Somatic and Neuroendocrine Changes in Response to Chronic Corticosterone Exposure During Adolescence in Male and Female Rats.

Prolonged stress and repeated activation of the hypothalamic-pituitary-adrenal axis can result in many sex-dependent behavioural and metabolic changes in rats, including alterations in feeding behaviour and reduced body weight. In adults, these effects of stress can be mimicked by corticosterone, a major output of the hypothalamic-pituitary-adrenal axis, and recapitulate the stress-induced sex difference, such that corticosterone-treated males show greater weight loss than females. Similar to adults, chronic stress during adolescence leads to reduced weight gain, particularly in males. However, it is currently unknown whether corticosterone mediates this somatic change and whether additional measures of neuroendocrine function are affected by chronic corticosterone exposure during adolescence in a sex-dependent manner. Therefore, we examined the effects of non-invasively administered corticosterone (150 or 300 µg/ml) in the drinking water of male and female rats throughout adolescent development (30-58 days of age). We found that adolescent animals exposed to chronic corticosterone gain significantly less weight than controls, which may be partly mediated by the effects of corticosterone on food consumption, fluid intake and gonadal hormone function. Our data further show that, despite similar circulating corticosterone levels, males demonstrate a greater sensitivity to these changes than females. We also found that Npy1 and Npy5 receptor mRNA expression, genes implicated in appetite regulation, was significantly reduced in the ventral medial hypothalamus of corticosterone-treated males and females compared to controls. Finally, parameters of gonadal function, such as plasma sex steroid concentrations and weight of reproductive tissues, were reduced by adolescent corticosterone treatment, although only in males. The data obtained in the present study indicate that chronic corticosterone exposure throughout adolescent development results in significant and sex-dependent somatic and neuroendocrine changes, and the results also provide an experimental framework for further investigating the impact of corticosterone on metabolic and neuroendocrine function during adolescence.

Stress and the developing adolescent brain.

Adolescence is a time of continued brain maturation, particularly in limbic and cortical regions, which undoubtedly plays a role in the physiological and emotional changes coincident with adolescence. An emerging line of research has indicated that stressors experienced during this crucial developmental stage may affect the trajectory of this neural maturation and contribute to the increase in psychological morbidities, such as anxiety and depression, often observed during adolescence. In this review, we discuss the short- and long-term effects of periadolescent stress exposure on the structure and function of the brain. More specifically, we examine how stress at prepubertal and early adolescent stages of development affects the morphological plasticity of limbic and cortical brain regions, as well as the enduring effects of adolescent stress exposure on these brain regions in adulthood. We suggest that, due to a number of converging factors during this period of maturation, the adolescent brain may be particularly sensitive to stress-induced neurobehavioral dysfunctions with important consequences on an individual's immediate and long-term health and well-being.

Estradiol acts via estrogen receptors alpha and beta on pathways important for synaptic plasticity in the mouse hippocampal formation.

Estradiol affects hippocampal-dependent spatial memory and underlying structural and electrical synaptic plasticity in female mice and rats. Using estrogen receptor (ER) alpha and beta knockout mice and wild-type littermates, we investigated the role of ERs in estradiol effects on multiple pathways important for hippocampal plasticity and learning. Six hours of estradiol administration increased immunoreactivity for phosphorylated Akt throughout the hippocampal formation, whereas 48 h of estradiol increased immunoreactivity for phosphorylated TrkB receptor. Estradiol effects on phosphorylated Akt and TrkB immunoreactivities were abolished in ER alpha and ER beta knockout mice. Estradiol also had distinct effects on immunoreactivity for post-synaptic density 95 (PSD-95) and brain derived-neurotrophic factor (BDNF) mRNA in ER alpha and beta knockout mice. Thus, estradiol acts through both ERs alpha and beta in several subregions of the hippocampal formation. The different effects of estradiol at 6 and 48 h indicate that several mechanisms of estrogen receptor signaling contribute to this female hormone's influence on hippocampal synaptic plasticity. By further delineating these mechanisms, we will better understand and predict the effects of endogenous and exogenous ovarian steroids on mood, cognition, and other hippocampal-dependent behaviors.

