The inflammatory response to birth requires MyD88 and is driven by both mother and offspring.

Birth is an inflammatory event for the newborn, characterized by elevations in interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α peripherally and/or centrally, as well as changes in brain microglia. However, the mechanism(s) underlying these responses is unknown. Toll-like receptors (TLRs) play crucial roles in innate immunity and initiate inflammatory cascades upon recognition of endogenous or exogenous antigens. Most TLR signaling depends on the adaptor molecule myeloid differentiation primary response 88 (MyD88). We independently varied MyD88 gene status in mouse dams and their offspring to determine whether the inflammatory response to birth depends on MyD88 signaling and, if so, whether that signaling occurs in the offspring, the mother, or both. We find that the perinatal surges in plasma IL-6 and brain expression of TNF-α depend solely on MyD88 gene status of the offspring, whereas postnatal increases in plasma IL-10 and TNF-α depend on MyD88 in both the pup and dam. Interestingly, MyD88 genotype of the dam primarily drives differences in offspring brain microglial density and has robust effects on developmental neuronal cell death. Milk cytokines were evaluated as a possible source of postnatal maternal influence; although we found high levels of CXCL1/GROα and several other cytokines in ingested post-partum milk, their presence did not require MyD88. Thus, the inflammatory response previously described in the late-term fetus and newborn depends on MyD88 (and, by extension, TLRs), with signaling in both the dam and offspring contributing. Unexpectedly, naturally-occuring neuronal cell death in the newborn is modulated primarily by maternal MyD88 gene status.

Birth triggers an inflammatory response in the neonatal periphery and brain.

Birth is preceded by inflammation at the fetal/maternal interface. Additionally, the newborn experiences stimuli that under any other circumstance could elicit an immune response. It is unknown, however, whether birth elicits an inflammatory response in the newborn that extends to the brain. Moreover, it is unknown whether birth mode may alter such a response. To study these questions, we first measured corticosterone and pro- and anti-inflammatory cytokines in plasma of mouse offspring at several timepoints spaced closely before and after a vaginal or Cesarean birth. We found highest levels of IL-6 one day before birth and surges in corticosterone and IL-10 just after birth, regardless of birth mode. We next examined the neuroimmune response by measuring cytokine mRNA expression and microglial number and morphology in the paraventricular nucleus of the hypothalamus and hippocampus around the time of birth. We found a marked increase in TNF-α expression in both brain regions a day after birth, and rapid increases in microglial cell number in the first three days postnatal, with subtle differences by birth mode. To test whether the association between birth and cytokine production or expansion of microglia is causal, we manipulated birth timing. Remarkably, advancing birth by a day advanced the increases in all of the markers tested. Thus, birth triggers an immune response in the body and brain of offspring. Our results may provide a mechanism for effects of birth (e.g., acute changes in cell death and neural activation) previously reported in the newborn brain.

DNA Methylation and Demethylation Underlie the Sex Difference in Estrogen Receptor Alpha in the Arcuate Nucleus.

INTRODUCTION: Neurons expressing estrogen receptor (ER) ɑ in the arcuate (ARC) and ventromedial (VMH) nuclei of the hypothalamus sex-specifically control energy homeostasis, sexual behavior, and bone density. Females have more ERɑ neurons in the VMH and ARC than males, and the sex difference in the VMH is eliminated by neonatal treatment with testosterone or a DNA methylation inhibitor. OBJECTIVE: Here, we tested the roles of testosterone and DNA methylation/demethylation in development of ERɑ in the ARC. METHODS: ERɑ was examined at birth and weaning in mice that received vehicle or testosterone subcutaneously, and vehicle or DNA methyltransferase inhibitor intracerebroventricularly, as neonates. To examine effects of DNA demethylation on the ERɑ cell number in the ARC, mice were treated neonatally with small interfering RNAs against ten-eleven translocase enzymes. The methylation status of the ERɑ gene (Esr1) was determined in the ARC and VMH using pyrosequencing of bisulfite-converted DNA. RESULTS: A sex difference in ERɑ in the ARC, favoring females, developed between birth and weaning and was due to programming effects of testosterone. Neonatal inhibition of DNA methylation decreased ERɑ in the ARC of females, and an inhibition of demethylation increased ERɑ in the ARC of males. The promoter region of Esr1 exhibited a small sex difference in percent of total methylation in the ARC (females > males) that was opposite to that in the VMH (males > females). CONCLUSION: DNA methylation and demethylation regulate ERɑ cell number in the ARC, and methylation correlates with activation of Esr1 in this region.

