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Puberty is an important phase of development when the neural circuit organization is transformed by sexual hormones, inducing sexual dimorphism in adult behavioural responses. The principal brain area responsible for the control of the receptive component of female sexual behaviour is the ventrolateral division of the ventromedial nucleus of the hypothalamus (VMHvl), which is known for its dependency on ovarian hormones. Inputs to the VMHvl originating from the medial preoptic nucleus (MPN) are responsible for conveying essential information that will trigger such behaviour. Here, we investigated the pattern of the projection of the MPN to the VMHvl in rats ovariectomized at the onset of puberty. Sprague Dawley rats were ovariectomized (OVX) at puberty and then subjected to iontophoretic injections of the neuronal anterograde tracer Phaseolus vulgaris leucoagglutinin into the MPN once they reached 90 days of age. This study analysed the connectivity pattern established between the MPN and the VMH that is involved in the neuronal circuit responsible for female sexual behaviour in control and OVX rats. The data show the changes in the organization of the connections observed in the OVX adult rats that displayed a reduced axonal length for the MPN fibres reaching the VMHvl, suggesting that peripubertal ovarian hormones are relevant to the organization of MPN connections with structures involved in the promotion of female sexual behaviour., (© 2020 Anatomical Society.)

Polycystic ovary syndrome (PCOS) is the most common form of infertility in women. The causes of PCOS are not yet understood and both genetics and early-life exposure have been considered as candidates. With regard to the latter, circulating androgens are elevated in mid-late gestation in women with PCOS, potentially exposing offspring to elevated androgens in utero; daughters of women with PCOS are at increased risk for developing this disorder. Consistent with these clinical observations, prenatal androgenization (PNA) of several species recapitulates many phenotypes observed in PCOS. There is increasing evidence that symptoms associated with PCOS, including elevated luteinizing hormone (LH) (and presumably gonadotropin-releasing hormone [GnRH]) pulse frequency emerge during the pubertal transition. We utilized translating ribosome affinity purification coupled with ribonucleic acid (RNA) sequencing to examine GnRH neuron messenger RNAs from prepubertal (3 weeks) and adult female control and PNA mice. Prominent in GnRH neurons were transcripts associated with protein synthesis and cellular energetics, in particular oxidative phosphorylation. The GnRH neuron transcript profile was affected more by the transition from prepuberty to adulthood than by PNA treatment; however, PNA did change the developmental trajectory of GnRH neurons. This included families of transcripts related to both protein synthesis and oxidative phosphorylation, which were more prevalent in adults than in prepubertal mice but were blunted in PNA adults. These findings suggest that prenatal androgen exposure can program alterations in the translatome of GnRH neurons, providing a mechanism independent of changes in the genetic code for altered expression., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neural systems underlying important behaviors are usually highly conserved across species. The medial preoptic area (MPOA) has been demonstrated to play a crucial role in reward associated with affiliative, nonsexual, social communication in songbirds. However, the role of MPOA in affiliative, rewarding social behaviors (eg, social play behavior) in mammals remains largely unknown. Here we applied our insights from songbirds to rats to determine whether opioids in the MPOA govern social play behavior in rats. Using an immediate early gene (ie, Egr1, early growth response 1) expression approach, we identified increased numbers of Egr1-labeled cells in the MPOA after social play in adolescent male rats. We also demonstrated that cells expressing mu opioid receptors (MORs, gene name Oprm1) in the MPOA displayed increased Egr1 expression when adolescent rats were engaged in social play using double immunofluorescence labeling of MOR and Egr1. Furthermore, using short hairpin RNA-mediated gene silencing we revealed that knockdown of Oprm1 in the MPOA reduced the number of total play bouts and the frequency of pouncing. Last, RNA sequencing differential gene expression analysis identified genes involved in neuronal signaling with altered expression after Oprm1 knockdown, and identified Egr1 as potentially a key modulator for Oprm1 in the regulation of social play behavior. Altogether, these results show that the MPOA is involved in social play behavior in adolescent male rats and support the hypothesis that the MPOA is part of a conserved neural circuit across vertebrates in which opioids act to govern affiliative, intrinsically rewarded social behaviors., (© 2020 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Gamma-aminobutyric acid (GABA) plays a critical role in regulation of gonadotropin-releasing hormone (GnRH) through GABA A receptor (GABA A R). Nitric oxide (NO) production has correlation with GABA and regulates GnRH secretion. This study was performed to examine the mechanisms by which manganese (Mn) accelerate puberty onset involves GABA A R/NO pathway in the preoptic area-anterior hypothalamus (POA-AH) in immature female rats. First, female rats received daily dose of MnCl 2 0 (saline), 2.5, 5 and 10 mg/kg b.w by oral gavage during postnatal day (PND) 21-32. Animals administered with 10 mg/kg MnCl 2 exhibited earlier puberty onset age and advanced ovary and uterus development than these in saline-treatment group. Furthermore, we found that decrease of GABA A R result in elevated production of nitric oxide synthase1 (NOS1), NO and GnRH in the POA-AH. Second, we recorded the neuronal spikes alternation after perfusion with GABA A R inhibitor bicuculline (BIC), GABA A R agonist isoguvacine (isog), and MnCl 2 from the POA-AH in acute brain slices of PND21 rats. Spontaneous firing revealed a powerful GABA A R-mediated action on immature POA-AH and confirm that MnCl 2 has a significant effect on GABA A R. Third, we revealed that decrease in NOS1 and NO production by treatment with isog-alone or isog+MnCl 2 contribute to the decrease of GnRH in the POA-AH and a delayed puberty onset age compared to treatment with MnCl 2 -alone. Together, these results suggested that excessive exposure to MnCl 2 stimulates NO production through decreased GABA A R in the POA-AH to advance puberty onset in immature female rats., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Testosterone activates singing within days in castrated male songbirds but full song quality only develops after a few weeks. Lesions of the medial preoptic nucleus (POM) inhibit while stereotaxic testosterone implants into this nucleus increase singing rate suggesting that this site plays a key role in the regulation of singing motivation. Testosterone action in the song control system works in parallel to control song quality. Accordingly, systemic testosterone increases POM volume within 1-2 days in female canaries, while the increase in volume of song control nuclei takes at least 2 weeks. The current study tested whether testosterone action is associated with similar differences in latencies in males. Photosensitive castrated male canaries were implanted with testosterone-filled Silastic™ implants and control castrates received empty implants, while simultaneously the photoperiod was switched from short- to long-days. Brains were collected from all subjects two days later. Plasma testosterone was elevated in testosterone-treated but not in controls. HVC volumes were not affected, but testosterone significantly increased the POM volume as identified by the dense group of aromatase-immunoreactive neurons, the number and somal area of these neurons and the fractional area they cover in POM. Testosterone-treated females from a previous experiment had a smaller POM volume in similar conditions suggesting the existence of a stable sex difference potentially affecting singing behavior. Thus testosterone induces male POM growth and aromatase expression in this nucleus within two days without affecting HVC size, further supporting the notion that testosterone increases singing motivation via its action in POM., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The delicate balance of excitation and inhibition is crucial for proper function of the cerebral cortex, relying on the accurate number and subtype composition of inhibitory gamma-aminobutyric (GABA)-expressing interneurons. Various intrinsic and extrinsic factors precisely orchestrate their multifaceted development including the long-range migration from the basal telencephalon to cortical targets as well as interneuron survival throughout the developmental period. Particularly expressed guidance receptors were described to channel the migration of cortical interneurons deriving from the medial ganglionic eminence (MGE) and the preoptic area (POA) along distinct routes. Hence, unveiling the regulatory genetic networks controlling subtype-specific gene expression profiles is key to understand interneuron-specific developmental programs and to reveal causes for associated disorders. In contrast to MGE-derived interneurons, little is known about the transcriptional networks in interneurons born in the POA. Here, we provide first evidence for the LIM-homeobox transcription factor LHX1 as a crucial key player in the post-mitotic development of POA-derived cortical interneurons. By transcriptional regulation of related genes, LHX1 modulates their survival as well as the subtype-specific expression of guidance receptors of the Eph/ephrin family, thereby affecting directional migration and layer distribution in the adult cortex., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The paraventricular nucleus (PVN) of the hypothalamus harbors diverse neurosecretory cells with critical physiological roles for the homeostasis. Decades of research in rodents have provided a large amount of information on the anatomy, development, and function of this important hypothalamic nucleus. However, since the hypothalamus lies deep within the brain in mammals and is difficult to access, many questions regarding development and plasticity of this nucleus still remain. In particular, how different environmental conditions, including stress exposure, shape the development of this important nucleus has been difficult to address in animals that develop in utero. To address these open questions, the transparent larval zebrafish with its rapid external development and excellent genetic toolbox offers exciting opportunities. In this review, we summarize recent information on the anatomy and development of the neurosecretory preoptic area (NPO), which represents a similar structure to the mammalian PVN in zebrafish. We will then review recent studies on the development of different cell types in the neurosecretory hypothalamus both in mouse and in fish. Lastly, we discuss stress-induced plasticity of the PVN mainly discussing the data obtained in rodents, but pointing out tools and approaches available in zebrafish for future studies. This review serves as a primer for the currently available information relevant for studying the development and plasticity of this important brain region using zebrafish.

