Steroid-induced developmental plasticity in hypothalamic astrocytes: implications for synaptic patterning.

We have previously demonstrated that astrocytes in the developing arcuate nucleus of the rat hypothalamus exhibit a sexually dimorphic morphology as a result of differential exposure to gonadal steroids. Testosterone via its aromatized byproduct, estrogen, induces arcuate astrocytes to undergo differentiation during the first few days of life. These differentiated astrocytes exhibit a stellate morphology. Coincident with the steroid-induced increase in astrocyte differentiation is a reduction of dendritic spines on arcuate neurons. As a result, the arcuate nucleus of males has fewer axodendritic spine synapses than females and this dimorphism is retained throughout life. In the immediately adjacent ventromedial nucleus, neonatal astrocytes are immature and unresponsive to steroids. Neurons in this region show no change in dendritic spines in the first few days of life but do exhibit increased dendritic branching as a result of testosterone exposure. These findings illustrate the importance of distinct populations of astrocytes in restricted brain regions and their potential importance to the establishment of regionally specific synaptic patterning. Conflicting reports leave the site of steroid-mediated astrocyte responsiveness in the arcuate nucleus unresolved: Are gonadal steroids acting directly on astrocytes or are steroid-concentrating neurons mediating astrocytic responsiveness? In this review, we discuss the current understanding of astrocyte-neuron interactions and the possible mechanisms for steroid-mediated, astrocyte-directed synaptic patterning in the developing hypothalamus.     

Evidence for Sexual Differentiation of Glia in Rat Brain

It is well established that gonadal steroids mediate sexual differentiation of the brain via direct effects on neurons during a restricted critical period. In addition, estrogen can influence glial morphology in the adult brain, andin vitrostudies suggest estrogen induces glial differentiation. However, there is a lack ofin vivoevidence for steroid effects on glia during the critical period. We report here a hormone-mediated sexual differentiation of arcuate glia as early as Postnatal Day 1. Using glial fibrillary acidic protein immunoreactivity (GFAP-ir), we compared the responsiveness of astroglia in the rat arcuate nucleus among five hormonally different groups. The results indicate increased GFAP-ir cell surface area 24 hr after hormonal manipulation in castrate males compared to intact males, intact females (ANOVA;P< 0.01), and females injected with testosterone propionate (50 μg; ANOVA;P< 0.05). However, astroglia in intact males extended their processes significantly greater distances from the cell body compared to all other treatment groups (ANOVA;P< 0.01). The GFAP-ir cells were categorized into four distinct classes ranging from a simple bipolar to a fully stellate morphology. The frequency distribution of classes varied between groups with more stellate cells found in intact males. Finally, these sex differences in arcuate glia persisted into adulthood. We hypothesize that during the critical period, testosterone, or its metabolite estrogen, induce sexual differentiation of glia. We further hypothesize that in females glial cells remain partially undifferentiated and this may be important to glial plasticity seen in adult female arcuate.     

Role of astroglia in estrogen regulation of synaptic plasticity and brain repair.

Astroglia are targets for estrogen and testosterone and are apparently involved in the action of sex steroids on the brain. Sex hormones induce changes in the expression of glial fibrillary acidic protein, the growth of astrocytic processes, and the degree of apposition of astroglial processes to neuronal membranes in the rat hypothalamus. These changes are linked to modifications in the number of synaptic inputs to hypothalamic neurons. These findings suggest that astrocytes may participate in the genesis of androgen-induced sex differences in synaptic connectivity and in estrogen-induced synaptic plasticity in the adult brain. Astrocytes and tanycytes may also participate in the cellular effects of sex steroids by releasing neuroactive substances and by regulating the local accumulation of specific growth factors, such as insulin-like growth factor-I, that are involved in estrogen-induced synaptic plasticity and estrogen-mediated neuroendocrine control. Astroglia may also be involved in regenerative and neuroprotective effects of sex steroids, since astroglia formation after brain injury or after peripheral nerve axotomy is regulated by sex hormones. Furthermore, the expression of aromatase, the enzyme that produces estrogen, is induced de novo in astrocytes in lesioned brain areas of adult male and female rodents. Since astroglia do not express aromatase under normal circumstances, the induction of this enzyme may be part of the program of glial activation to cope with the new conditions of the neural tissue after injury. Given the neuroprotective and growth-promoting effects of estrogen after injury, the local production of this steroid may be a relevant component of the reparative process.     