Cesarean birth elicits long-term effects on vasopressin and oxytocin neurons in the hypothalamic paraventricular nucleus of mice.

Birth is an extraordinary event for placental mammals and occurs at a time when key developmental processes are shaping the brain. Remarkably, little is known about the contributions of birth to brain development and whether birth mode (vaginal vs. Cesarean) alters neurodevelopmental trajectories. We previously reported that Cesarean birth reduces vasopressin (VP) neuron number in the hypothalamic paraventricular nucleus (PVN) of mice at weaning. In this study, we investigated whether this effect extends to adulthood and whether birth mode affects oxytocin (OT) neurons, which are another prominent population in the PVN. We found that Cesarean-born adults had fewer VP neurons in the PVN, specifically in magnocellular regions. Interestingly, these regions also had more dying cells following a Cesarean birth, suggesting that cell death may be the underlying mechanism. The PVN of Cesarean-born adults also had smaller VP neuron somas and reduced VP efferent projections. Additionally, Cesarean-born mice showed fewer and smaller OT neurons in the PVN, but these effects were less robust than for VP neurons. We also examined VP and OT neuron number in the supraoptic and suprachiasmatic nuclei but found no effect of birth mode in these regions. Thus, Cesarean birth causes long-term effects on the VP and, to a lesser extent, OT systems in the PVN, suggesting that this region is particularly sensitive to the effects of birth mode. Our findings may help explain the social deficits reported for Cesarean-born mice, and are also of clinical significance given the widespread practice of Cesarean births across the world.

Adult Neural Plasticity in Naked Mole-Rats: Implications of Fossoriality, Longevity and Sociality on the Brain's Capacity for Change.

Naked mole-rats (Heterocephalus glaber) are small African rodents that have many unique behavioral and physiological adaptations well-suited for testing hypotheses about mammalian neural plasticity. In this chapter, we focus on three features of naked mole-rat biology and how they impact neural plasticity in this species: (1) their fossorial lifestyle, (2) their extreme longevity with a lack of demonstrable senescence, and (3) their unusual social structure. Critically, each of these features requires some degree of biological flexibility. First, their fossorial habitat situates them in an environment with characteristics to which the central nervous system is particularly sensitive (e.g., oxygen content, photoperiod, spatial complexity). Second, their long lifespan requires adaptations to combat senescence and declines in neural functioning. Finally, their extreme reproductive skew and sustained ability for release from reproductive suppression indicates remarkable neural sensitivity to the sociosexual environment that is distinct from chronological age. These three features of naked mole-rat life are not mutually exclusive, but they do each offer unique considerations for the possibilities, constraints, and mechanisms associated with adult neural plasticity.

Birth delivery mode alters perinatal cell death in the mouse brain.

Labor and a vaginal delivery trigger changes in peripheral organs that prepare the mammalian fetus to survive ex utero. Surprisingly little attention has been given to whether birth also influences the brain, and to how alterations in birth mode affect neonatal brain development. These are important questions, given the high rates of cesarean section (C-section) delivery worldwide, many of which are elective. We examined the effect of birth mode on neuronal cell death, a widespread developmental process that occurs primarily during the first postnatal week in mice. Timed-pregnant dams were randomly assigned to C-section deliveries that were yoked to vaginal births to carefully match gestation length and circadian time of parturition. Compared with rates of cell death just before birth, vaginally-born offspring had an abrupt, transient decrease in cell death in many brain regions, suggesting that a vaginal delivery is neuroprotective. In contrast, cell death was either unchanged or increased in C-section-born mice. Effects of delivery mode on cell death were greatest for the paraventricular nucleus of the hypothalamus (PVN), which is central to the stress response and brain-immune interactions. The greater cell death in the PVN of C-section-delivered newborns was associated with a reduction in the number of PVN neurons expressing vasopressin at weaning. C-section-delivered mice also showed altered vocalizations in a maternal separation test and greater body mass at weaning. Our results suggest that vaginal birth acutely impacts brain development, and that alterations in birth mode may have lasting consequences.