The development of the neocortex requires co-ordination between proliferation and differentiation, as well as the precise orchestration of neuronal migration. Eph/ephrin signaling is crucial in guiding neurons and their projections during embryonic development. In adult ephrin-A2 knockout mice we consistently observed focal patches of disorganized neocortical laminar architecture, ranging in severity from reduced neuronal density to a complete lack of neurons. Loss of ephrin-A2 in the pre-optic area of the diencephalon reduced the migration of neocortex-bound interneurons from this region. Furthermore, ephrin-A2 participates in the creation of excitatory neurons by inhibiting apical progenitor proliferation in the ventricular zone, with the disruption of ephrin-A2 signaling in these cells recapitulating the abnormal neocortex observed in the knockout. The disturbance to the architecture of the neocortex observed following deletion of ephrin-A2 signaling shares many similarities with defects found in the neocortex of children diagnosed with autism spectrum disorder.

Paracetamol/acetaminophen (N-Acetyl-p-Aminophenol; APAP) is the preferred analgesic for pain relief and fever during pregnancy. It has therefore caused concern that several studies have reported that prenatal exposure to APAP results in developmental alterations in both the reproductive tract and the brain. Genitals and nervous system of male mammals are actively masculinised during foetal development and early postnatal life by the combined actions of prostaglandins and androgens, resulting in the male-typical reproductive behaviour seen in adulthood. Both androgens and prostaglandins are known to be inhibited by APAP. Through intrauterine exposure experiments in C57BL/6 mice, we found that exposure to APAP decreased neuronal number in the sexually dimorphic nucleus (SDN) of the preoptic area (POA) in the anterior hypothalamus of male adult offspring. Likewise, exposure to the environmental pollutant and precursor of APAP, aniline, resulted in a similar reduction. Decrease in neuronal number in the SDN-POA is associated with reductions in male sexual behaviour. Consistent with the changes, male mice exposed in uteri to APAP exhibited changes in urinary marking behaviour as adults and had a less aggressive territorial display towards intruders of the same gender. Additionally, exposed males had reduced intromissions and ejaculations during mating with females in oestrus. Together, these data suggest that prenatal exposure to APAP may impair male sexual behaviour in adulthood by disrupting the sexual neurobehavioral programming. These findings add to the growing body of evidence suggesting the need to limit the widespread exposure and use of APAP by pregnant women., (© 2017 Society for Reproduction and Fertility.)

The male bias in autism spectrum disorder incidence is among the most extreme of all neuropsychiatric disorders, yet the origins of the sex difference remain obscure. Developmentally, males are exposed to high levels of testosterone and its byproduct, estradiol. Together these steroids modify the course of brain development by altering neurogenesis, cell death, migration, differentiation, dendritic and axonal growth, synaptogenesis, and synaptic pruning, all of which can be deleteriously impacted during the course of developmental neuropsychiatric disorders. Elucidating the cellular mechanisms by which steroids modulate brain development provides valuable insights into how these processes may go awry. An emerging theme is the role of inflammatory signaling molecules and the innate immune system in directing brain masculinization, the evidence for which we review here. Evidence is also emerging that the neuroimmune system is overactivated in individuals with autism spectrum disorder. These combined observations lead us to propose that the natural process of brain masculinization puts males at risk by moving them closer to a vulnerability threshold that could more easily be breached by inflammation during critical periods of brain development. Two brain regions are highlighted: the preoptic area and the cerebellum. Both are developmentally regulated by the inflammatory prostaglandin E2, but in different ways. Microglia, innate immune cells of the brain, and astrocytes are also critical contributors to masculinization and illustrate the importance of nonneuronal cells to the health of the developing brain., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Sleep homeostasis in rats undergoes significant maturational changes during postweaning development, but the underlying mechanisms of this process are unknown. In the present study we tested the hypothesis that the maturation of sleep is related to the functional emergence of adenosine (AD) signaling in the brain. We assessed postweaning changes in 1) wake-related elevation of extracellular AD in the basal forebrain (BF) and adjacent lateral preoptic area (LPO), and 2) the responsiveness of median preoptic nucleus (MnPO) sleep-active cells to increasing homeostatic sleep drive. We tested the ability of exogenous AD to augment homeostatic responses to sleep deprivation (SD) in newly weaned rats. In groups of postnatal day (P)22 and P30 rats, we collected dialysate from the BF/LPO during baseline (BSL) wake-sleep, SD, and recovery sleep (RS). HPLC analysis of microdialysis samples revealed that SD in P30 rats results in significant increases in AD levels compared with BSL. P22 rats do not exhibit changes in AD levels in response to SD. We recorded neuronal activity in the MnPO during BSL, SD, and RS at P22/P30. MnPO neurons exhibited adult-like increases in waking neuronal discharge across SD on both P22 and P30, but discharge rates during enforced wake were higher on P30 vs. P22. Central administration of AD (1 nmol) during SD on P22 resulted in increased sleep time and EEG slow-wave activity during RS compared with saline control. Collectively, these findings support the hypothesis that functional reorganization of an adenosinergic mechanism of sleep regulation contributes to the maturation of sleep homeostasis., New & Noteworthy: Brain mechanisms that regulate the maturation of sleep are understudied. The present study generated first evidence about a potential mechanistic role for adenosine in the maturation of sleep homeostasis. Specifically, we demonstrate that early postweaning development in rats, when homeostatic response to sleep loss become adult like, is characterized by maturational changes in wake-related production/release of adenosine in the brain. Pharmacologically increased adenosine signaling in developing brain facilitates homeostatic responses to sleep deprivation.

It is well established that sexually dimorphic brain regions, which are critical for reproductive physiology and behavior, are organized by steroid hormones during the first 2 weeks after birth in the rodents. In our recent observation, neonatal exposure to estradiol-17β (E2) in the female rat revealed increase in cyclooxygenase 2 (COX-2) level, sexually dimorphic nucleus (SDN)-pre-optic area (POA) size and down-regulation of synaptogenesis related genes in POA in the adult stage. In the present study, using the same animal model, the protein profile of control and neonatally E2-treated POA was compared by 1D-SDS-PAGE, and the protein that shows a change in abundance was identified by LC-MS/MS analysis. Results indicated that there was a single protein band, which was down-regulation in E2-treated POA and it was identified as spectrin alpha chain, non-erythrocytic 1 (SPTAN1). Consistently, the down-regulation of SPTAN1 expression was also confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The SPTAN1 was identified as a cytoskeletal protein that is involved in stabilization of the plasma membrane and organizes intracellular organelles, and it has been implicated in cellular functions including DNA repair and cell cycle regulation. The evidence shows that any mutation in spectrins causes impairment of synaptogenesis and other neurological disorders. Also, protein-protein interaction analysis of SPTAN1 revealed a strong association with proteins such as kirrel, actinin, alpha 4 (ACTN4) and vinculin (VCL) which are implicated in sexual behavior, masculinization and defeminization. Our results indicate that SPTAN1 expression in the developing rat brain is sexually dimorphic, and we suggest that this gene may mediate E2-17β-induced masculinization and defeminization, and disrupted reproductive function in the adult stage.