Pubertal-related changes in hypothalamic-pituitary-adrenal axis reactivity and cytokine secretion in response to an immunological stressor.

Pubertal development is marked by profound changes in stress reactivity. For example, following a brief stressor, such as foot shock, ether inhalation or restraint, prepubertal rats display a prolonged adrenocorticotrophic hormone (ACTH) and corticosterone response that takes twice as long to return to baseline compared to adults. Pubertal-related differences in the recovery of the hormonal stress response following a more protracted systemic stressor, such as an immunological challenge, have not yet been investigated. Moreover, it is unclear whether an immunological stressor leads to a differential cytokine response in animals before and after pubertal maturation. To examine these issues, we used a single injection of lipopolysaccharide (LPS; 0.1 mg/kg) to induce a hormonal stress and innate immune response and measured plasma ACTH, corticosterone, and the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 in prepubertal and adult male rats 0, 2, 4, 6, 8, or 24 h after LPS exposure. In a follow-up experiment, we assessed neural activation, as indexed by FOS immunohistochemistry, in the paraventricular nucleus of the hypothalamus (PVN) in prepubertal and adult males 0, 4, 8, or 24 h after a 0.1 mg/kg injection of LPS. By contrast to the prolonged response observed in prepubertal animals following a variety of acute stressors, we found that corticosterone and IL-6 responses induced by LPS recover toward baseline faster in prepubertal compared to adult rats. Along with these different peripheral responses, we also found that LPS-induced neural activation in the PVN of prepubertal animals showed a faster return to baseline compared to adults. Together, these data indicate that prepubertal and adult animals react in distinct ways, both peripherally and centrally, to an immunological stressor.

Ultrastructural evidence that androgen receptors are located at extranuclear sites in the rat hippocampal formation.

Like estrogens in female rats, androgens can affect dendritic spine density in the CA1 subfield of the male rat hippocampus [J Neurosci 23:1588 (2003)]. Previous light microscopic studies have shown that androgen receptors (ARs) are present in the nuclei of CA1 pyramidal cells. However, androgens may also exert their effects through rapid non-genomic mechanisms, possibly by binding to membranes. Thus, to investigate whether ARs are at potential extranuclear sites of ARs, antibodies to ARs were localized by light and electron microscopy in the male rat hippocampal formation. By light microscopy, AR immunoreactivity (-ir) was found in CA1 pyramidal cell nuclei and in disperse, punctate processes that were most dense in the pyramidal cell layer. Additionally, diffuse AR-ir was found in the mossy fiber pathway. Ultrastructural analysis revealed AR-ir at several extranuclear sites in all hippocampal subregions. AR-ir was found in dendritic spines, many arising from pyramidal and granule cell dendrites. AR-ir was associated with clusters of small, synaptic vesicles within preterminal axons and axon terminals. Labeled preterminal axons were most prominent in stratum lucidum of the CA3 region. AR-containing terminals formed asymmetric synapses or did not form synaptic junctions in the plane of section analyzed. AR-ir also was detected in astrocytic profiles, many of which apposed terminals synapsing on unlabeled dendritic spines or formed gap junctions with other AR-labeled or unlabeled astrocytes. Collectively, these results suggest that ARs may serve as both a genomic and non-genomic transducer of androgen action in the hippocampal formation.

Estradiol affects spinophilin protein differently in gonadectomized males and females.