The microbiota influences cell death and microglial colonization in the perinatal mouse brain.

The mammalian fetus develops in a largely sterile environment, and direct exposure to a complex microbiota does not occur until birth. We took advantage of this to examine the effect of the microbiota on brain development during the first few days of life. The expression of anti- and pro-inflammatory cytokines, developmental cell death, and microglial colonization in the brain were compared between newborn conventionally colonized mice and mice born in sterile, germ-free (GF) conditions. Expression of the pro-inflammatory cytokines interleukin 1ß and tumor necrosis factor α was markedly suppressed in GF newborns. GF mice also had altered cell death, with some regions exhibiting higher rates (paraventricular nucleus of the hypothalamus and the CA1 oriens layer of the hippocampus) and other regions exhibiting no change or lower rates (arcuate nucleus of the hypothalamus) of cell death. Microglial labeling was elevated in GF mice, due to an increase in both microglial cell size and number. The changes in cytokine expression, cell death and microglial labeling were evident on the day of birth, but were absent on embryonic day 18.5, approximately one-half day prior to expected delivery. Taken together, our results suggest that direct exposure to the microbiota at birth influences key neurodevelopmental events and does so within hours. These findings may help to explain some of the behavioral and neurochemical alterations previously seen in adult GF mice.

Effects of sex and prenatal androgen manipulations on Onuf's nucleus of rhesus macaques.

The role of gonadal steroids in sexual differentiation of the central nervous system (CNS) is well established in rodents, but no study to date has manipulated androgens prenatally and examined their effects on any CNS structure in a primate. Onuf's nucleus is a column of motoneurons in the sacral spinal cord that innervates the striated perineal muscles. This cell group is larger in males than in females of many species, due to androgens acting during a sensitive perinatal period. Here, we examined Onuf's nucleus in 21 adult rhesus monkeys, including control males and females, as well as males whose mothers had been treated with an anti-androgen or testosterone during gestation. We found a robust sex difference, with more motoneurons in control males than in females. The soma size of Onuf's nucleus motoneurons was also marginally larger in males. Treatment with the anti-androgen flutamide for 35-40 days during early gestation partially blocked masculinization of Onuf's nucleus: motoneuron number in flutamide-treated males was decreased relative to control and testosterone-treated males, but remained greater than in females, with no effect on cell size. A control motor nucleus that innervates foot muscles (Pes9) showed no difference in motoneuron number or size between control males and females. Prenatal testosterone treatment of males did not alter Onuf's nucleus motoneuron number, but did increase the size of both Onuf's and Pes9 motoneurons. Thus, prenatal androgen manipulations cause cellular-level changes in the primate CNS, which may underlie previously observed effects of these manipulations on behavior.

Cellular and molecular mechanisms of sexual differentiation in the mammalian nervous system.

Neuroscientists are likely to discover new sex differences in the coming years, spurred by the National Institutes of Health initiative to include both sexes in preclinical studies. This review summarizes the current state of knowledge of the cellular and molecular mechanisms underlying sex differences in the mammalian nervous system, based primarily on work in rodents. Cellular mechanisms examined include neurogenesis, migration, the differentiation of neurochemical and morphological cell phenotype, and cell death. At the molecular level we discuss evolving roles for epigenetics, sex chromosome complement, the immune system, and newly identified cell signaling pathways. We review recent findings on the role of the environment, as well as genome-wide studies with some surprising results, causing us to re-think often-used models of sexual differentiation. We end by pointing to future directions, including an increased awareness of the important contributions of tissues outside of the nervous system to sexual differentiation of the brain.

Plasma 25-hydroxyvitamin D and risk of premenstrual syndrome in a prospective cohort study.

