increased PR immunoreactivity in the BST and CeA contrasted to vehicle treatment (P < 0.05, Fig. 1). Interestingly, SKF 38393 and cAMP was without effect on PR cell number within the mPOA and the VMH. In contrast, estradiol induced PR immunoreactivity in the mPOA, and VMH as well as the CeA (P < 0.05). Estradiol treatment did not statistically alter PR cell number within the BST; however; this is consistent with findings by Greco et al. (44).
Experiment 2: can SKF 38393 induction of PR be blocked by an ER antagonist
As expected, neonatal treatment with SKF 38393 increased the number of cells expressing PR immunoreactivity only within the BST and CeA. More importantly, prior treatment with the ER antagonist, tamoxifen, completely blocked SKF 38393-induced PR immunoreactivity within the BST and CeA (P < 0.05, Fig. 2). Interestingly, whereas SKF 38393 was without effect on PR immunoreactivity within the mPOA, neonatal treatment with tamoxifen lead to a small but statistically significant increase in PR immunoreactivity contrasted to vehicle-only-treated animals within the mPOA (P < 0.05). This indicates that tamoxifen acts as a weak agonist within this particular brain region. No significant effects were present in the VMH.
Experiment 3: does neonatal treatment with estradiol alter social play behavior
As expected, control males exhibited higher levels of social play than control females (P < 0.001, Fig. 3 and Table 1). Treatment of females with estradiol increased the frequency of social play behavior contrasted to control females (P < 0.0001). Estradiol-treated females engaged in social play at levels statistically similar to those of control males. The estradiol-induced increase in social play was completely blocked by prior treatment with the ER antagonist, tamoxifen, indicating that ERs are required for estrogen regulation of social play behavior.
Experiment 4: does neonatal treatment with SKF 38393 alter social play behavior
Neonatal treatment with SKF 38393 dramatically increased juvenile social play behavior contrasted to control animals (P = 0.004, Fig. 3).
Experiment 5: can the SKF 38393-induced increase in social play be blocked by tamoxifen
As expected, neonatal treatment with SKF 38393 increased the instances of juvenile social play behavior on PN25-PN29 contrasted to controls (P < 0.001, Fig. 3). More importantly, prior treatment with the ER antagonist, tamoxifen, completely blocked SKF 38393-induced social play behavior, indicating ERs are required for SKF 38393 regulation of social play behavior. Tamoxifen treatment alone was without effect on social play behavior.
Experiment 6: does SKF 38393 alter serum estradiol concentrations
Although estradiol treatment dramatically increased serum estradiol concentrations above control levels (P = 0.002, Fig. 4), SKF 38393 treatment did not alter serum estradiol concentrations.
Discussion
We report that ERs in developing brain are not only sensitive to changes in serum estradiol levels but also can be activated by increased dopaminergic transmission. To determine whether dopamine activates ERs in developing brain, we used immunocytochemistry to examine whether stimulation of dopamine D1-like receptors increased the expression of an ER-regulated gene, PR. Neonatal female rats were treated with the dopamine D1-like receptor agonist SKF 38393, cAMP, estradiol benzoate, or vehicle and examined for PR protein expression 24 h later. As expected, estradiol increased PR expression in the CeA, mPOA, and VMH, areas that have all been shown to contain both ER and ER during development (45). Estradiol treatment did not alter PR cell number within the BST, even though this region contains high levels of both ER and ER during development (45); however, this finding is consistent with those published previously by Greco et al. (44). Interestingly, treatment with SKF 38393 and cAMP increased PR only within the BST and CeA contrasted to control females. SKF 38393 had no effect on PR expression within the mPOA or VMH (Fig. 1). These data suggest the possibility that ligand-independent and ligand activation of ER are functionally distinct at least with respect to PR expression in some areas. This idea is supported by recent in vitro studies indicating that ligand-independent activation of ER by protein kinase A occurs through a different pathway and has a different functional consequence on ER expression than does ligand activation (46).
Because estradiol is known to regulate PR expression during development (47) and the expression of PR in developing brain is dependent on ER (48), the up-regulation of PR by cAMP and SKF 38393 suggests that dopamine can induce ER-regulated gene expression during brain development. To confirm that SKF 38393-induced PR expression was a result of ER activation, we treated female rats with the ER antagonist, tamoxifen, 2 h before injection of SKF 38393 or estradiol. We found that the SKF 38393 induction of PR in the BST and CeA was completely blocked by prior treatment with tamoxifen, indicating that ER is a critical component for this induction (Fig. 2). This suggests that stimulation of dopamine D1 receptors leads to the activation of ER in a ligand-independent manner in restricted regions of developing brain. These results are consistent with previous studies showing ligand-independent activation of ER by dopamine in vitro (15, 31, 32) and by IGF-I in the adult brain (30).