Estrogen (E) treatment of ovariectomized animals increases dendritic spines and/or synaptic protein expression in the hippocampus of female rats [J Neurosci 12 (1992) 2549; Endocrinology 142 (2001) 1284; Endocrinol Rev 20 (1999) 279; Annu Rev Pharmacol Toxicol 41 (2001) 569], mice [Proc Natl Acad Sci USA 101 (2004) 2185], rhesus monkeys [Proc Natl Acad Sci USA 98 (2001) 8071; Endocrinology 144 (2003) 4734; J Comp Neurol 465 (2003) 540] and hippocampal cells in vitro [J Neurosci 16 (1996) 4059; Neuroscience 124 (2004) 549]. The role of E in hippocampal synaptic structural plasticity in males is less well understood. In the present study, we have used a recently developed technique to count spinophilin immunogold-reactive (Ir) puncta as well as in situ hybridization to compare E effects on spinophilin-Ir and mRNA in gonadectomized female and male rats 48 h after E treatment. Spinophilin is an established spine marker, which interacts with several proteins (including actin and protein phosphatase 1) that are highly enriched in spines [Proc Natl Acad Sci USA 94 (1997) 9956; Proc Natl Acad Sci USA 97 (2000) 9287]. We report that E exerts sex-specific effects on dendritic spinophilin-labeled spines in the CA1 region: E treatment significantly increased spinophilin-Ir puncta, indicative of spines, in females, but led to a decrease in males. Furthermore, while hippocampal spinophilin mRNA changes could have occurred earlier, spinophilin mRNA levels were unchanged after 48 h of E in both males and females. This suggests the possibility that E regulates spinophilin protein expression and or stability within dendrites via post-transcriptional mechanisms.

Puberty: a period of both organizational and activational effects of steroid hormones on neurobehavioural development.

During perinatal development, steroid hormones act on the central nervous system (CNS) to organize neural circuits. These circuits remain relatively dormant until hormonal stimulation received in adulthood acts on the CNS to activate adult reproductive physiology and behaviour. In this review, the proposal is put forward that, in addition to perinatal development, puberty serves as another period of neural maturation mediated by both steroid-dependent and -independent events that further organize and shape the behavioural potential of the adult organism. In support of this thesis, data are summarized that clearly show the organizational effects of the pubertal rise in gonadal hormones on mating behaviour and other steroid-mediated behaviours exhibited in adulthood, and on the neural pathways that mediate these behaviours. The importance of determining whether this sensitive period of neural development during puberty is a 'critical period' is also discussed, as well as whether perturbations of the nervous system during pubertal development may result in negative behavioural and physiological outcomes in adulthood. It is concluded that puberty is not merely a time when increasing levels of gonadal steroids activate the neural circuits organized during perinatal development, but also a time of further organization of the CNS, which allows for appropriate behaviours to emerge in adulthood.

Dihydrotestosterone activates sexual behavior in adult male hamsters but not in juveniles.

The effect of an androgenic metabolite of testosterone, dihydrotestosterone (DHT), on reproductive behavior and brain androgen receptor (AR) immunoreactivity was compared in juvenile and adult male Syrian hamsters. Prepubertal and adult animals were castrated and treated with 0, 500, or 1000 microg of DHT daily for 1 week and then tested for their ability to engage in mating behavior. The 1000-microg dose of DHT activated intromissions in adult but not prepubertal males. Brains were collected immediately after the behavioral test to investigate whether the lack of a behavioral response to DHT prior to puberty is associated with fewer AR-immunoreactive (AR-ir) cells in the forebrain nuclei that mediate male sexual behavior. In four of the five nuclei within the behavioral circuit that were examined, the number of AR-containing cells was similar in prepubertal and adult males treated with 1000 microg of DHT. Only in the anterior medial amygdala (MeA) was there a greater number of AR-ir cells in adults. These data indicate that (1) DHT does not activate components of male reproductive behavior prior to puberty and (2) the lack of behavioral responsiveness to DHT in prepubertal males is most likely not related to an overall reduction in ARs within the forebrain circuit that mediates mating behavior.

Pubertal and seasonal plasticity in the amygdala.