BACKGROUND: Moderate to severe premenstrual syndrome (PMS) affects 8-20 percent of premenopausal women. Previous studies suggest that high dietary vitamin D intake may reduce risk. However, vitamin D status is influenced by both dietary vitamin D intake and sunlight exposure and the association of vitamin D status with PMS remains unclear. METHODS: We assessed the relation of plasma 25-hydroxyvitamin D (25OHD), total calcium and parathyroid hormone levels with risk of PMS and specific menstrual symptoms in a case-control study nested within the prospective Nurses' Health Study II. Cases were 401 women free from PMS at baseline who developed PMS during follow-up (1991-2005). Controls were women not experiencing PMS (1991-2005), matched 1:1 with cases on age and other factors. Timed luteal phase blood samples were collected between 1996 and 1999 from cases and controls. We used conditional logistic regression to model the relation of 25OHD levels with risk of PMS and individual menstrual symptoms. RESULTS: In analyses of all cases and controls, 25OHD levels were not associated with risk of PMS. However, results differed when the timing of blood collection vs. PMS diagnosis was considered. Among cases who had already been diagnosed with PMS at the time of blood collection (n = 279), 25OHD levels were positively associated with PMS, with each 10 nmol/L change in 25OHD associated with a 13% higher risk. Among cases who developed PMS after blood collection (n = 123), 25OHD levels were unrelated to risk of PMS overall, but inversely related to risk of specific menstrual symptoms. For example, each 10 nmol/L increase was associated with a significant 21% lower risk of breast tenderness (P = 0.02). Total calcium or parathyroid hormone levels were unrelated to PMS. CONCLUSIONS: 25OHD levels were not associated with overall risk of PMS. The positive association observed among women already experiencing PMS at the time of 25OHD measurement is likely due to confounding by indication related to use of dietary supplements to treat menstrual symptoms. Results from prospective analyses, which were less likely influenced by this bias, suggest that higher 25OHD levels may be inversely related to the development of specific menstrual symptoms.

Neuronal scaling in the olfactory system of bats.

Comparative studies are a common way to address large-scale questions in sensory biology. For studies that investigate olfactory abilities, the most commonly used metric is olfactory bulb size. However, recent work has called into question the broad-scale use of olfactory bulb size. In this paper, we use three neuroanatomical measures with a more mechanistic link to olfactory function (number of olfactory sensory neurons (OSNs), number of mitral cells (MCs), and number of glomeruli) to ask how species with different diets may differ with respect to olfactory ability. We use phyllostomid bats as our study system because behavioral and physiological work has shown that fruit- and nectar-feeding phyllostomids rely on odors for detecting, localizing, and assessing potential foods, while insect-eating species do not. Therefore, we predicted that fruit- and nectar-feeding bats would have larger numbers of these three neuroanatomical measures than insect-eating species. In general, our results supported the predictions. We found that fruit-eaters had greater numbers of OSNs and glomeruli than insect-eaters, but we found no difference between groups in number of MCs. We also examined the allometric relationship between the three neuroanatomical variables and olfactory bulb volume, and we found isometry in all cases. These findings lend support to the notion that neuroanatomical measures can offer valuable insights into comparative olfactory abilities, and suggest that the size of the olfactory bulb may be an informative parameter to use at the whole-organism level.

Brain effects of gestating germ-free persist in mouse neonates despite acquisition of a microbiota at birth.

At birth, mammals experience a massive colonization by microorganisms. We previously reported that newborn mice gestated and born germ-free (GF) have increased microglial labeling and alterations in developmental neuronal cell death in the hippocampus and hypothalamus, as well as greater forebrain volume and body weight when compared to conventionally colonized (CC) mice. To test whether these effects are solely due to differences in postnatal microbial exposure, or instead may be programmed in utero, we cross-fostered GF newborns immediately after birth to CC dams (GF→CC) and compared them to offspring fostered within the same microbiota status (CC→CC, GF→GF). Because key developmental events (including microglial colonization and neuronal cell death) shape the brain during the first postnatal week, we collected brains on postnatal day (P) 7. To track gut bacterial colonization, colonic content was also collected and subjected to 16S rRNA qPCR and Illumina sequencing. In the brains of GF→GF mice, we replicated most of the effects seen previously in GF mice. Interestingly, the GF brain phenotype persisted in GF→CC offspring for almost all measures. In contrast, total bacterial load did not differ between the CC→CC and GF→CC groups on P7, and bacterial community composition was also very similar, with a few exceptions. Thus, GF→CC offspring had altered brain development during at least the first 7 days after birth despite a largely normal microbiota. This suggests that prenatal influences of gestating in an altered microbial environment programs neonatal brain development.