Gonadal steroids and astroglial plasticity

Summary 1. Recent evidence indicates that astroglia participate in the metabolism of gonadal hormones, in the synthesis of neurosteroids, and in the plastic responses of neurons to gonadal steroids. The role of astroglia on plastic responses of neural tissue to gonadal hormones and neurosteroids is examined in this review.2. Gonadal steroids and neurosteroids promote astroglia plasticity in several areas of the central nervous system, including the hypothalamus, the striatum, and the hippocampus.3. Gonadal steroids and neurosteroids modulate astroglia proliferation and the formation of reactive astroglia after brain injury.4. Astroglia is a source of trophic factors that may mediate effects of gonadal steroids on neural tissue.5. Astroglia is involved in the promotion of synaptic plastic changes by gonadal hormones.6. The effect of gonadal hormones on astroglial plasticity is dependent on specific membrane interactions with neurons and on the expression of the embryonic highly polysialylated isoform of the neural cell adhesion molecule on neuronal membranes.7. In conclusion, coordinated responses of neurons and astroglia appear to be involved in the modulation of neural function and response to injury by gonadal hormones and neurosteroids.     

Neuronal interactions between neuropeptide Y (NPY) and catecholaminergic systems in the rat arcuate nucleus as shown by dual immunocytochemistry

Several recent studies have suggested interactions between catecholamine (CA) and neuropeptide Y (NPY) neuronal systems in the rat brain. In order to obtain morphological evidence for such CA/NPY interactions in the arcuate nucleus, we have used a double immunostaining procedure using an anti-tyrosine hydroxylase (TH) antiserum as a marker for catecholamine neurons and an anti-NPY antiserum. This double staining, where the first staining is silver-gold intensified, was detectable at both light and electron microscopic levels. In semi-thin sections, a substantial overlap and close proximity of TH-immunopositive neurons and NPY neuronal elements could be seen within the arcuate nucleus. At the electron microscopic level, direct appositions between TH- and NPY-immunoreactive structures could be detected. These appositions were of axosomatic, axodendritic or axoaxonic types without any synaptic membrane differentiation. Moreover, direct appositions between NPY-immunoreactive structures have also been observed. This morphological study showing appositions between TH and NPY neuronal systems suggest direct interactions between these two systems in the arcuate nucleus.     

Potential neuronal mechanisms of estrogen actions in synaptogenesis and synaptic plasticity

Summary 1. Studies conducted on the rat arcuate nucleus, an area involved in the development and control of LH and FSH secretion, have shown the existence of hormonally regulated developmental sex differences in synaptic patterns and estrogen-induced synaptic plasticity during adult life. Several questions raised by these findings are examined in this review:2. The mechanisms of estrogen-regulated developmental synaptogenesis. These include the role of glycocalyx glycoproteins in neuronal membranes, neural cell adhesion molecules, and insulin-like growth factor I.3. The relationship among circulating estrogen, gonadotropin levels, and hypothalamic synaptic plasticity. Recent evidence for the role of GABAergic and dopaminergic synaptic inputs and POMC projections from the arcuate nucleus to the GnRH cells is discussed.4. The synaptologic basis of age-related failure of positive feedback. The role of the cumulative effect of repeated preovulatory synaptic retraction and reapplication cycles on sensescent constant estrus is analyzed.     

Estrogen effects on the synaptology and neural membranes of the rat hypothalamic arcuate nucleus

The concept of estrogen-induced "sexual differentiation of the brain" has been a useful focus for investigation. However, it has become clear that the action of estrogen on the rat brain is lifelong, including effects on neurogenesis in the fetus, synaptogenesis in the newborn, and synaptic remodeling in the adult. Estrogen imparts sex differences in the rat's brain by shaping synaptology, postsynaptic membranes, and glia within the arcuate nucleus. These effects of estrogen on the arcuate nucleus also could underlie sexual maturation in both sexes and the development of senescent constant estrus in females.     