The present experiments investigated the effects of pubertal maturation and photoperiod on the size of brain regions that mediate mating behavior in the male Syrian hamster. We hypothesized that the low levels of reproductive behavior exhibited by prepubertal and photoinhibited males would be correlated with morphological changes in the neural circuit that mediates mating behavior. We found that the Nissl-stained cross-sectional area of the posterodorsal subdivision of the medial amygdala was significantly smaller in prepubertal and photoinhibited males compared to photostimulated adult males. These differences appear to be caused by a decrease in somal size of individual cells in the ventral aspect of this nucleus. We also found that prepubertal males have a larger anterior subdivision of the medial amygdala (MeA) compared to adults. This difference in the MeA does not appear to be caused by alteration in somal size since somal size did not differ significantly between juveniles and adults. It is concluded that the neural circuit that mediates male mating behavior in this species is capable of significant morphological plasticity during both pubertal development and in adulthood. Furthermore, these alterations may reflect underlying mechanisms of the deficits in sexual behavior exhibited by prepubertal and photoinhibited males.

Effects of gonadal steroids during pubertal development on androgen and estrogen receptor-alpha immunoreactivity in the hypothalamus and amygdala.

Perinatal development is often viewed as the major window of time for organization of steroid-sensitive neural circuits by steroid hormones. Behavioral and neuroendocrine responses to steroids are dramatically different before and after puberty, suggesting that puberty is another window of time during which gonadal steroids affect neural development. In the present study, we investigated whether the presence of gonadal hormones during pubertal development affects the number of androgen receptor and estrogen receptor alpha-immunoreactive (AR-ir and ER alpha-ir, respectively) cells in limbic regions. Male Syrian hamsters were castrated either before or after pubertal development, and 4 weeks later they received a single injection of testosterone or oil vehicle 4 h prior to tissue collection. Immunocytochemistry for AR and ER alpha was performed on brain sections from testosterone-treated and oil-treated males, respectively. Adult males that had been castrated before puberty had a greater number of AR-ir cells in the medial preoptic nucleus than adult males that had been castrated after puberty. There were no significant differences in ER alpha-ir cell number in any of the brain regions examined. The demonstration that exposure to gonadal hormones during pubertal development is associated with reduced AR-ir in the medial preoptic nucleus indicates that puberty is a period of neural development during which hormones shape steroid-sensitive neural circuits.

GnRH mRNA increases with puberty in the male Syrian hamster brain.

Puberty in the male Syrian hamster (Mesocricetus auratus) is characterized by decreased responsiveness to testosterone mediated negative feedback, but the neural mechanism for this change remains elusive. We hypothesized that decreased inhibition of the gonadotropin-releasing hormone (GnRH) system results in increased neurosecretory activity, which includes an increase in GnRH gene expression. This study examined GnRH mRNA in male hamsters before and after puberty, and sought to determine if any increase in mRNA was specific to particular subpopulations of GnRH neurones. Brains were collected from 21-day-old prepubertal males (n = 5) and 56-day-old postpubertal males (n = 5). Alternate 10 microm coronal sections from fresh-frozen brains were collected throughout the septo-hypothalamic region, and 25% of those sections were processed for in-situ hybridization histochemistry using an 35S-riboprobe complementary to hamster GnRH. No differences were observed in the number of GnRH mRNA expressing cells in any region, but in the diagonal band of Broca (DBB)/organum vasculosum of the lamina terminalis (OVLT) there was a significant increase in labelling intensity (defined as area of the cell occupied by silver grains) in postpubertal males. A second analysis compared the frequency distributions of cells based on labelling intensity between prepubertal and postpubertal males. This analysis revealed significant differences between the two frequency distributions in all areas analysed (DBB/OVLT, medial septum (MS), and preoptic area (POA)). Furthermore, examining the distribution of cells in these regions revealed a shift to the right in the postpubertal population of cells, which indicated an increased number of GnRH neurones with greater labelling intensity. These data clearly demonstrate increased GnRH mRNA during puberty. Furthermore, they suggest that the previous observation of brain region specific pubertal decreases in GnRH-immunoreactivity only within the DBB/OVLT and MS but not the POA are not due to differential levels of GnRH gene expression, but could indicate increased release from these neurones during puberty.