Effects of Bax gene deletion on social behaviors and neural response to olfactory cues in mice.

Bax is a pro-death protein that plays a crucial role in developmental neuronal cell death. Bax(-/-) mice exhibit increased neuron number and lack several neural sex differences. Here we examined the effects of Bax gene deletion on social behaviors (olfactory preference, social recognition, social approach and aggression) and the neural processing of olfactory cues. Bax deletion eliminated the normal sex difference in olfactory preference behavior. In the social recognition test, both genotypes discriminated a novel conspecific, but wild-type males and Bax(-/-) animals of both sexes spent much more time than wild-type females investigating stimulus animals. Similarly, Bax(-/-) mice were more sociable than wild-type mice in a social approach test. Bax deletion had no effect on aggression in a resident/intruder paradigm where males, regardless of genotype, exhibited a shorter latency to attack. Thus, the prevention of neuronal cell death by Bax gene deletion results in greater sociability as well as the elimination of sex differences in some social behaviors. To examine olfactory processing of socially relevant cues, we counted c-Fos-immunoreactive (Fos-ir) cells in several nodes of the accessory olfactory pathway after exposure to male-soiled or control bedding. In both genotypes, exposure to male-soiled bedding increased Fos-ir cells in the posterodorsal medial amygdala, principal nucleus of the bed nucleus of the stria terminalis and medial preoptic nucleus (MPN), and the response in the MPN was greater in females than in males. However, a reduction in Fos-ir cells was seen in the anteroventral periventricular nucleus of Bax(-/-) mice.

Social status and sex effects on neural morphology in Damaraland mole-rats, Fukomys damarensis.

We previously reported that in a eusocial rodent, the naked mole-rat (Heterocephalus glaber), traditional neural sex differences were absent; instead, neural dimorphisms were associated with breeding status. Here we examined the same neural regions previously studied in naked mole-rats in a second eusocial species, the Damaraland mole-rat (Fukomys damarensis). Damaraland mole-rats live in social groups with breeding restricted to a small number of animals. However, colony sizes are much smaller in Damaraland mole-rats than in naked mole-rats and there is consequently less reproductive skew. In this sense, Damaraland mole-rats may be considered intermediate in social organization between naked mole-rats and more traditional laboratory rodents. We report that, as in naked mole-rats, breeding Damaraland mole-rats have larger volumes of the principal nucleus of the bed nucleus of the stria terminalis and paraventricular nucleus of the hypothalamus than do subordinates, with no effect of sex on these measures. Thus, these structures may play special roles in breeders of eusocial species. However, in contrast to what was seen in naked mole-rats, we also found sex differences in Damaraland mole-rats: volume of the medial amygdala and motoneuron number in Onuf's nucleus were both greater in males than in females, with no significant effect of breeding status. Thus, both sex and breeding status influence neural morphology in Damaraland mole-rats. These findings are in accord with the observed sex differences in body weight and genitalia in Damaraland but not naked mole-rats. We hypothesize that the increased sexual dimorphism in Damaraland mole-rats relative to naked mole-rats is related to reduced reproductive skew.

First Encounters: Effects of the Microbiota on Neonatal Brain Development.