The region-specific induction of PR by SKF 38393 in developing brain appears to correlate with the distribution of dopaminergic innervation. SKF 38393 increases PR within the BST and CeA but not the mPOA or VMH. This pattern correlates with the expression of the dopamine D1 receptor marker, DARPP-32. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, is found primarily within cells containing dopamine D1 receptors (49) and is expressed at high levels within the BST and CeA. In contrast, low levels of DARPP-32 immunoreactivity are found within the mPOA and VMH (50). This suggests that the lack of SKF 38393-induced PR in the mPOA and VMH may be due to lower levels of dopamine D1 receptors within these regions. The BST and CeA also express more dopamine D1 receptors and tyrosine hydroxylase immunoreactivity than the mPOA or VMH (51, 52). Because tyrosine hydroxylase is involved in the synthesis of dopamine, these data indicate that the BST and CeA are more heavily innervated by dopamine than the mPOA and VMH.
Hormonal activation of steroid receptors during development is known to organize sex differences in the BST (2) and CeA (53). Both of these regions are involved in development of social behavior (54, 55, 56), with the CeA being critical for sexual differentiation of juvenile social play (54). The region-specific induction of PR by SKF 38393 in the CeA and BST suggests that ligand-independent activation of ER by dopamine during development may alter behaviors associated with these brain regions later in life. For example, the BST and CeA modulate numerous nonsexual social behaviors (e.g. social play behavior), whereas the mPOA and VMH modulate sexual behavior. Because the BST and CeA are targets of dopaminergic-induced ligand-independent activation of ER, we examined the ability of the dopamine D1-like receptor agonist, SKF 38393, to alter the development of prepubertal social play behavior by treating neonatal female rats with SKF 38393 or vehicle. As expected, animals treated with SKF 38393 showed higher levels of social play behavior, contrasted to vehicle-treated animals. These results are consistent with previous studies showing masculinization of rough-and-tumble play in response to neonatal treatment with the dopamine agonist lisuride (36, 37), indicating dopamine modifies the development of social play behavior. To determine whether the increase in social play by SKF 38393 was due to ER activation, we attempted to block the effect with tamoxifen. We found that prior treatment with the ER antagonist, tamoxifen, completely blocked the SKF 38393-induced increase in social play, indicating that ER is a critical component in mediating dopaminergic regulation of social play behavior (Fig. 3). These data suggest that stimulation of dopamine D1 receptors can activate ER in a ligand-independent manner within developing brain and have lasting consequences on social behavior.
The development of social play behavior is known to be sexually dimorphic, with males engaging in more social play behavior contrasted to females (9). The sexually dimorphic patterns of social play are organized by exposure to testosterone during the perinatal period. Castration of neonatal males before PN6 reduces the frequency of social play to female-typical levels (9, 57). Additionally, peripheral testosterone (58) treatment and implants of testosterone into the amygdala (43, 59) during the early neonatal period both masculinize the social play behavior of females. The effects of testosterone on the sexual differentiation of social play have been attributed to activation of androgen receptors. Meaney and Stewart (9) found that although peripheral treatment with testosterone and its androgenic metabolite, dihydrotestosterone, masculinized social play behavior, 5 μg EB had no effect. It is possible that the lack of effect of EB was due to the dosage. Recent data indicate that it takes 100 μg peripheral EB to reach male-typical levels of estradiol in the brain (4), leaving open the possibility that estradiol may influence social play behavior. Furthermore, males rendered insensitive to androgens by the testicular feminization mutation showed decreased levels of play behavior (60). Although testicular feminization mutation males showed lower frequencies of play behavior contrasted to normal males, they did tend to engage in play more frequently than females, providing support for the idea that ERs may play a role in differentiating social play behavior. We examined the ability of a male-like dose of estradiol to influence the development of social play behavior by treating neonatal female rats with 100 μg estradiol or vehicle. Control males engaged in significantly more social play contrasted to control females. This is consistent with previously reported sex differences in social play behavior (9). Estradiol-treated females engaged in significantly more social play behavior contrasted to vehicle-treated females. The levels of social play exhibited by estradiol-treated females were statistically similar to those exhibited by control males. More importantly, the estradiol-induced increase in social play behavior was completely blocked by prior treatment with tamoxifen, indicating that ER is required for estradiol regulation of social play.