Kisspeptin, a regulator of reproductive function and puberty in mammals, is expressed in the rostral (anteroventral) periventricular nucleus (AVPV) and arcuate nucleus (Arc), and its expression is at least partially regulated by estradiol in rodents. The aim of the present study was to determine contributions of genetic factors and gonadal steroid hormones to the sexual differentiation of kisspeptin-immunoreactive (kisspeptin-ir) cell populations in the AVPV and Arc during postnatal development using agonadal steroidogenic factor 1 (SF-1) knockout (KO) mice. To examine the effects of gonadal hormones on pubertal development of kisspeptin neurons, SF-1 KO mice were treated with estradiol benzoate (EB) from postnatal day (P)25 to P36, and their brains were examined at P36. No sex differences were observed in the SF-1 KO mice during postnatal development and after treatment with EB - which failed to increase the number of kisspeptin-ir cells at P36 to the levels found in wild-type (WT) control females. This suggests that specific time periods of estradiol actions or other factors are needed for sexual differentiation of the pattern of immunoreactive kisspeptin in the AVPV. Kisspeptin immunoreactivity in the Arc was significantly higher in gonadally intact WT and SF-1 KO females than in male mice at P36 during puberty. Further, in WT and SF-1 KO females, but not in males, adult levels were reached at P36. This suggests that maturation of the kisspeptin system in the Arc differs between sexes and is regulated by gonad-independent mechanisms., (© 2015 S. Karger AG, Basel.)

Aims: Administration of estradiol or compounds with estrogenic activity to newborn female rats results in irreversible masculinization as well as defeminization in the brain and the animals exhibit altered reproductive behavior as adults. The cellular and molecular mechanism involved in inducing the irreversible changes is largely unknown. In the present study, we have monitored the changes in the expression of selected synaptogenesis related genes in the sexually dimorphic brain regions such as POA, hypothalamus and pituitary following 17β-estradiol administration to neonatal female rats., Main Methods: Female Wistar rats which were administered 17β-estradiol on day 2 and 3 after birth were sacrificed 120days later and the expression levels of genes implicated in synaptogenesis were monitored by semi-quantitative reverse transcription PCR. Since estradiol induced up-regulation of COX-2 in POA is a marker for estradiol induced masculinization as well as defeminization, in the present study only animals in which the increase in expression of COX-2 gene was observed in POA were included in the study., Key Findings: Down-regulation of genes such as NMDA-2B, NETRIN-1, BDNF, MT-5 MMP and TNF-α was observed in the pre-optic area of neonatally E2 treated female rat brain but not in hypothalamus and pituitary compared to the vehicle- treated controls as assessed by RT-PCR and Western blot analysis., Significance: Our results suggest a possibility that down-regulation of genes associated with synaptogenesis in POA, may be resulting in disruption of the cyclical regulation of hormone secretion by pituitary the consequence of which could be infertility and altered reproductive behavior., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study examined developmental changes and sexual dimorphisms in hypothalamic microRNAs, and whether gestational exposures to environmental endocrine-disrupting chemicals (EDCs) altered their expression patterns. Pregnant rat dams were treated on gestational days 16 and 18 with vehicle, estradiol benzoate, or a mixture of polychlorinated biphenyls. Male and female offspring were euthanized on postnatal days (P) 15, 30, 45, or 90, and microRNA and mRNA targets were quantified in the medial preoptic nucleus (MPN) and ventromedial nucleus (VMN) of the hypothalamus. MicroRNAs showed robust developmental changes in both regions, and were sexually dimorphic in the MPN, but not VMN. Importantly, microRNAs in females were up-regulated by EDCs at P30, and down-regulated in males at P90. Few changes in mRNAs were found. Thus, hypothalamic microRNAs are sensitive to prenatal EDC treatment in a sex-, developmental age-, and brain region-specific manner., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Dopaminergic (DA) neurons located in the preoptico-hypothalamic region of the brain exert a major neuroendocrine control on reproduction, growth, and homeostasis by regulating the secretion of anterior pituitary (or adenohypophysis) hormones. Here, using a retrograde tract tracing experiment, we identified the neurons playing this role in the zebrafish. The DA cells projecting directly to the anterior pituitary are localized in the most anteroventral part of the preoptic area, and we named them preoptico-hypophyseal DA (POHDA) neurons. During development, these neurons do not appear before 72 hours postfertilization (hpf) and are the last dopaminergic cell group to differentiate. We found that the number of neurons in this cell population continues to increase throughout life proportionally to the growth of the fish. 5-Bromo-2'-deoxyuridine incorporation analysis suggested that this increase is due to continuous neurogenesis and not due to a phenotypic change in already-existing neurons. Finally, expression profiles of several genes (foxg1a, dlx2a, and nr4a2a/b) were different in the POHDA compared with the adjacent suprachiasmatic DA neurons, suggesting that POHDA neurons develop as a distinct DA cell population in the preoptic area. This study offers some insights into the regional identity of the preoptic area and provides the first bases for future functional genetic studies on the development of DA neurons controlling anterior pituitary functions.

The paraventricular nucleus (PVN) in mammals is the main hypothalamic nucleus controlling hormone release in the pituitary and plays pivotal roles in homeostasis. While the location of a PVN-homologous region has been described in adult fish as the neurosecretory preoptic area (NPO), this region has not been clearly defined in larval zebrafish due to the difficulty in defining cytoarchitectonic nuclear boundaries in the larval brain. Here we identify the precise location of the larval zebrafish NPO using conserved transcription factor and neuropeptide gene expressions. Our results identify the dorsal half of the preoptic-hypothalamic orthopedia a (otpa) domain as the larval NPO and the homologous region to the mammalian PVN. Further, by reconstructing the locations of cells producing zebrafish neuropeptides found in the mammalian PVN (CCK, CRH, ENK, NTS, SS, VIP, OXT, AVP), we provide the first 3D arrangement map of NPO neuropeptides in the larval zebrafish brain. Our results show striking conservation of transcription factor expression (otp, arx, dlx5a, isl1) in and around the NPO/PVN together with neuropeptide expression within it. Finally, we describe the exact anatomical location of cells producing Oxt and Avp in the adult zebrafish. Thus, our results identify the definitive borders and extent of the PVN homologous region in larval zebrafish and serve as an important basis for cross-species comparisons of PVN/NPO structure and function., (Copyright © 2013 Wiley Periodicals, Inc.)

Brain structures related to reproduction are thought to depend on the action of gonadal steroids acting either during early life (organizing irreversible effects) or adulthood (activating transient effects). More recently puberty has become a focus of attention and it was demonstrated that action of sex steroid hormones at this time plays a critical role in the final organization of brain and behavior. We studied by BrdU immunohistochemistry the ontogeny from hatching to sexual maturity of a previously identified cell population in the preoptic area labeled by a BrdU injection at the end of embryonic period (E12) of sexual differentiation in male and female Japanese quail. After an initial increase between E12 and hatching, the density of BrdU-immunoreactive cells decreased until the beginning of puberty but then increased again during sexual maturation in the caudal preoptic area specifically. Divisions of these cells took place in the brain parenchyma as indicated by the large numbers of pairs of labeled cells. No sex difference affecting these processes could be detected at any stage of development. Large numbers of new cells thus arise around puberty in the caudal preoptic area and presumably contribute to the reorganization of this structure that precedes the emergence of adult reproductive behaviors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In female sheep, estradiol (E2) stimulates the preovulatory GnRH/LH surge and receptive behavior, whereas progesterone blocks these effects. Prenatal exposure to testosterone disrupts both the positive feedback action of E2 and sexual behavior although the mechanisms remain unknown. The current study tested the hypothesis that both prenatal and postnatal steroids are required to organize the surge and sex differences in reproductive behavior. Our approach was to characterize the LH surge and mating behavior in prenatally untreated (Control) and testosterone-treated (T) female sheep subsequently exposed to one of three postnatal steroid manipulations: endogenous E2, excess E2 from a chronic implant, or no E2 due to neonatal ovariectomy (OVX). All females were then perfused at the time of the expected surge and brains processed for estrogen receptor and Fos immunoreactivity. None of the T females exposed postnatally to E2 exhibited an E2-induced LH surge, but a surge was produced in five of six T/OVX and all Control females. No surges were produced when progesterone was administered concomitantly with E2. All Control females were mounted by males, but significantly fewer T females were mounted by a male, including the T/OVX females that exhibited LH surges. The percentage of estrogen receptor neurons containing Fos was significantly influenced in a brain region-, developmental stage-, and steroid-specific fashion by testosterone and E2 treatments. These findings support the hypothesis that the feedback controls of the GnRH surge are sensitive to programming by prenatal and postnatal steroids in a precocial species.