The microbiota plays important roles in host metabolism and immunity, and its disruption affects adult brain physiology and behavior. Although such findings have been attributed to altered neurodevelopment, few studies have actually examined microbiota effects on the developing brain. This review focuses on developmental effects of the earliest exposure to microbes. At birth, the mammalian fetus enters a world teeming with microbes which colonize all body sites in contact with the environment. Bacteria reach the gut within a few hours of birth and cause a measurable response in the intestinal epithelium. In adults, the gut microbiota signals to the brain via the vagus nerve, bacterial metabolites, hormones, and immune signaling, and work in perinatal rodents is beginning to elucidate which of these signaling pathways herald the very first encounter with gut microbes in the neonate. Neural effects of the microbiota during the first few days of life include changes in neuronal cell death, microglia, and brain cytokine levels. In addition to these effects of direct exposure of the newborn to microbes, accumulating evidence points to a role for the maternal microbiota in affecting brain development via bacterial molecules and metabolites while the offspring is still in utero. Hence, perturbations to microbial exposure perinatally, such as through C-section delivery or antibiotic treatment, alter microbiota colonization and may have long-term neural consequences. The perinatal period is critical for brain development and a close look at microbiota effects during this time promises to reveal the earliest, most primary effects of the microbiota on neurodevelopment.

Birth elicits a conserved neuroendocrine response with implications for perinatal osmoregulation and neuronal cell death.

Long-standing clinical findings report a dramatic surge of vasopressin in umbilical cord blood of the human neonate, but the neural underpinnings and function(s) of this phenomenon remain obscure. We studied neural activation in perinatal mice and rats, and found that birth triggers activation of the suprachiasmatic, supraoptic, and paraventricular nuclei of the hypothalamus. This was seen whether mice were born vaginally or via Cesarean section (C-section), and when birth timing was experimentally manipulated. Neuronal phenotyping showed that the activated neurons were predominantly vasopressinergic, and vasopressin mRNA increased fivefold in the hypothalamus during the 2-3 days before birth. Copeptin, a surrogate marker of vasopressin, was elevated 30-to 50-fold in plasma of perinatal mice, with higher levels after a vaginal than a C-section birth. We also found an acute decrease in plasma osmolality after a vaginal, but not C-section birth, suggesting that the difference in vasopressin release between birth modes is functionally meaningful. When vasopressin was administered centrally to newborns, we found an ~ 50% reduction in neuronal cell death in specific brain areas. Collectively, our results identify a conserved neuroendocrine response to birth that is sensitive to birth mode, and influences peripheral physiology and neurodevelopment.

The epigenetics of sex differences in the brain.

Epigenetic changes in the nervous system are emerging as a critical component of enduring effects induced by early life experience, hormonal exposure, trauma and injury, or learning and memory. Sex differences in the brain are largely determined by steroid hormone exposure during a perinatal sensitive period that alters subsequent hormonal and nonhormonal responses throughout the lifespan. Steroid receptors are members of a nuclear receptor transcription factor superfamily and recruit multiple proteins that possess enzymatic activity relevant to epigenetic changes such as acetylation and methylation. Thus steroid hormones are uniquely poised to exert epigenetic effects on the developing nervous system to dictate adult sex differences in brain and behavior. Sex differences in the methylation pattern in the promoter of estrogen and progesterone receptor genes are evident in newborns and persist in adults but with a different pattern. Changes in response to injury and in methyl-binding proteins and steroid receptor coregulatory proteins are also reported. Many steroid-induced epigenetic changes are opportunistic and restricted to a single lifespan, but new evidence suggests endocrine-disrupting compounds can exert multigenerational effects. Similarly, maternal diet also induces transgenerational effects, but the impact is sex specific. The study of epigenetics of sex differences is in its earliest stages, with needed advances in understanding of the hormonal regulation of enzymes controlling acetylation and methylation, coregulatory proteins, transient versus stable DNA methylation patterns, and sex differences across the epigenome to fully understand sex differences in brain and behavior.

Neuroendocrinology and sexual differentiation in eusocial mammals.