Although the neonatal ovary does not begin producing steroid hormones until PN8 (61), we needed to confirm that treatment with SKF 38393 did not result in early maturation of the ovary. We tested this by measuring serum estradiol concentrations 6 h after treatment with SKF 38393, estradiol, or saline vehicle. We found that SKF 38393 did not alter serum levels of estradiol, indicating that the changes in gene expression and behavior by SKF 38393 occurred independently of changes in serum estradiol concentrations. This is consistent with previous work demonstrating that cAMP did not alter neonatal ovarian synthesis of estrogens before PN14 (43). Furthermore, if SKF 38393 was causing peripheral release of estradiol, then this released estradiol would have increased PR expression within the mPOA and VMH; however, SKF 38393 treatment altered only PR expression within the BST and the CeA. Alternatively, a localized increase in estradiol levels may have occurred within the BST and CeA but not the mPOA and VMH. Recent data suggest that synthesis of estradiol can occur in newborn rat brain (4). Therefore, we cannot rule out the possibility that SKF 38393 stimulated local synthesis of estradiol.
In summary, our data indicate that the dopamine receptor agonist, SKF 38393, activates ER in a ligand-independent manner within developing brain to induce gene expression and subsequently alters social play behavior. Because there are no clear data on the role of PRs in the development of social play behavior, it is likely that the induction of PR expression and the regulation of social play behavior by ER are separate phenomena. However, these data do suggest that the activity of ER in developing brain can be regulated by both steroid hormones and dopamine. Our data illustrate a potential pathway by which endogenous or exogenous signals from the environment, leading to changes in dopamine transmission, can activate ERs and have lasting consequences on the development of social behavior.
Acknowledgments
We thank Elizabeth Zao for her assistance.
Footnotes
This work was supported by funding from National Institutes of Health Grant K01MH002035 (to A.P.A.).
Abbreviations: BST, Bed nucleus of the stria terminalis; CeA, central amygdala; EB, estradiol benzoate; EGF, epidermal growth factor; ER, estrogen receptor; mPOA, medial preoptic area; PN, postnatal day; PR, progestin receptor; TBS, Tris-buffered saline; TTBS, TBS containing Triton X-100; VMH, ventromedial hypothalamus.
References
Cooke B, Hegstrom CD, Villeneuve LS, Breedlove SM 1998 Sexual differentiation of the vertebrate brain: principles and mechanisms. Front Neuroendocrinol 19:323–362
Simerly RB 2002 Wired for reproduction: organization and development of sexually dimorphic circuits in the mammalian forebrain. Annu Rev Neurosci 25:507–536
Weisz J, Ward IL 1980 Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106:306–316
Amateau SK, Alt JJ, Stamps CL, McCarthy MM 2004 Brain estradiol content in newborn rats: sex differences, regional heterogeneity, and possible de novo synthesis by the female telencephalon. Endocrinology 145:2906–2917
Beach F, Holz AM 1946 Mating behavior in male rats castrated at various ages and injected with androgen. J Exp Zool 101:91–142
Whalen RE, Edwards DA 1967 Hormonal determinants of the development of masculine and feminine behavior in male and female rats. Anat Rec 157:173–180
Feder HH, Phoenix CH, Young WC 1966 Suppression of feminine behaviour by administration of testosterone propionate to neonatal rats. J Endocrinol 34:131–132
Pellis SM 2002 Sex differences in play fighting revisited: traditional and nontraditional mechanisms of sexual differentiation in rats. Arch Sex Behav 31:17–26
Meaney MJ, Stewart J 1981 Neonatal-androgens influence the social play of prepubescent rats. Horm Behav 15:197–213
Olioff M, Stewart J 1978 Sex differences in the play behavior of prepubescent rats. Physiol Behav 20:113–115
Mong JA, McCarthy MM 1999 Steroid-induced developmental plasticity in hypothalamic astrocytes: implications for synaptic patterning. J Neurobiol 40:602–619
Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486