Estradiol induces hypothalamic dendritic spines by enhancing glutamate release: a mechanism for organizational sex differences

The naturally occurring sex difference in dendritic spine number on hypothalamic neurons offers a unique opportunity to investigate mechanisms establishing synaptic patterning during perinatal sensitive periods. A major advantage of the rat as a model of sexual differentiation is that treatment of neonatal females with estradiol will permanently induce the male phenotype. During the development of other systems, exuberant innervation is followed by activity-dependent pruning necessary for elimination of spurious synapses. In contrast, we demonstrate that estradiol-induced organization in the hypothalamus involves the induction of new synapses on dendritic spines. Activation of estrogen receptors by estradiol triggers a nongenomic activation of PI3 kinase that results in enhanced glutamate release from presynaptic neurons. Subsequent activation of ionotropic glutamate receptors activates MAP kinases, thereby inducing dendritic spine formation. These results reveal a transneuronal mechanism by which estradiol acts during a sensitive period to establish a profound and lasting sex difference in hypothalamic synaptic patterning.     

Glutamate AMPA/kainate receptors, not GABA(A) receptors, mediate estradiol-induced sex differences in the hypothalamus

Sex differences in brain morphology underlie physiological and behavioral differences between males and females. During the critical perinatal period for sexual differentiation in the rat, gonadal steroids act in a regionally specific manner to alter neuronal morphology. Using Golgi-Cox impregnation, we examined several parameters of neuronal morphology in postnatal day 2 (PN2) rats. We found that in the ventromedial nucleus of the hypothalamus (VMN) and in areas just dorsal and just lateral to the VMN that there was a sex difference in total dendritic spine number (males greater) that was abolished by treating female neonates with exogenous testosterone. Dendritic branching was similarly sexually differentiated and hormonally modulated in the VMN and dorsal to the VMN. We then used spinophilin, a protein that positively correlates with the amount of dendritic spines, to investigate the mechanisms underlying these sex differences. Estradiol, which mediates most aspects of masculinization and is the aromatized product of testosterone, increased spinophilin levels in female PN2 rats to that of males. Muscimol, an agonist at GABA(A) receptors, did not affect spinophilin protein levels in either male or female neonates. Kainic acid, an agonist at glutamatergic AMPA/kainate receptors, mimicked the effect of estradiol in females. Antagonizing AMPA/kainate receptors with NBQX prevented the estradiol-induced increase in spinophilin in females but did not affect spinophilin level in males.     

Steroid modulation of astrocytes in the neonatal brain: implications for adult reproductive function

There is a growing appreciation for the importance of astrocytes, a type of nonneuronal glial cell, to overall brain functioning. The ability of astrocytes to respond to gonadal steroid hormones with changes in morphology has been well documented in the adult brain. It is also apparent that astrocytes of the developing brain are permanently differentiated by the neonatal hormonal milieu, in particular by estradiol, resulting in sexually dimorphic cell morphology, synaptic patterning, and density in males and females. The mechanisms of hormonally mediated astrocyte differentiation are likely to be region specific. In the arcuate nucleus of the hypothalamus, neuron-to-astrocyte signaling appears to play a critical role in estradiol-induced astrocyte differentiation during the first few days of life. Gamma aminobutyric acid (GABA) is an amino acid neurotransmitter that is synthesized and released exclusively by neurons. The levels of GABA are increased in the arcuate nucleus of neonatal males versus females. Preventing the increase in males or mimicking GABA action in females modulates astrocytes accordingly. Speculation about and evidence in support of the functional significance of this dimorphism to adult reproductive functioning is the topic of this review.     

Physiological role of metastin/kisspeptin in regulating gonadotropin-releasing hormone (GnRH) secretion in female rats

Various studies have attempted to unravel the physiological role of metastin/kisspeptin in the control of gonadotropin-releasing hormone (GnRH) release. A number of evidences suggested that the population of metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) is involved in generating a GnRH surge to induce ovulation in rodents, and thus the target of estrogen positive feedback. Females have an obvious metastin/kisspeptin neuronal population in the AVPV, but males have only a few cell bodies in the nucleus, suggesting that the absence of the surge-generating mechanism or positive feedback action in males is due to the limited AVPV metastin/kisspeptin neuronal population. On the other hand, the arcuate nucleus (ARC) metastin/kisspeptin neuronal population is considered to be involved in the regulation of tonic GnRH release. The ARC metastin/kisspeptin neurons show no sex difference in their expression, which is suppressed by gonadal steroids in both sexes. Thus, the ARC population of metastin/kisspeptin neurons is a target of estrogen negative feedback action on tonic GnRH release. The lactating rat model provided further evidence indicating that ARC metastin/kisspeptin neurons are involved in GnRH pulse generation, because pulsatile release of luteinizing hormone (LH) is profoundly suppressed by suckling stimulus and the LH pulse suppression is well associated with the suppression of ARC metastin/kisspeptin and KiSS-1 gene expression in lactating rats.     