During puberty, the brain goes through extensive remodeling, involving the addition of new neurons and glia to brain regions beyond the canonical neurogenic regions (i.e., dentate gyrus and olfactory bulb), including limbic and hypothalamic cell groups associated with sex-typical behavior. Whether these pubertally born cells become functionally integrated into neural circuits remains unknown. To address this question, we gave male Syrian hamsters daily injections of the cell birthdate marker bromodeoxyuridine throughout puberty (postnatal day 28-49). Half of the animals were housed in enriched environments with access to a running wheel to determine whether enrichment increased the survival of pubertally born cells compared with the control environment. At 4 wk after the last BrdU injection, animals were allowed to interact with a receptive female and were then killed 1 h later. Triple-label immunofluorescence for BrdU, the mature neuron marker neuronal nuclear antigen, and the astrocytic marker glial fibrillary acidic protein revealed that a proportion of pubertally born cells in the medial preoptic area, arcuate nucleus, and medial amygdala differentiate into either mature neurons or astrocytes. Double-label immunofluorescence for BrdU and the protein Fos revealed that a subset of pubertally born cells in these regions is activated during sociosexual behavior, indicative of their functional incorporation into neural circuits. Enrichment affected the survival and activation of pubertally born cells in a brain region-specific manner. These results demonstrate that pubertally born cells located outside of the traditional neurogenic regions differentiate into neurons and glia and become functionally incorporated into neural circuits that subserve sex-typical behaviors.

Brain sexual differentiation in rodents results from the perinatal testicular androgen surge. In the preoptic area (POA), estradiol aromatized from testosterone upregulates the production of the proinflammatory molecule, prostaglandin E(2) (PGE(2)) to produce sex-specific brain development. PGE(2) produces a two-fold greater density of dendritic spines in males than in females and masculinizes adult copulatory behavior. One neonatal dose of PGE(2) masculinizes the POA and behavior, and simultaneous treatment with an inhibitor of additional prostaglandin synthesis prevents this masculinization, indicating a positive feedforward process that leads to sustained increases in PGE(2). The mechanisms underlying this feedforward process were unknown. Microglia, the primary immunocompetent cells in the brain, are active neonatally, contribute to normal brain development, and both produce and respond to prostaglandins. We investigated whether there are sex differences in microglia in the POA and whether they influence developmental masculinization. Neonatal males had twice as many ameboid microglia as females and a more activated morphological profile, and both estradiol and PGE(2) masculinized microglial number and morphology in females. Microglial inhibition during the critical period for sexual differentiation prevented sex differences in microglia, estradiol-induced masculinization of dendritic spine density, and adult copulatory behavior. Microglial inhibition also prevented the estradiol-induced upregulation of PGE(2), indicating that microglia are essential to the feedforward process through which estradiol upregulates prostaglandin production. These studies demonstrate that immune cells in the brain interact with the nervous and endocrine systems during development, and are crucial for sexual differentiation of brain and behavior.

Reproductive function is highly dynamic during postnatal developmental. Here, we performed molecular profiling of gene expression patterns in the hypothalamus of developing male and female rats to identify which genes are sexually dimorphic, to gain insight into a more complex network of hypothalamic genes, and to ascertain dynamic changes in their relationships with one another and with sex steroid hormones during development. Using a low-density PCR platform, we quantified mRNA levels in the preoptic area (POA) and medial basal hypothalamus (MBH), and assayed circulating estradiol, testosterone, and progesterone at six ages from birth through adulthood. Numerous genes underwent developmental change, particularly postnatal increases, decreases, or peaks/plateaus at puberty. Surprisingly, there were few sex differences; only Esr1, Kiss1, and Tac2 were dimorphic (higher in females). Cluster analysis of gene expression revealed sexually dimorphic correlations in the POA but not the MBH from P30 (Postnatal Day 30) to P60. Hormone measurements showed few sex differences in developmental profiles of estradiol; higher levels of progesterone in females only after P30; and a developmental pattern of testosterone with a nadir at P30 followed by a dramatic increase through P60 (males). Furthermore, bionetwork analysis revealed that hypothalamic gene expression profiles and their relationships to hormones undergo dynamic developmental changes that differ considerably from adults. These data underscore the importance of developmental stage in considering the effects of hormones on the regulation of neuroendocrine genes in the hypothalamus. Moreover, the finding that few neuroendocrine genes are sexually dimorphic highlights the need to consider postnatal development from a network approach that allows assessment of interactions and patterns of expression.

Perinatal treatment with relatively high doses of bisphenol A (BPA) appears to have little effect on volume of the rodent sexually dimorphic nucleus of the preoptic area (SDN-POA). However, doses more relevant to human exposures have not been examined. Here, effects of pre- and post-natal treatment with low BPA doses on SDN-POA volume of postnatal day (PND) 21 Sprague-Dawley rats were evaluated. Pregnant rats were orally gavaged with vehicle, 2.5 or 25.0 μg/kg BPA, or 5.0 or 10.0 μg/kg ethinyl estradiol (EE₂) on gestational days 6-21. Beginning on the day after birth, offspring were orally treated with the same dose their dam had received. On PND 21, offspring (n=10-15/sex/group; 1/sex/litter) were perfused and volume evaluation was conducted blind to treatment. SDN-POA outline was delineated using calbindin D28K immunoreactivity. Pairwise comparisons of the significant treatment by sex interaction indicated that neither BPA dose affected female volume. However, females treated with 5.0 or 10.0 μg/kg EE₂ exhibited volumes that were larger than same-sex controls, respectively (p<0.001). Males treated with either BPA dose or 10.0 μg/kg/day EE₂ had larger volumes than same-sex controls (p<0.006). These data indicate that BPA can have sex-specific effects on SDN-POA volume and that these effects manifest as larger volumes in males. Sensitivity of the methodology as well as the treatment paradigm was confirmed by the expected EE₂-induced increase in female volume. These treatment effects might lead to organizational changes within sexually dimorphic neuroendocrine pathways which, if persistent, could theoretically alter adult reproductive physiology and socio-sexual behavior in rats., (Published by Elsevier Inc.)

Prenatal stress disrupts normal sexual differentiation and behavior with concomitant alterations in brain development; however, its effects on the cytoarchitecture of neurons in the sexually dimorphic medial preoptic area (mPOA) of the hypothalamus is not known. Morphometric analysis of the mPOA of adult rats showed sex differences as neurons from control females had significantly greater numbers of basal dendritic branches and cumulative basal dendritic length as compared to control male neurons. Prenatal stress significantly altered these sexual dimorphisms, as prenatally stressed (P-S) males had increased measures of cell body area, perimeter, cumulative basal dendritic length, and branch point numbers as compared to control males. Prenatal stress also altered the cytoarchitecture in the female mPOA neurons as P-S female neurons had significantly greater measures for primary dendritic branch number and a trend towards significance for several additional measures as compared to control females. Therefore, there are significant effects of both sex and prenatal stress on neuronal architecture in the mPOA that may help to explain the well-documented alterations in reproductive behaviors observed in P-S animals., (Copyright © 2011 Wiley Periodicals, Inc.)

