Feeding neurons integrate metabolic and reproductive states in mice.

Balance between metabolic and reproductive processes is important for survival, particularly in mammals that gestate their young. How the nervous system coordinates this balance is an active area of study. Herein, we demonstrate that somatostatin (SST) neurons of the tuberal hypothalamus alter feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of SST neurons increased food intake across sexes, ablation decreased food intake only in female mice during proestrus. This ablation effect was only apparent in animals with low body mass. Fat transplantation and bioinformatics analysis of SST neuronal transcriptomes revealed white adipose as a key modulator of these effects. These studies indicate that SST hypothalamic neurons integrate metabolic and reproductive cues by responding to varying levels of circulating estrogens to modulate feeding differentially based on energy stores. Thus, gonadal steroid modulation of neuronal circuits can be context dependent and gated by metabolic status.

Feeding Neurons Integrate Metabolic and Reproductive States in Mice.

Trade-offs between metabolic and reproductive processes are important for survival, particularly in mammals that gestate their young. Puberty and reproduction, as energetically taxing life stages, are often gated by metabolic availability in animals with ovaries. How the nervous system coordinates these trade-offs is an active area of study. We identify somatostatin neurons of the tuberal nucleus (TNSST) as a node of the feeding circuit that alters feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of TNSST neurons increased food intake across sexes, selective ablation decreased food intake only in female mice during proestrus. Interestingly, this ablation effect was only apparent in animals with a low body mass. Fat transplantation and bioinformatics analysis of TNSST neuronal transcriptomes revealed white adipose as a key modulator of the effects of TNSST neurons on food intake. Together, these studies point to a mechanism whereby TNSST hypothalamic neurons modulate feeding by responding to varying levels of circulating estrogens differentially based on energy stores. This research provides insight into how neural circuits integrate reproductive and metabolic signals, and illustrates how gonadal steroid modulation of neuronal circuits can be context-dependent and gated by metabolic status.

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.

Adolescent stress during, but not after, pubertal onset impairs indices of prepulse inhibition in adult rats.

Exposure to stress during adolescence is a risk factor for developing several psychiatric disorders, many of which involve prefrontal cortex (PFC) dysfunction. The human PFC and analogous rodent medial prefrontal cortex (mPFC) continue to mature functionally and anatomically during adolescence, and some of these maturational events coincide with pubertal onset. As developing brain regions are more susceptible to the negative effects of stress, this may make puberty especially vulnerable. To test this, we exposed male and female rats to isolation and restraint stress during the onset of puberty or during the post-pubertal period of adolescence. In young adulthood, both stressed groups and an unstressed control group underwent testing on a battery of tasks to assess emotional and cognitive behaviors, and the volume of the mPFC was quantified postmortem. Factor analysis revealed only subjects stressed peri-pubertally showed a long-term deficiency compared to controls in prepulse inhibition. Additionally, both sexes showed volumetric mPFC decreases following adolescent stress, and these losses were most pronounced in females. Our findings suggest that pubertal onset may be a vulnerable window wherein adolescents are most susceptible to the negative consequences of stress exposure. Furthermore, it highlights the importance of accounting for pubertal status when studying adolescents.

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.

Extended access self-administration of methamphetamine is associated with age- and sex-dependent differences in drug taking behavior and recognition memory in rats.

Individuals who begin drug use during early adolescence experience more adverse consequences compared to those initiating later, especially if they are female. The mechanisms for these age and gender differences remain obscure, but studies in rodents suggest that psychostimulants may disrupt the normal ontogeny of dopamine and glutamate systems in the prefrontal cortex (PFC). Here, we studied Sprague-Dawley rats of both sexes who began methamphetamine (METH, i.v.) self-administration in adolescence (postnatal [P] day 41) or adulthood (P91). Rats received seven daily 2-h self-administration sessions with METH or saccharin as the reinforcer, followed by 14 daily long access (LgA; 6 h) sessions. After 7 and 14 days of abstinence, novel object (NOR) or object-in-place (OiP) recognition was assessed. PFC and nucleus accumbens were collected 7 days after the final cognitive test and NMDA receptor subunits and dopamine D1 receptor expression was measured. We found that during LgA sessions, adolescent-onset rats escalated METH intake more rapidly than adult-onset rats, with adolescent-onset females earning the most infusions. Adolescent-onset rats with a history of METH self-administration exhibited modest deficits in OiP compared to their adult-onset counterparts, but there was no sex difference and self-administration groups did not differ from naïve control rats. All rats displayed intact novel object recognition memory. We found no group differences in D1 and NMDA receptor expression, suggesting no long-lasting alteration of ontogenetic expression profiles. Our findings suggest that adolescent-onset drug use is more likely to lead to compulsive-like patterns of drug-taking and modest dysfunction in PFC-dependent cognition.