The cellular effects of estrogens on neuroendocrine tissues

Estrogen action on sensitive neurons in the rat diencephalon has been studied by morphologic techniques; evidence of estrogen action at every level is presented, including tracts, cells, circuitry and subcellular organelles. The demonstration in the arcuate nucleus of estrogen-induced synaptic remodelling, estrogen-induced postsynaptic membrane phenotypes, changes in intracellular membranes and rapid estrogen actions on neuronal endo-exocytosis indicates that cellular estrogen actions may underlie the neuronal control of reproduction.     

Sex-dependent loss of projection neurons involved in avian song learning.

In zebra finches, only males sing, and the neural regions controlling song exhibit prominent, hormone-induced sex differences in neuron number. In order to understand how sexual differentiation regulates neuron number within one song nucleus, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), we studied the development of sex differences among IMAN neurons that project to the robust nucleus of the archistriatum (RA). The IMAN is implicated in song learning, and previous ontogenetic studies have indicated that males lose over 50% of their IMAN neurons during the juvenile song learning period. Based on developmental changes in both the extent of androgen accumulation within the IMAN and its appearance in Nissl-stained tissue, it had been hypothesized that IMAN neuron loss was even greater in young females, resulting in sex differences in neuron number. However, this hypothesis has not been tested directly because the Nissl-stained boundaries of the IMAN sometimes are ambiguous in young animals, and are not evident at all in adult females. To circumvent these problems, we employed the retrograde tracer fast blue to study the development of IMAN neurons defined on the basis of their projections to the RA. We find that the number of these IMAN-RA projection neurons is much greater in adult males than in females, and that this sex difference develops during the juvenile period of sexual differentiation and song learning because a significant number of these neurons are lost in females but not in males. With respect to sexual differentiation, we conclude that masculinization (which is stimulated by the hormone estradiol) promotes the retention of IMAN-RA projection neurons. In addition, our results indicate that any loss of IMAN neurons that may occur in young males does not include cells projecting to the RA.     

Sex-dependent loss of projection neurons involved in avian song learning

In zebra finches, only males sing, and the neural regions controlling song exhibit prominent, hormone-induced sex diffences in neuron number. In order to understand how sexual differentiation regulates neuron number within one song nucleus, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), we studied the development of sex differences among IMAN neurons that project to the robust nucleus of the archistriatum (RA). The IMAN is implicated in song learning, and previous ontogenetic studies have indicated that males lose over 50% of their IMAN neurons during the juvenile song learning period. Based on developmental changes in both the extent of androgen accumulation within the IMAN and its appearance in Nissl-stained tissue, it had been hypothesized that IMAN neuron loss was even greater in young females, resulting in sex differences in neuron number. However, this hypothesis has not been tested directly because the Nissl-stained boundaries of the IMAN sometimes are ambiguous in young animals, and are not evident at all in adult females. To circumvent these problems, we employed the retrograde tracer fast blue to study the development of IMAN neurons defined on the basis of their projections to the RA. We find that the number of these IMAN-RA projection neurons is much greater in adult males than in females, and that this sex difference develops during the juvenile period of sexual differentiation and song learning because a significant number of these neurons are lost in females but not in males. With respect to sexual differentiation, we conclude that masculinization (which is stimulated by the hormone estradiol) promotes the retention of IMAN-RA projection neurons. In addition, our results indicate that any loss of IMAN neurons that may occur in young males does not include cells projecting to the RA. © 1992 John Wiley & Sons, Inc.     

Steroid-mediated differentiation of neural/neuronal cells from epithelial ovarian precursors in vitro