Auger AP, Tetel MJ, McCarthy MM 2000 Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior. Proc Natl Acad Sci USA 97:7551–7555
Auger AP, Perrot-Sinal TS, Auger CJ, Ekas LA, Tetel MJ, McCarthy MM 2002 Expression of the nuclear receptor coactivator, cAMP response element-binding protein, is sexually dimorphic and modulates sexual differentiation of neonatal rat brain. Endocrinology 143:3009–3016
Power RF, Mani SK, Codina J, Conneely OM, O’Malley BW 1991 Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 254:1636–1639
Denner LA, Weigel NL, Maxwell BL, Schrader WT, O’Malley BW 1990 Regulation of progesterone receptor-mediated transcription by phosphorylation. Science 250:1740–1743
Waring DW, Turgeon JL 1992 A pathway for luteinizing hormone releasing-hormone self-potentiation: cross-talk with the progesterone receptor. Endocrinology 130:3275–3282
Turgeon JL, Waring DW 1994 Activation of the progesterone receptor by the gonadotropin-releasing hormone self-priming signaling pathway. Mol Endocrinol 8:860–869
Mani SK, Allen JMC, Clark JH, Blaustein JD, O’Malley BW 1994 Convergent pathways for steroid hormone-and neurotransmitter-induced rat sexual behavior. Science 265:1246–1249
Beyer C, Gonzalez-Flores O, Gonzalez-Mariscal G 1997 Progesterone receptor participates in the stimulatory effect of LHRH, prostaglandin E2, and cyclic AMP on lordosis and proceptive behaviors in rats. J Neuroendocrinol 9:609–614
Auger AP, Moffatt CA, Blaustein JD 1997 Progesterone-independent activation of rat brain progestin receptors by reproductive stimuli. Endocrinology 138:511–514
Curtis SW, Washburn T, Sewall C, DiAugustine R, Lindzey J, Couse JF, Korach KS 1996 Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. Proc Natl Acad Sci USA 93:12626–12630
El-Tanani MK, Green CD 1997 Two separate mechanisms for ligand-independent activation of the estrogen receptor. Mol Endocrinol 11:928–937
Aronica SM, Katzenellenbogen BS 1993 Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen. Mol Endocrinol 7:743–752
Schlegel A, Wang C, Katzenellenbogen BS, Pestell RG, Lisanti MP 1999 Caveolin-1 potentiates estrogen receptor (ER) signaling. caveolin-1 drives ligand-independent nuclear translocation and activation of ER. J Biol Chem 274:33551–33556
Cho H, Katzenellenbogen BS 1993 Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. Mol Endocrinol 7:441–452
Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658–4662
Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP, Korach KS 2002 Requirement of estrogen receptor- in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. J Biol Chem 277:8531–8537
Apostolakis EM, Garai J, Lohmann JE, Clark JH, O’Malley BW 2000 Epidermal growth factor activates reproductive behavior independent of ovarian steroids in female rodents. Mol Endocrinol 14:1086–1098
Perez-Martin M, Azcoitia I, Trejo JL, Sierra A, Garcia-Segura LM 2003 An antagonist of estrogen receptors blocks the induction of adult neurogenesis by insulin-like growth factor-I in the dentate gyrus of adult female rat. Eur J Neurosci 18:923–930
Smith CL, Conneely OM, O’Malley BW 1993 Modulation of the ligand-independent activation of the human estrogen receptor by hormone and antihormone. Proc Natl Acad Sci USA 90:6120–6124
Gangolli EA, Conneely OM, O’Malley BW 1997 Neurotransmitters activate the human estrogen receptor in a neuroblastoma cell line. J Steroid Biochem Mol Biol 61:1–9
Lesage J, Bernet F, Montel V, Dupouy JP 1996 Hypothalamic metabolism of neurotransmitters (serotonin, norepinephrine, dopamine) and NPY, and gonadal and adrenal activities, during the early postnatal period in the rat. Neurochem Res 21:87–96
Hull EM, Nishita JK, Bitran D, Dalterio S 1984 Perinatal dopamine-related drugs demasculinize rats. Science 224:1011–1013
Gonzales FG, Ortega JG, Salazar M 2000 Effect of neonatal administration of an antidopaminergic drug (metoclopramide) on sexual behavior of male rats. Arch Androl 45:137–142
Tonjes R, Gotz F, Maywald J, Dorner G 1989 Influence of a dopamine agonist (lisuride) on sex-specific behavioural patterns in rats. II. Long-term effects. Exp Clin Endocrinol 94:48–54
Gotz F, Tonjes R, Maywald J, Dorner G 1991 Short- and long-term effects of a dopamine agonist (lisuride) on sex-specific behavioural patterns in rats. Exp Clin Endocrinol 98:111–121