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.

Neonatal Inhibition of DNA Methylation Disrupts Testosterone-Dependent Masculinization of Neurochemical Phenotype.

Many neural sex differences are differences in the number of neurons of a particular phenotype. For example, male rodents have more calbindin-expressing neurons in the medial preoptic area (mPOA) and bed nucleus of the stria terminalis (BNST), and females have more neurons expressing estrogen receptor alpha (ERα) and kisspeptin in the ventromedial nucleus of the hypothalamus (VMH) and the anteroventral periventricular nucleus (AVPV), respectively. These sex differences depend on neonatal exposure to testosterone, but the underlying molecular mechanisms are unknown. DNA methylation is important for cell phenotype differentiation throughout the developing organism. We hypothesized that testosterone causes sex differences in neurochemical phenotype via changes in DNA methylation, and tested this by inhibiting DNA methylation neonatally in male and female mice, and in females given a masculinizing dose of testosterone. Neonatal testosterone treatment masculinized calbindin, ERα and kisspeptin cell number of females at weaning. Inhibiting DNA methylation with zebularine increased calbindin cell number only in control females, thus eliminating sex differences in calbindin in the mPOA and BNST. Zebularine also reduced the sex difference in ERα cell number in the VMH, in this case by increasing ERα neuron number in males and testosterone-treated females. In contrast, the neonatal inhibition of DNA methylation had no effect on kisspeptin cell number. We conclude that testosterone normally increases the number of calbindin cells and reduces ERα cells in males through orchestrated changes in DNA methylation, contributing to, or causing, the sex differences in both cell types.

Does Gender Leave an Epigenetic Imprint on the Brain?

The words "sex" and "gender" are often used interchangeably in common usage. In fact, the Merriam-Webster dictionary offers "sex" as the definition of gender. The authors of this review are neuroscientists, and the words "sex" and "gender" mean very different things to us: sex is based on biological factors such as sex chromosomes and gonads, whereas gender has a social component and involves differential expectations or treatment by conspecifics, based on an individual's perceived sex. While we are accustomed to thinking about "sex" and differences between males and females in epigenetic marks in the brain, we are much less used to thinking about the biological implications of gender. Nonetheless, careful consideration of the field of epigenetics leads us to conclude that gender must also leave an epigenetic imprint on the brain. Indeed, it would be strange if this were not the case, because all environmental influences of any import can epigenetically change the brain. In the following pages, we explain why there is now sufficient evidence to suggest that an epigenetic imprint for gender is a logical conclusion. We define our terms for sex, gender, and epigenetics, and describe research demonstrating sex differences in epigenetic mechanisms in the brain which, to date, is mainly based on work in non-human animals. We then give several examples of how gender, rather than sex, may cause the brain epigenome to differ in males and females, and finally consider the myriad of ways that sex and gender interact to shape gene expression in the brain.

Effects of Perinatal Exposure to Phthalates and a High-Fat Diet on Maternal Behavior and Pup Development and Social Play.

Humans are ubiquitously exposed to many phthalates, a class of endocrine-disrupting chemicals commonly used in many consumer goods, and diet, especially fatty food, is presumed to be a major source of exposure. Here, we use a rat model of human prenatal exposure to investigate the potential interactive effects of an environmentally relevant mixture of phthalates and a maternal high-fat diet (HFD). From gestation through postnatal day (P)10, dams consumed the mixture of phthalates (0, 200, or 1000 µg/kg/d) and were fed a control diet or HFD. In males, perinatal exposure to the mixture of phthalates decreased prepubertal body weight and, in a dose-specific manner, periadolescent social play behavior. A dose-specific effect from phthalates with HFD was also seen in increased time alone in females during social play. HFD resulted in dams consuming more calories, having greater gestational weight gain, and licking and nursing their pups more, such that an early postnatal HFD generally increased pup body weight. There also was a tendency for increased oxidative stress markers at P10 within the medial prefrontal cortex of males exposed to the relatively high dose of phthalates and HFD. Effects on gene expression were inconsistent at P10 and P90 in both the medial prefrontal cortex and hypothalamus. Overall, this study demonstrates that phthalates and a maternal HFD only rarely interacted, except in oxidative stress markers in males. Additionally, perinatal exposure to an environmentally relevant mixture of phthalates can have a modest, but lasting, impact on social behaviors in both males and females.