We reported earlier that occasional neurons evolve in human cultures of pluripotent ovarian epithelial stem cells. In subsequent experiments, frequent transdifferentiation into neural stem cells (NSC) and differentiating neurons was observed in human ovarian epithelial stem cells and porcine granulosa cells after exposure to certain combinations of sex steroids. Testosterone (TS), progesterone (PG) or estradiol (E2) alone do not increase the emergence of neurons. However, a mixture of TS+PG after E2 pretreatment converted a majority of ovarian epithelial stem cells or porcine granulosa cells into NSC and differentiating neuronal cells within one to three hours. Cultured neurons manifested an interconnectivity resembling primitive neuronal pathways in culture. These converted cells expressed the cell markers SSEA-1, SSEA-4, NCAM, and Thy-1 glycoconjugates of NSC and neurons, and differentiating cells showed characteristic neuronal morphology. Emergence of NSC and neuronal cells was associated with significant cellular depletion of L-glutamic acid (glutamate), which serves as the major excitatory neurotransmitter in the vertebrate CNS and its fast removal is essential for preventing glutamate excitotoxicity. These observations suggest that certain sequential systemic treatment with common sex steroids and their mixture might be effective in the treatment or prevention of degenerative CNS disorders. The ovarian stem cell cultures readily obtainable from human ovaries regardless of the woman's age have the potential to produce NSC for autologous regenerative treatment of neurologic diseases in aging women. Finally, the proper combination of sex steroids could possibly be employed for transdifferentiation of adult bone marrow stem cells or mobilized peripheral blood cells into autologous NSC and stimulate their neuronal differentiation after homing in the CNS.     

Effects of neonatal gonadal steroids on adult CA3 pyramidal neuron dendritic morphology and spatial memory in rats

The hippocampus is implicated in spatial cognition, which is sexually dimorphic and developmentally sensitive to gonadal steroids. Previously we have shown a sex difference in CA3 pyramidal cell layer volume and neuronal soma size that was reversible with neonatal castration in males or prenatal treatment of females with either testosterone propionate (TP) or a nonaromatizable androgen, dihydrotestosterone propionate, but not estradiol benzoate, all of which correlated with adult water maze navigation. The present study further investigates developmental androgen sensitivity of CA3 pyramidal neurons by measuring dendritic morphology and its relation to adult spatial ability. Female rats were injected with TP on postnatal day (P) 3 and P5 or ovariectomized (OVX) on P2, and male rats were castrated on P2, with or without testosterone replacement (Cas+T). Sham surgery controls were also included. Animals were tested on a water maze in adulthood, sacrificed, and CA3 pyramidal neurons were Golgi-stained and reconstructed in three dimensions using a computer-interfaced morphometry system. High-androgen groups (control males, Cas+T, TP females) performed better in spatial navigation and exhibited CA3 neurons with longer dendrites, a larger number of dendritic branches, and volumes of influence compared to low-androgen groups (control females, castrated males, OVX). Collectively, these findings indicate that the critical time period for organizational effects of androgens on the CA3 pyramidal neurons includes both prenatal and postnatal life, during which time androgens regulate developmental events such as somal growth and neuronal differentiation, all of which significantly contribute to establishing the sex difference in adult spatial navigation.     

Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.     

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.     

Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals

Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.     

Female mice deficient in alpha-fetoprotein show female-typical neural responses to conspecific-derived pheromones

The neural mechanisms controlling sexual behavior are sexually differentiated by the perinatal actions of sex steroid hormones. We recently observed using female mice deficient in alpha-fetoprotein (AFP-KO) and which lack the protective actions of AFP against maternal estradiol, that exposure to prenatal estradiol completely defeminized the potential to show lordosis behavior in adulthood. Furthermore, AFP-KO females failed to show any male-directed mate preferences following treatment with estradiol and progesterone, indicating a reduced sexual motivation to seek out the male. In the present study, we asked whether neural responses to male- and female-derived odors are also affected in AFP-KO female mice. Therefore, we compared patterns of Fos, the protein product of the immediate early gene, c-fos, commonly used as a marker of neuronal activation, between wild-type (WT) and AFP-KO female mice following exposure to male or estrous female urine. We also tested WT males to confirm the previously observed sex differences in neural responses to male urinary odors. Interestingly, AFP-KO females showed normal, female-like Fos responses, i.e. exposure to urinary odors from male but not estrous female mice induced equivalent levels of Fos protein in the accessory olfactory pathways (e.g. the medial part of the preoptic nucleus, the bed nucleus of the stria terminalis, the amygdala, and the lateral part of the ventromedial hypothalamic nucleus) as well as in the main olfactory pathways (e.g. the piriform cortex and the anterior cortical amygdaloid nucleus), as WT females. By contrast, WT males did not show any significant induction of Fos protein in these brain areas upon exposure to either male or estrous female urinary odors. These results thus suggest that prenatal estradiol is not involved in the sexual differentiation of neural Fos responses to male-derived odors.