Sexual differentiation of the mammalian nervous system has been studied intensively for over 25 years. Most of what we know, however, comes from work on relatively non-social species in which direct reproduction (i.e., production of offspring) is virtually the only route to reproductive success. In social species, an individual's inclusive fitness may include contributions to the gene pool that are achieved by supporting the reproductive efforts of close relatives; this feature is most evident in eusocial organisms. Here, we review what is known about neuroendocrine mechanisms, sexual differentiation, and effects of social status on the brain and spinal cord in two eusocial mammals: the naked mole-rat and Damaraland mole-rat. These small rodents exhibit the most rigidly organized reproductive hierarchy among mammals, with reproduction suppressed in a majority of individuals. Our findings suggest that eusociality may be associated with a relative lack of sex differences and a reduced influence of gonadal hormones on some functions to which these hormones are usually tightly linked. We also identify neural changes accompanying a change in social and reproductive status, and discuss the implications of our findings for understanding the evolution of sex differences and the neuroendocrinology of reproductive suppression.

Control of cell number in the bed nucleus of the stria terminalis of mice: role of testosterone metabolites and estrogen receptor subtypes.

INTRODUCTION: The bed nucleus of the stria terminalis (BNST) exhibits several sex differences that may be related to male sexual behavior and gender identity. In mice and rats, sex differences in the principal nucleus of the BNST (BNSTp) are due to sexually dimorphic cell death during perinatal life. Although testosterone treatment of newborn female rats increases BNSTp cell number, the relevant hormone metabolite(s) are not known, and the effect of testosterone on the development of BNSTp cell number in mice has not been examined. AIM: To identify the sex hormone metabolites and receptors controlling cell number, volume, and cell size in the BNSTp of mice. METHODS: In the first experiment, C57BL/6J male mice were injected on the day of birth with peanut oil; females were injected with testosterone propionate (TP), estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), or oil alone, and the BNSTp of all animals was examined in adulthood. In the second experiment, to compare effects of EB to the effects of estrogen receptor subtype specific agonists, newborn female mice were injected with EB, propyl-pyrazole-triol (PPT, a selective estrogen receptor alpha [ERalpha] agonist), or diarylpropionitrile (DPN, a selective estrogen receptor beta [ERbeta] agonist). MAIN OUTCOME MEASURES: Nuclear volume measurements and stereological cell counts in the BNSTp in adulthood. RESULTS: TP treatment of newborn females completely masculinized both BNSTp volume and cell number. EB masculinized neuron number, whereas DHTP had no effect on volume or cell number. In the second experiment, EB again fully masculinized neuron number in the BNSTp and in this study also masculinized BNSTp volume. PPT and DPN each significantly increased cell number, but neither completely mimicked the effects of EB. CONCLUSIONS: We conclude that estrogenic metabolites of testosterone control sexually dimorphic cell survival in the BNSTp and that activation of both ERalpha and ERbeta may be required for complete masculinization of this brain region.

Developmental changes and sex differences in DNA methylation and demethylation in hypothalamic regions of the mouse brain.

DNA methylation is dynamically modulated during postnatal brain development, and plays a key role in neuronal lineage commitment. This epigenetic mark has also recently been implicated in the development of neural sex differences, many of which are found in the hypothalamus. The level of DNA methylation depends on a balance between the placement of methyl marks by DNA methyltransferases (Dnmts) and their removal, which is catalyzed by ten-eleven translocation (Tet) methylcytosine dioxygenases. Here, we examined developmental changes and sex differences in the expression of Tet and Dnmt enzymes from birth to adulthood in two hypothalamic regions (the preoptic area and ventromedial nucleus) and the hippocampus of mice. We found highest expression of all Tet enzymes (Tet1, Tet2, Tet3) and Dnmts (Dnmt1, Dnmt3a, Dnmt3b) in newborns, despite the fact that global methylation and hydroxymethylation were at their lowest levels at birth. Expression of the Dnmt co-activator, Dnmt3l, followed a pattern opposite to that of the canonical Dnmts (i.e., was very low in newborns and increased with age). Tet enzyme activity was much higher at birth than at weaning in both the hypothalamus and hippocampus, mirroring developmental changes in gene expression. Sex differences in Tet enzyme expression were seen in all brain regions examined during the first week of life, whereas Dnmt expression was more balanced between the sexes. Neonatal testosterone treatment of females only partially masculinized enzyme expression. Thus, Tet expression and activity are elevated during neonatal brain development, and may play important roles in sexual differentiation of the brain.