Innervation of the medial prefrontal cortex by tyrosine hydroxylase immunoreactive fibers during adolescence in male and female rats.

Adolescence is associated with continued maturation of the cerebral cortex, particularly the medial prefrontal cortex (mPFC). We have previously documented pruning in the number of neurons, dendrites, and synapses in the rat mPFC from preadolescence to adulthood, with the period of pubertal onset being particularly important. We hypothesized that dopaminergic innervation of this region, critical for executive functions, would also be influenced by pubertal onset. Here, we measured changes in the volume of tyrosine hydroxylase (TH) immunoreactive axons in all layers of the male and female mPFC from preadolescence to adulthood (postnatal Day (P) 25, 35, 45, 60, and 90) as a marker of dopaminergic innervation. Assessing both total fiber volume and length, TH fibers were quantified by multiplying the mPFC volume by fiber density. While there were subtle layer-specific changes, TH fiber volume and length increased between P25 and P90 in both males and females. Contrary to our hypothesis, a role for pubertal onset in TH innervation of this region was not discernable. In summary, axons immunoreactive for TH increase with similar trajectories in the mPFC of male and female rats from pre-puberty to young adulthood.

Neonatal Inhibition of DNA Methylation Alters Cell Phenotype in Sexually Dimorphic Regions of the Mouse Brain.

Many of the best-studied neural sex differences relate to differences in cell number and are due to the hormonal control of developmental cell death. However, several prominent neural sex differences persist even if cell death is eliminated. We hypothesized that these may reflect cell phenotype "decisions" that depend on epigenetic mechanisms, such as DNA methylation. To test this, we treated newborn mice with the DNA methyltransferase (DNMT) inhibitor zebularine, or vehicle, and examined two sexually dimorphic markers at weaning. As expected, control males had more cells immunoreactive for calbindin-D28k (CALB) in the medial preoptic area (mPOA) and fewer cells immunoreactive for estrogen receptor α (ERα) in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl) and the mPOA than did females. Neonatal DNMT inhibition markedly increased CALB cell number in both sexes and ERα cell density in males; as a result, the sex differences in ERα in the VMHvl and mPOA were completely eliminated in zebularine-treated animals. Zebularine treatment did not affect developmental cell death or the total density of Nissl-stained cells at weaning. Thus, a neonatal disruption of DNA methylation apparently has long-term effects on the proportion of cells expressing CALB and ERα, and some of these effects are sex specific. We also found that sex differences in CALB in the mPOA and ERα in the VMHvl persist in mice with a neuron-specific depletion of either Dnmt1 or Dnmt3b, indicating that neither DNMT alone is likely to be required for the sexually dimorphic expression of these markers.

A role for puberty in water maze performance in male and female rats.

Adolescence is characterized by neuroanatomical changes that coincide with increased cognitive performance. This developmental period is particularly important for the medial prefrontal cortex (mPFC), which mediates higher-order cognitive functioning. The authors' laboratory has shown that puberty is associated with sex-specific changes in neuron number and the dendritic tree in the rat mPFC, but the effects of pubertal onset on cognitive performance remain relatively unexplored. Here, we use a water maze task to assess spatial memory for the location of an escape platform, followed by a test of reversal learning, when the platform is moved to an alternate quadrant in the maze. For both males and females, 2 groups of prepubertal animals were tested (postnatal day [P]30 and P33 for females, P40 and P43 for males), along with 1 group of newly (2 days) postpubertal animals and 1 group of young adults (P60). There were no group differences in learning the initial location of the platform or when the platform location changed, although grouping pre- and postpubertal ages did result in significantly better performance in postpubertal animals. In addition after the platform location changed, individual prepubertal males and females spent a significantly greater percentage of time in the quadrant of the maze where the platform was formerly located than the postpubertal animals. This collectively implies that pubertal onset in both males and females coincides with improved performance on a reversal task, which may be linked with the neuroanatomical changes occurring in the mPFC during this time. (PsycINFO Database Record