Weaver DR, Reppert SM 1995 Definition of the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the rat suprachiasmatic nucleus. Mol Brain Res 33:136–148
Vancutsem PM, Roessler ML 1997 Neonatal treatment with tamoxifen causes immediate alterations of the sexually dimorphic nucleus of the preoptic area and medial preoptic area in male rats. Teratology 56:220–228
Paxinos G, Watson C 1997 The rat brain in stereotaxic coordinates. Compact ed. 3rd ed. Sydney, Australia: Academic Press
Casto JM, Ward OB, Bartke A 2003 Play, copulation, anatomy, and testosterone in gonadally intact male rats prenatally exposed to flutamide. Physiol Behav 79:633–641
Meaney MJ, McEwen BS 1986 Testosterone implants into the amygdala during the neonatal period masculinize the social play of juvenile female rats. Brain Res 398:324–328
Weniger JP, Zeis A, Chouraqui J 1993 Estrogen production by fetal and infantile rat ovaries. Reprod Nutr Dev 33:129–136
Greco B, Allegretto EA, Tetel MJ, Blaustein JD 2001 Coexpression of ER with ER and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment. Endocrinology 142:5172–5181
Perez SE, Chen EY, Mufson EJ 2003 Distribution of estrogen receptor and immunoreactive profiles in the postnatal rat brain. Brain Res Dev Brain Res 145:117–139
Tsai HW, Katzenellenbogen JA, Katzenellenbogen BS, Shupnik MA 2004 Protein kinase A activation of estrogen receptor transcription does not require proteasome activity and protects the receptor from ligand-mediated degradation. Endocrinology 145:2730–2738
Quadros PS, Pfau JL, Goldstein AY, De Vries GJ, Wagner CK 2002 Sex differences in progesterone receptor expression: a potential mechanism for estradiol-mediated sexual differentiation. Endocrinology 143:3727–3739
Wagner CK, Pfau JL, De Vries GJ, Merchenthaler IJ 2001 Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor expression. J Neurobiol 47:176–182
Schalling M, Djurfeldt M, Hokfelt T, Ehrlich M, Kurihara T, Greengard P 1990 Distribution and cellular localization of DARPP-32 mRNA in rat brain. Brain Res Mol Brain Res 7:139–149
Ouimet CC 1991 DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, is present in corticothalamic neurons of the rat cingulate cortex. Brain Res 562:85–92
Weiner DM, Levey AI, Sunahara RK, Niznik HB, O’Dowd BF, Seeman P, Brann MR 1991 D1 and D2 dopamine receptor mRNA in rat brain. Proc Natl Acad Sci USA 88:1859–1863
Lazarov NE, Schmidt U, Wanner I, Pilgrim C 1998 Mapping of D1 dopamine receptor mRNA by non-radioactive in situ hybridization. Histochem Cell Biol 109:271–279
Staudt J, Dorner G 1976 Structural changes in the medial and central amygdala of the male rat, following neonatal castration and androgen treatment. Endokrinologie 67:296–300
Meaney MJ, Dodge AM, Beatty WW 1981 Sex-dependent effects of amygdaloid lesions on the social play of prepubertal rats. Physiol Behav 26:467–472
Daenen EW, Wolterink G, Gerrits MA, Van Ree JM 2002 The effects of neonatal lesions in the amygdala or ventral hippocampus on social behaviour later in life. Behav Brain Res 136:571–582
De Vries GJ, Simerly RB 2002 Anatomy, development, and function of sexually dimorphic neural circuits in the mammalian brain. In: Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, eds. Hormones, brain and behavior. San Diego: Academic Press; 137–191
Beatty WW, Dodge AM, Traylor KL, Meaney MJ 1981 Temporal boundary of the sensitive period for hormonal organization of social play in juvenile rats. Physiol Behav 26:241–243
Thor DH, Holloway Jr WR 1986 Social play soliciting by male and female juvenile rats: effects of neonatal androgenization and sex of cagemates. Behav Neurosci 100:275–279
Tonjes R, Docke F, Dorner G 1987 Effects of neonatal intracerebral implantation of sex steroids on sexual behaviour, social play behaviour and gonadotrophin secretion. Exp Clin Endocrinol 90:257–263
Meaney MJ, Stewart J, Poulin P, McEwen BS 1983 Sexual differentiation of social play in rat pups is mediated by the neonatal androgen-receptor system. Neuroendocrinology 37:85–90
Sokka TA, Huhtaniemi IT 1995 Functional maturation of the pituitary-gonadal axis in the neonatal female rat. Biol Reprod 52:1404–1409(Kristin M. Olesen, Heathe)