Fetal alcohol exposure blocks full masculinization of the dorsolateral nucleus in rat spinal cord.

Male rats prenatally exposed to a combination of stress and ethanol show severely impaired ejaculatory patterns. This study examined two sexually dimorphic nuclei in the lumbar spinal cord implicated in the control of male copulatory reflexes in rats whose mothers were exposed to alcohol, to stress, or to both treatments during pregnancy. Alcohol exposure led to a marked decrease (22%) in the number of motor neurons in the dorsolateral nucleus (DLN) of the adult male offspring, but no significant change in cell count was detectable in the sexually dimorphic nucleus of the bulbocavernosus (SNB). The combination of alcohol and stress did not enhance the effect on the DLN above that produced by alcohol alone. Somal sizes in the DLN and SNB were not altered by any of the treatment conditions. Alcohol exposure probably leads to incomplete masculinization of the DLN in male rats by decreasing testicular steroidogenesis during the fetal stage(s) when sexual differentiation is ongoing in that CNS structure.

Estrogen receptor immunoreactivity in prepubertal and adult male Syrian hamsters.

Estrogen and estrogen receptors (ER) are involved in the expression of steroid-dependent male sexual behavior and negative feedback regulation of the hypothalamic-pituitary-gonadal axis. The purpose of the present experiment was to determine whether there are pubertal changes in ER expression in brain that are correlated with pubertal changes in responsiveness to steroid negative feedback and behavioral activation. We found equivalent numbers of ER-immunoreactive (ER-ir) cells in castrated prepubertal and adult male hamsters in nuclei that comprise the neural circuit that mediate male sexual behavior. Therefore, increases in the number of cells in these nuclei that express ER are not correlated with the increased behavioral responsiveness to steroid hormone shown by hamsters after puberty. The number of ER-ir cells in the ventral medial hypothalamus was less in adults compared with juveniles. This pubertal decrease in ER expression is correlated with the decreased responsiveness to steroid negative feedback in the adult.

Regional changes in GnRH immunoreactivity with puberty in the male Syrian hamster.

Gonadotropin-releasing-hormone immunopositive (GnRH+) neurons were identified in juvenile and adult male Syrian hamsters. There were significantly fewer GnRH+ cells in the diagonal band of Broca/organum vasculosum of the lamina terminalis (DBB/OVLT) and medial septum (MS) in adults as compared to juvenile males, while no cell number difference was found in the preoptic area (POA). The decrease in cell number likely reflects reduced somal stores of GnRH in DBB/OVLT and MS, suggesting that these subpopulations promote increased GnRH release during pubertal maturation in male hamsters.

Androgenic regulation of hypothalamic aromatase activity in prepubertal and postpubertal male golden hamsters.

Doses of testosterone that fully activate male reproductive behavior in castrated adult male hamsters fail to elicit mounting and intromissions in prepubertal castrates, even when circulating levels of testosterone are equivalent in the two age groups. We hypothesize that this differential responsiveness to testosterone is mediated at least in part by the efficacy with which testosterone in the hypothalamus is aromatized to estradiol, an important hormone mediating male sexual behavior. Therefore, hypothalamic aromatase activity, as measured by the conversion of [3H]testosterone to [3H]estradiol in tissue homogenates, was assessed in four separate experiments: 1) intact prepubertal and adult male golden hamsters, 2 and 3) castrated adult or prepubertal males that received either a 0- or 2.5-mg dose of testosterone, and 4) castrated adult and prepubertal males treated with the 2.5-mg dose oftestosterone. These studies demonstrate that hypothalamic aromatase activity is significantly higher in adult males compared with prepubertal males, and that hypothalamic aromatase activity is increased by testosterone to the same extent in both the adult and prepubertal male hamster. Therefore, the failure of testosterone-treated castrated prepubertal male hamsters to engage in the full suite of male reproductive behaviors is not due to the inability of testosterone to be converted into estradiol in the hypothalamus. Differences in the ability of testosterone to increase aromatase activity in other brain regions, or differences in the action of testosterone and/or estradiol on other cellular processes must account for the inability of testosterone to facilitate male reproductive behavior in juvenile males.