The number of progesterone receptors is greater in the male than female neonatal rat hypothalamus. The aims of the present study were to determine developmental effects of progesterone on the expression of adult male sexual behaviour and whether changes in behaviour were reflected by altered gene expression within the hypothalamic preoptic area (POA) or medial amygdala. Male rats were treated with progesterone (40 µg kg(-1), i.p.), the progesterone receptor antagonist RU486 (40 µg kg(-1), i.p.) or an equal volume of vehicle (10% ethanol, 90% corn oil) on postnatal Days 1-5. Treatment with either progesterone or RU486 inhibited (P ≤ 0.07) the initial expression of consummatory sexual behaviour at 10.5 weeks of age without influencing growth or serum concentrations of testosterone. Sexual interest, as measured by latency to exhibiting mounting behaviour or the number of mounts achieved, was not influenced by treatment with either progesterone or RU486. The effects of treatment with progesterone or RU486 on sexual behaviour were diminished by experience. Microarray analysis of the POA indicated 61 genes that were upregulated and 49 that were downregulated (P ≤ 0.01) following RU486 treatment of male rats. However, the altered expression of selected genes was not confirmed by real-time reverse transcription-polymerase chain reaction. The expression of targeted genes within the amygdala was not influenced by treatment with either progesterone or RU486. Neonatal treatment with RU486, but not progesterone, decreased testes weight (P=0.02) without affecting testes morphology. The results indicate that altering the progesterone environment during a critical developmental period affects the expression of behaviour, but that changes in behaviour are not mirrored by the altered expression of selected genes.

Epigenetic histone modifications are emerging as important mechanisms for conveyance of and maintenance of effects of the hormonal milieu to the developing brain. We hypothesized that alteration of histone acetylation status early in development by sex steroid hormones is important for sexual differentiation of the brain. It was found that during the critical period for sexual differentiation, histones associated with promoters of essential genes in masculinization of the brain (estrogen receptor α and aromatase) in the medial preoptic area, an area necessary for male sexual behavior, were differentially acetylated between the sexes. Consistent with these findings, binding of histone deacetylase (HDAC) 2 and 4 to the promoters was higher in males than in females. To examine the involvement of histone deacetylation on masculinization of the brain at the behavioral level, we inhibited HDAC in vivo by intracerebroventricular infusion of the HDAC inhibitor trichostatin A or antisense oligodeoxynucleotide directed against the mRNA for HDAC2 and -4 in newborn male rats. Aspects of male sexual behavior in adulthood were significantly reduced by administration of either trichostatin A or antisense oligodeoxynucleotide. These results demonstrate that HDAC activity during the early postnatal period plays a crucial role in the masculinization of the brain via modifications of histone acetylation status.

There is little debate that mammalian sexual differentiation starts from the perspective of two primary sexes that correspond to differential sex chromosomes (X versus Y) that lead to individuals with sex typical characteristics. Sex steroid hormones account for most aspects of brain sexual differentiation, however, a growing literature has raised important questions about the role of sex chromosomal genes separate from sex steroid actions. Several important model animals are being used to address these issues and, in particular, they are taking advantage of molecular genetic approaches using different mouse strains. The current review examines the cooperation of genetic and endocrine influences from the perspective of behavioral and morphological hypothalamic sexual differentiation, first in adults and then in development. In the final analysis, there is an ongoing need to account for the influence of hormones in the context of underlying genetic circumstances and null hormone conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Now under intense scrutiny, due to its endocrine disrupting properties, the potential threat the plastics component bisphenol-A (BPA) poses to human health remains unclear. Found in a multitude of polycarbonate plastics, food and beverage containers, and medical equipment, BPA is thought to bind to estrogen receptors (ERs), thereby interfering with estrogen-dependent processes. Our lab has previously shown that exposure to BPA (50mg/kg bw or 50μg/kg bw) during the neonatal critical period is associated with advancement of puberty, early reproductive senescence and ovarian malformations in female Long Evans rats. Here, using neural tissue obtained from the same animals, we explored the impact of neonatal BPA exposure on the development of sexually dimorphic hypothalamic regions critical for female reproductive physiology and behavior. Endpoints included quantification of oxytocin-immunoreactive neurons (OT-ir) in the paraventricular nucleus (PVN), serotonin (5-HT-ir) fiber density in the ventrolateral subdivision of the ventromedial nucleus (VMNvl) as well as ERα-ir neuron number in the medial preoptic area (MPOA), the VMNvl, and the arcuate nucleus (ARC). Both doses of BPA increased the number of OT-ir neurons within the PVN, but no significant effects were seen on 5-HT-ir fiber density or ERα-ir neuron number in any of the areas analyzed. In addition to hypothalamic development, we also assessed female sex behavior and body weight. No effect of BPA on sexual receptivity or proceptive behavior in females was observed. Females treated with BPA, however, weighed significantly more than control females by postnatal day 99. This effect of BPA on weight is critical because alterations in metabolism, are frequently associated with reproductive dysfunction. Collectively, the results of this and our prior study indicate that the impact of neonatal BPA exposure within the female rat hypothalamus is region specific and support the hypothesis that developmental BPA exposure may adversely affect reproductive development in females., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The birth date of neurons comprising the sexually dimorphic nucleus of the rat preoptic area (SDN-POA) was determined by bromodeoxyuridine (BrdU) injections at a prescribed time during the embryonic period. Calbindin immunostaining was used as a marker to identity the SDN-POA. The animals were bred from dams injected with BrdU on days 14, 16 or 18 of pregnancy (fertilization defined as day 1). On day 15 after birth (PD), all offspring were euthanized and brain sections were prepared for histology. Neurogenesis in the SDN-POA began around embryonic day (ED) 14 and culminated on ED 18, whereas the preoptic neurons surrounding the SDN-POA generated earlier than did those of the SDN-POA. Although the SDN-POA was significantly larger in males than in females at PD15, the total numbers of neurons comprising the SDN-POA were not significantly different between sexes. Similar aggregates of somatostatin mRNA-positive cells in the central portion of the SDN-POA were observed in both sexes at PD8. On PD15, the aggregates became scattered in males, whereas the aggregates in females remained congested. These data suggest that sexual dimorphism in the SDN-POA results from male-specific postnatal radial spreading of cells rather than cell proliferation during embryonic neurogenesis., (2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Genomic imprinting results in preferential expression of the paternal or maternal allele of certain genes. We have performed a genome-wide characterization of imprinting in the mouse embryonic and adult brain. This approach uncovered parent-of-origin allelic effects of more than 1300 loci. We identified parental bias in the expression of individual genes and of specific transcript isoforms, with differences between brain regions. Many imprinted genes are expressed in neural systems associated with feeding and motivated behaviors, and parental biases preferentially target genetic pathways governing metabolism and cell adhesion. We observed a preferential maternal contribution to gene expression in the developing brain and a major paternal contribution in the adult brain. Thus, parental expression bias emerges as a major mode of epigenetic regulation in the brain.

Sex differences in the brain are largely organized by a testicular hormone surge that occurs in males shortly after birth. Although this hormone surge is transient, sex differences in brain and behavior are lasting. Here we describe a sex difference in DNA methylation of the estrogen receptor-alpha (ERalpha) promoter region within the developing rat preoptic area, with males exhibiting more DNA methylation within the ERalpha promoter than females. More importantly, we report that simulating maternal grooming, a form of maternal interaction that is sexually dimorphic with males experiencing more than females during the neonatal period, effectively masculinizes female ERalpha promoter methylation and gene expression. This suggests natural variations in maternal care that are directed differentially at males vs. females can influence sex differences in the brain by creating sexually dimorphic DNA methylation patterns. We also find that the early estradiol exposure may contribute to sex differences in DNA methylation patterns. This suggests that early social interaction and estradiol exposure may converge at the genome to organize lasting sex differences in the brain via epigenetic differentiation.