Pheromones elicit equivalent levels of Fos-immunoreactivity in prepubertal and adult male Syrian hamsters.

Male reproductive behavior in the Syrian hamster is dependent on both pheromones from the female and the presence of gonadal steroid hormones. The pheromones are contained within female hamster vaginal secretions (FHVS) and stimulate anogenital investigation and mounting by the male. Administration of testosterone to castrated male hamsters facilitates anogenital investigation, mounts, and intromissions in adults, but elicits only anogenital investigation in prepubertal males. One hypothesis for why the full complement of reproductive behaviors is not activated by testosterone in prepubertal males is that the neural processing of pheromonal cues encountered during anogenital investigation is different in juveniles and adults. In the present experiment, we investigated the influence of sexual maturity on Fos expression in response to FHVS in the male Syrian hamster. We predicted a greater increase in Fos-immunoreactivity after exposure to FHVS within the neural circuit mediating male reproductive behaviors in adult compared to prepubertal males. Intact adult and prepubertal males were exposed to either a clean cotton swab or a swab containing FHVS. We found that, compared to animals exposed to a clean cotton swab, both prepubertal and adult males exposed to FHVS have a greater amount of Fos-immunoreactivity within several brain nuclei comprising the neural circuit mediating male reproductive behavior. Furthermore, this Fos response was equivalent in the two age groups. These results suggest that the inability of the prepubertal male hamster to perform the full repertoire of male reproductive behaviors is not due to a lack of a neuronal activation in response to the pheromonal cues present in FHVS.

Actions of testosterone in prepubertal and postpubertal male hamsters: dissociation of effects on reproductive behavior and brain androgen receptor immunoreactivity.

This study was conducted to determine whether there is a increase in responsiveness to the activating effects of testosterone on male reproductive behavior during puberty in male golden hamsters and whether responsiveness to behavioral actions of testosterone is correlated with the ability of testosterone to upregulate brain androgen receptor immunoreactivity (AR-ir). Sexually naive male hamsters were castrated at 21 or 42 days of age and implanted subcutaneously with a pellet containing 0, 2.5, or 5 mg of testosterone. One week later, males were given a 10-min mating test with a receptive female. Animals were euthanized 1 hr after the behavioral test, and blood samples and brains were collected. Plasma testosterone levels were equivalent in prepubertal and adult males that had been administered the same dose of testosterone. However, adult males exhibited more mounts, intromissions, and ejaculations than prepubertal males, demonstrating that postpubertal males are more responsive than prepubertal males to the effects of testosterone on sexual behavior. In both age groups, testosterone increased the number of AR-ir cells per unit area in several brain regions involved in male sexual behavior, including the medial preoptic nucleus (MPN), medial amygdala, posteromedial bed nucleus of the stria terminalis, and magnocellular preoptic nucleus (MPNmag). Surprisingly, testosterone increased AR-ir in the latter three regions to a greater extent in prepubertal males than in adults. Thus, prepubertal males are more responsive to the effects of testosterone on AR-ir in these regions. In a separate experiment, a pubertal increase in the number of AR-ir cells per unit area was found in both the MPN and MPNmag of intact male hamsters. These results indicate that a testosterone-dependent increase in brain AR during puberty may be necessary, but is not sufficient, to induce an increase in behavioral responsiveness to testosterone.