The development of dopaminergic and noradrenergic neurons has received much attention based on their modulatory effect on many behavioral circuits and their involvement in neurodegenerative diseases. The zebrafish (Danio rerio) has emerged as a new model organism with which to study development and function of catecholaminergic systems. Tyrosine hydroxylase is the entry enzyme into catecholamine biosynthesis and is frequently used as a marker for catecholaminergic neurons. A genome duplication at the base of teleost evolution resulted in two paralogous zebrafish tyrosine hydroxylase-encoding genes, th1 and th2, the expression of which has been described previously only for th1. Here we investigate the expression of th2 in the brain of embryonic and juvenile zebrafish. We optimized whole-mount in situ hybridization protocols to detect gene expression in the anatomical three-dimensional context of whole juvenile brains. To confirm whether th2-expressing cells may indeed use dopamine as a neurotransmitter, we also included expression of dopamine beta hydroxylase, dopa decarboxylase, and dopamine transporter in our analysis. Our data provide the first complete account of catecholaminergic neurons in the zebrafish embryonic and juvenile brain. We identified four major th2-expressing neuronal groups that likely use dopamine as transmitter in the zebrafish diencephalon, including neurons of the posterior preoptic nucleus, the paraventricular organ, and the nuclei of the lateral and posterior recesses in the caudal hypothalamus. th2 expression in the latter two groups resolves a previously reported discrepancy, in which strong dopamine but little tyrosine hydroxylase immunoreactivity had been detected in the caudal hypothalamus. Our data also confirm that there are no mesencephalic DA neurons in zebrafish., (2009 Wiley-Liss, Inc.)

Thermal control set point is regulated by thermosensitive neurons of the preoptic anterior hypothalamus (PO/AH) and completes its development during postnatal critical sensory period. External stimuli, like increase in environmental temperature, influence the neuronal protein repertoire and, ultimately, cell properties via activation or silencing of gene transcription, both of which are regulated by the "histone code.''" Here, we demonstrated an increase in global histone H3 lysine 9 (H3K9) acetylation as well as H3K9 dimethylation in chick PO/AH during heat conditioning at the critical period of sensory development. In contrast to the global profile of H3K9 modifications, acetylation and dimethylation patterns of H3K9 at the promoter of the catalytic subunit of eukaryotic translation initiation factor 2B (Eif2b5) were opposite to each other. During heat conditioning, there was an increase in H3K9 acetylation at the Eif2b5 promoter, simultaneously with decrease in H3K9 dimethylation. These alterations coincided with Eif2b5 mRNA induction. Moreover, exposure to excessive heat during the critical period resulted in long-term effect on both H3K9 tagging at the Eif2b5 promoter and Eif2b5 mRNA expression. These data suggest a role for dynamic H3K9 post-translational modifications in global translation regulation during the thermal control establishment.

Following transcription of the SRY gene on the Y chromosome of genetic males, a cascade of genomic and biochemical events causes the developing brain to be influenced by two testosterone metabolites, the potent androgen dihydrotestosterone and the aromatization product estradiol (E2). These steroid hormones binding to their cognate nuclear receptors produce differential gene expression profiles between male and female brains, and as a result, male-typical sex behaviors appear in adulthood and female-typical sex behaviors are suppressed. Although anatomical and cellular substrates underlying sexually dimorphic brain and behavior have been identified, still very little information is available about the molecular mechanisms involved. Microarray technology is a powerful technique that can be a used to assess the changes in thousands of gene transcripts simultaneously. Thus such high-throughput screening may be a useful initial step in the identification of estrogen-responsive genes involved in the sexual differentiation of brain., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In most species, the effects of oxytocin (OT) on female reproductive behavior are dependent upon estrogen, which increases both OT and OT receptor expression. It is also becoming apparent that OT neurotransmission can influence estrogen signaling, especially during development, as neonatal OT manipulations in prairie voles alter ERalpha expression and estrogen-dependent behaviors. We tested the hypothesis that OT developmentally programs ERalpha expression and estrogen sensitivity in female Sprague-Dawley rats, a species previously used to establish the estrogen-dependence of OT signaling in adulthood. OT treatment for the first postnatal week significantly increased ERalpha-immunoreactivity in the ventromedial nucleus of the hypothalamus (VMH), but not in the medial preoptic area (MPOA). Conversely, neonatal OT antagonist (OTA) treatment significantly reduced ERalpha-immunoreactivity in the MPOA, but not in the VMH. Both treatments increased OT-immunoreactivity in the paraventricular nucleus of the hypothalamus (PVN) and reduced estrogen sensitivity, indicated by reduced sexual receptivity following chronic estradiol benzoate (EB) administration. Behavioral deficits in OTA-treated females were apparent during both paced and non-paced tests with 0.5 microg EB (but not 5.0 or 10.0 microg EB), whereas deficits in OT-treated females were only observed during the initial paced test with 0.5 and 5.0 microg EB (but not 10.0 microg EB). The current results demonstrate that OT can positively regulate ERalpha expression within the MPOA and VMH during development; however, endogenous OT selectively programs ERalpha expression within the MPOA. Thus, exogenous OT or OTA exposure during development may have long-term consequences on behavior through stable changes in ERalpha and OT expression.

Amylin, or islet amyloid polypeptide, is known as a satiating signal expressed in pancreatic beta-cells but not in the brain. In this study, regulations of postpartum mRNA expressions were investigated in the preoptic area of the hypothalamus. mRNA levels of lactating dams and mothers whose pups were removed immediately after delivery were compared in microarray experiments. There were 20 genes identified with significantly increased and 14 with decreased expression 9 days postpartum. Amylin mRNA level demonstrated the largest change, a 25.7 times increase. Quantitative RT-PCR measurements validated the increase in the mRNA level of amylin in the preoptic area of lactating dams while the expression level of other members of the calcitonin gene-related peptide family did not change. In situ hybridization histochemistry for amylin further verified its induction in lactating mothers and demonstrated the distribution of amylin mRNA in the medial preoptic nucleus, parts of the medial preoptic area, and the ventral part of the bed nucleus of the stria terminalis but nowhere else in the rat brain. Immunolabeling verified the postpartum induction of amylin in the preoptic area at the peptide level, as well. The results suggest that amylin may play a part in maternal regulations.

Prostaglandin E(2) (PGE(2)) mediates the masculinization of adult sex behavior in rats in response to the surge in serum testosterone at approximately birth. Measures of behavioral masculinization correlate with a twofold increase in spinophilin protein and the density of dendritic spines in the medial preoptic area (POA). Of the four receptors for PGE(2), EP(2) and EP(4) are required for the masculinization of behavior by PGE(2). EP(2) and EP(4) couple to G(s)-proteins, activating protein kinase A (PKA). By using H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl) and Ht31, disruptors of PKA signaling, we have determined that PKA signaling is required for the masculinization of behavior by PGE(2). Glutamatergic signaling often mediates PGE(2) signaling; therefore, we tested whether inhibition of AMPA/kainate and metabotropic glutamate receptor (mGluR) signaling prevents PGE(2)-induced behavioral masculinization and whether activation of glutamate receptors mimics PGE(2). Females treated neonatally with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) plus LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] combined (AMPA/kainate and mGluR inhibitors, respectively) before PGE(2) did not exhibit as many mounts or intromission-like behaviors or initiate these behaviors as quickly as animals treated with PGE(2) alone. Animals neonatally treated with kainate, (+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) (type I mGluR agonist), or the two combined mounted as frequently and initiated mounting behavior as quickly as those given PGE(2). Ht31 does not prevent the masculinization of behavior by ACPD plus kainate cotreatment; rather, the coadministration of NBQX plus LY341495 prevents the forskolin-induced formation of POA dendritic spine-like processes. We conclude that PKA, AMPA/kainate, and metabotropic glutamate receptor signaling are necessary for the effects of PGE(2), that each receptor individually suffices to organize behavior, and that PKA is upstream of the glutamate receptors.

Using morphological criteria we describe the postnatal ontogeny of gonadotrophin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) of the GnRH prohormone in the ovine preoptic area (POA)-hypothalamus. The immunohistochemical GnRH- and GAP-neuroanatomy was investigated in infantile (12-week-old weanlings), prepubertal (15-, 22-week-old juveniles), peripubertal (30-, 52-week-old during maturational quiescence) and pubertal (63-week-old) females born after the summer solstice, thus undergoing delayed puberty. The changes observed in the medial POA included a postinfantile increase in the number of multipolar GnRH-immunoreactive (ir) perikarya until late prepuberty (p < 0.05). Whereas in peripubertal lambs were observed both an increase in the number of GnRH-ir and GAP-ir cell clusters in the medial POA and an increase in the mediobasal hypothalamus (MBH) subpopulation of GnRH-ir and GAP-ir neurons (p < 0.05). The GnRH-ir perikarya, nerve fibers and terminals showed a progressive increase in numbers until peripuberty. Conversely, the acute postinfantile alterations of decrease and then increase in the population of GAP-ir nerve elements were observed in the POA-hypothalamus until late prepuberty (p < 0.05). The preoptico-hypothalamic ir GAP and GnRH were of comparable abundance in both peripubertal groups and scarce during puberty. In conclusion, the infantile/prepubertal transition period changes GnRH posttranslational biosynthesis and GnRH-GAP-neuroanatomy from the infantile to the pubertal pattern, which allows the inauguration of the breeding season. The postinfantile MBH distribution of GnRH-containing neurons and increase in the POA population of GnRH-GAP neurons and their somatic appositions seem important for maturation. The nature of GnRH-GAP neurobiology during inauguration of puberty seems morphotic and in female sheep is seasonal.

The sexually dimorphic population of dopamine neurons in the anteroventral periventricular nucleus of the preoptic region of the hypothalamus (AVPV) develops postnatally under the influence of testosterone, which is aromatized to estrogen. There are fewer dopaminergic neurons labeled with tyrosine hydroxylase (TH) in the male AVPV than the female, and sex steroids determine this sex difference, yet the role of cell death in specifying numbers of dopaminergic neurons in the AVPV is unknown. Estradiol treatment of the AVPV, in vivo and in vitro, was used to manipulate TH-ir cell number. In vitro, concurrent treatment with the estrogen receptor antagonist ICI 182,780 rescued TH-ir cells. Cyclosporin A, an inhibitor of cell death dependent on the opening of a mitochondrial permeability transition pore also blocked TH-ir cell loss. In vivo, estradiol increased the number of apoptotic profiles, both TUNEL and Hoechst labeled nuclei, in the AVPV. This increased apoptosis was also dependent on the presence of the alpha form of the estrogen receptor. To test for caspase dependent TH-ir cell loss, the pancaspase inhibitor ZVAD (N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone) was used to rescue TH-ir cells from estradiol-mediated reduction in number. Together, these data suggest that an intrinsic cell death pathway is activated by estrogen to regulate TH-ir cell number. Thus, in contrast to the more widespread neuroprotective actions of sex steroids in the mammalian nervous system, in the AVPV estrogen regulates dopaminergic neuron number through a caspase-dependent mechanism of apoptotic cell death.

Gonadal steroids that establish sexually dimorphic characteristics of brain morphology and physiology act at a particular stage of ontogeny. Testosterone secreted by the testes during late gestational and neonatal periods causes significant brain sexual dimorphism in the rat. This results in both sex-specific behaviour and endocrinology in adults. Sexual differentiation may be due to neurogenesis, migration or survival. Each mechanism appears to be uniquely regulated in a site-specific manner. Thus, the volume of an aggregate of neurones in the rat medial preoptic area (POA), termed the sexually dimorphic nucleus of the POA (SDN-POA), is larger in males than in females. The anteroventral periventricular nucleus (AVPV) is packed with neurones containing oestrogen receptor (ER)beta in female rats but, in males, ERbeta-positive neurones scatter into the more lateral portion of the POA. POA neurones are born up to embryonic days 16-17 and not after parturition. Therefore, neurogenesis is unlikely to contribute to the larger SDN-POA in males. DNA microarray analysis for oestrogen-responsive genes and western blotting demonstrated site-specific regulation of apoptosis- and migration-related genes in the SDN-POA and AVPV.

Early-life events may induce alterations in neuronal function in adulthood. A crucial aspect in studying long-lasting effects induced by environmental interventions imposed to the animal several weeks before is finding a stable change that could be causally related to the phenotype observed in adulthood. In order to explain an adult trait, it seems necessary to look back to early life and establish a temporal line between events. The neonatal handling procedure is an experimental tool to analyze the long-lasting impact of early-life events. Aside from the neuroendocrine response to stress, neonatal handling also alters the functionality of the hypothalamus-pituitary-gonad (HPG) axis. Reductions in ovulation and surge of the luteinizing hormone (LH) on the proestrous day were shown in female rats. Considering the importance of the medial preoptic area (MPA) for the control of ovulation, the present study aimed to verify the effects of neonatal handling on the numerical density and cell size in the MPA in 11-day-old and 90-day-old female rats. Cellular proliferation was also assessed using BrdU (5-bromo-2'-deoxyuridine) in 11-day-old pups. Results showed that neonatal handling induces a stable reduction in the number of cells and in the size of the cell soma, which were lower in handled females than in nonhandled ones at both ages. Cellular proliferation in the MPA was also reduced 24 h after the last manipulation. The repeated mother-infant disruption imposed by the handling procedure "lesioned" the MPA. The dysfunction in the ovulation mechanisms induced by the handling procedure could be related to that neuronal loss. The study also illustrates the impact of an environmental intervention on the development of the brain.

Progesterone receptor (PR) expression is highly dependent on estradiol in the medial preoptic nucleus (MPN) and the ventromedial nucleus (VMN) of the adult rat brain. During development, males express high levels of PR in the MPN, whereas females have virtually no PR, a sex difference resulting entirely from differential exposure to estradiol. Because PR is also estradiol dependent in the adult VMN, the present study examined the regulation of PR immunoreactivity (PRir) in the developing VMN. Surprisingly, PRir was present at high levels in the VMN of both neonatal males and females. In the neonatal VMN, PR expression was dependent on gonadal hormones in males but not females. When females were ovariectomized and exposed to estradiol at various ages from neonatal to adulthood, estradiol reliably induced PRir in the MPN at postnatal d 7 but failed to induce PRir in the VMN of the same animals. Only later in development, around postnatal d 14, did estradiol increase PRir in the female VMN. There appears to be a developmental switch in the VMN when PR expression changes from estradiol independent to estradiol dependent. Furthermore, this switch is anatomically specific and does not exist in the MPN. The present results indicate that the regulation of PR expression by estradiol is dependent on age, sex, and brain region, suggesting that PR may play a critical but specific role in the normal development of these reproductively important brain areas. In addition, the neonatal female VMN may provide a unique model in which to examine the mechanisms underlying the specificity of steroid-induced gene expression.

Masculinization of the brain is dependent upon a perinatal surge in testosterone. It also requires a transient decrease in hypothalamic 5-HT concentration and turnover and an increase in androgen receptor (AR) expression during the second postnatal week. We have previously shown that increasing 5-HT activity over this period in male or androgenized female rats feminizes their adult behaviour and also feminizes the size of their anteroventral periventricular nucleus (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA). To investigate the role of 5-HT in sexual differentiation of the brain, 5-HT activity was raised over postnatal days 8-16 in male, female and androgenized female rats by daily administration of the 5-HT(2) receptor agonist (-)[2,5 dimethoxy-4-iodophenyl]-2-amino propane hydrochloride [(-)DOI]. By postnatal day 18, the size of the AVPV and SDN-POA was sexually dimorphic; their sizes were feminized by (-)DOI treatment. In the absence of (-)DOI treatment, there were significantly more AR-immunoreactive cells in the AVPV of males, and in the SDN-POA of males and androgenized females, than in those of females on postnatal day 18. (-)DOI treatment reduced the number of AR-immunoreactive cells in the AVPV and SDN-POA of males and androgenized females, but not of females, by postnatal day 18. These results suggest that 5-HT(2) receptor activation can influence sexual differentiation of the brain by controlling AR expression.

In the sexually dimorphic nucleus of the preoptic area (SDN-POA) of postnatal rats, apoptotic cells are detected more frequently in females than males. This sex difference is under the influence of aromatized androgen. We have reported that there are sex differences in the levels of Bcl-2 (femalemale) in the central division of the medial preoptic nucleus (MPNc), a significant component of the SDN-POA, followed by a sex difference in induction of apoptosis via caspase-3 activation (female>male). In the present study, we examined effects of estradiol benzoate (EB) on expression of Bcl-2 and Bax in the MPNc. Female rats were subcutaneously injected with EB (25 or 50 microg per head) on postnatal day 5. MPNc and caudate putamen (CP) tissues were obtained from EB-treated female and male rats on postnatal day 6. Protein levels of Bcl-2 and Bax were determined by Western blotting. In the MPNc of female rats, EB at a dose of 50 microg/head but not 25 microg/head significantly increased Bcl-2 protein level and decreased Bax protein level. The levels of Bcl-2 and Bax of female rats treated with 50 microg of EB were comparable to those of male rats. However, the protein levels of Bcl-2 and Bax in the CP did not change with EB treatment. These results suggest that estrogen up-regulates Bcl-2 expression and down-regulates Bax expression in the MPNc of postnatal rats. Effects of estrogen on the Bcl-2 family are presumably responsible for sex difference in postnatal apoptosis of the SDN-POA.

In vitro, the organophosphate insecticide fenitrothion is a potent competitive androgen receptor antagonist, whereas in vivo it affects the development of the male rat reproductive system. The purpose of this pilot study was to determine whether prenatal exposure to fenitrothion affects development of the rat sexually dimorphic nucleus of the medial preoptic area (SDN-POA). Pregnant rats (n = 5-6 litters/group) were orally dosed with corn oil (vehicle) or fenitrothion (20 or 25 mg kg(-1) day(-1)) from gestation day (GD) 12-21. Offspring were euthanized after reaching sexual maturity (females 60-65 days old and males 96-105 days old) and the SDN-POA volumes determined for two rats/sex/litter. Tremors, increased lacrimation and decreased body weight gain were observed in dams from both fenitrothion exposure groups. Reproductive effects in male offspring, including reduced anogenital distance on postnatal day (PND) 1 and increased retention of areolae (PND 13) were observed following fenitrothion exposure at these dose levels. These effects did not persist into adulthood. There was a dose-related increase in the SDN-POA volume in males and a dose-related decrease in SDN-POA volume in females exposed to fenitrothion. These SDN-POA volume changes contrast with those seen with flutamide, another potent anti-androgen, and suggest that fenitrothion may have mixed endocrine effects on the developing brain.

Steroid hormones act on developing neural circuits that regulate the hypothalamic-pituitary-gonadal axis and are involved in hormone-sensitive behaviours. To test the hypothesis that developmental exposure to oestradiol (E(2)) organises the quantity of adult oestrogen receptors (ERalpha and ERbeta), we used male mice with a targeted mutation of the aromatase enzyme gene (ArKO) and their wild-type (WT) littermates. These mice are unable to aromatise testosterone to E(2), but still express both ERalpha and beta. To evaluate adult responsiveness to E(2), gonadectomised males were implanted with Silastic capsules containing E(2), or an empty implant, 5 days prior to sacrifice. Immunoreactivity for ERalpha and ERbeta was quantified in the caudal ventromedial nucleus (VMN) and the medial preoptic area (POA). Regardless of genotype, adult treatment with E(2) reduced ERalpha-immunoreactive (ir) and ERbeta-ir cell numbers in the POA, as well as ERbeta-ir, but not ERalpha-ir, cell numbers in the VMN. Genotype, and thus endogenous exposure to E(2), produced opposite effects on ER expression in the two brain areas. In the VMN, ArKO males had more ERalpha-ir and ERbeta-ir cells than did WT males. In the POA, ArKO males had fewer ERalpha-ir and ERbeta-ir cells than did WT males. Thus, numbers of immunoreactive neurones containing both ERs in the adult ArKO male were enhanced in the POA, but decreased in the VMN, and most likely these patterns were established during the developmental critical period. Furthermore, although both ERalpha and beta-ir cell numbers are altered by the disruption of the aromatase gene, ERbeta is altered in a more robust and region-specific manner.

Throughout the hypothalamus there are several regions known to contain sex differences in specific cellular, neurochemical, or cell grouping characteristics. The current study examined the potential origin of sex differences in calbindin expression in the preoptic area and hypothalamus as related to sources of nitric oxide. Specific cell populations were defined by immunoreactive (ir) calbindin and neuronal nitric oxide synthase (nNOS) in the preoptic area/anterior hypothalamus (POA/AH), anteroventral periventricular nucleus (AVPv), and ventromedial nucleus of the hypothalamus (VMN). The POA/AH of adult mice was characterized by a striking sex difference in the distribution of cells with ir-calbindin. Examination of the POA/AH of androgen receptor deficient Tfm mice suggests that this pattern was in part androgen receptor dependent, since Tfm males had reduced ir-calbindin compared with wild-type males and more similar to wild-type females. At P0 ir-calbindin was more prevalent than in adulthood, with males having significantly more ir-calbindin and nNOS than have females. Cells that contained either ir-calbindin or ir-nNOS in the POA/AH were in adjacent cell groups, suggesting that NO derived from the enzymatic activity of nNOS may influence the development of ir-calbindin cells. In the region of AVPv, at P0, there was a sex difference with males having more ir-nNOS fibers than have females while ir-calbindin was not detected. In the VMN, at P0, ir-nNOS was greater in females than in males, with no significant difference in ir-calbindin. We suggest that NO as an effector molecule and calbindin as a molecular biomarker illuminate key aspects of sexual differentiation in the developing mouse brain.

Pervasive developmental disorder is a classification covering five related conditions including the neurodevelopmental disorder Rett syndrome (RTT) and autism. Of these five conditions, only RTT has a known genetic cause with mutations in Methyl-CpG-binding protein 2 (MeCP2), a global repressor of gene expression, responsible for the majority of RTT cases. However, recent evidence indicates that reduced MeCP2 expression or activity is also found in autism and other disorders with overlapping phenotypes. Considering the sex difference in autism diagnosis, with males diagnosed four times more often than females, we questioned if a sex difference existed in the expression of MeCP2, in particular within the amygdala, a region that develops atypically in autism. We found that male rats express significantly less mecp2 mRNA and protein than females within the amygdala, as well as the ventromedial hypothalamus (VMH), but not within the preoptic area (POA) on post-natal day 1 (PN1). At PN10 these differences were gone; however, on this day males had more mecp2 mRNA than females within the POA. The transient sex difference of mecp2 expression during the steroid-sensitive period of brain development suggests that mecp2 may participate in normal sexual differentiation of the rat brain. Considering the strong link between MeCP2 and neurodevelopmental disorders, the lower levels of mecp2 expression in males may also underlie a biological risk for mecp2-related neural disorders.

To investigate the potential role of kisspeptin in the advance onset of puberty in precocious puberty, model rats induced by danazol were used to study the developmental expression of hypothalamic kisspeptin. Kisspeptin immunoreactive cells were observed in the arcuate nucleus (ARC), periventricular nucleus (PeN) and preoptic area (POA) in model rats on the day of onset-puberty. On the day of post-puberty, however, the number of kisspeptin immunoreactive cells in ARC and PeN decreased while the number of those cells in POA increased. Kisspeptin immunoreactive cells were not detected in hypothalamus in both normal and model rats at their pre-puberty stages. Furthermore, the expression of hypothalamic Kiss-1 mRNA reached top on the day of onset-puberty in both of the normal and model rats, and the expression of Kiss-1 mRNA increased significantly in the model rats compared with those in the normal ones. Our results indicated that kisspeptin might involve in the advance onset of puberty in danazol induced female precocious model rats.