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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.
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Interleucina-10 , Fator 88 de Diferenciação Mieloide , Animais , Feminino , Camundongos , Gravidez , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Camundongos Endogâmicos C57BL , Mães , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
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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 (GFâCC) and compared them to offspring fostered within the same microbiota status (CCâCC, GFâGF). 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 GFâGF mice, we replicated most of the effects seen previously in GF mice. Interestingly, the GF brain phenotype persisted in GFâCC offspring for almost all measures. In contrast, total bacterial load did not differ between the CCâCC and GFâCC groups on P7, and bacterial community composition was also very similar, with a few exceptions. Thus, GFâCC 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.
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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.
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INTRODUCTION: Neurons expressing estrogen receptor (ER) É in the arcuate (ARC) and ventromedial (VMH) nuclei of the hypothalamus sex-specifically control energy homeostasis, sexual behavior, and bone density. Females have more ERÉ 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 ERÉ in the ARC. METHODS: ERÉ 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 ERÉ cell number in the ARC, mice were treated neonatally with small interfering RNAs against ten-eleven translocase enzymes. The methylation status of the ERÉ gene (Esr1) was determined in the ARC and VMH using pyrosequencing of bisulfite-converted DNA. RESULTS: A sex difference in ERÉ in the ARC, favoring females, developed between birth and weaning and was due to programming effects of testosterone. Neonatal inhibition of DNA methylation decreased ERÉ in the ARC of females, and an inhibition of
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Núcleo Arqueado do Hipotálamo , Receptor alfa de Estrogênio , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Metilação de DNA , Desmetilação , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Masculino , Camundongos , Caracteres Sexuais , Testosterona/farmacologiaRESUMO
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.
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Ocitocina , Núcleo Hipotalâmico Paraventricular , Animais , Feminino , Mamíferos/metabolismo , Camundongos , Neurônios/metabolismo , Ocitocina/farmacologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Placenta/metabolismo , Gravidez , Vasopressinas/metabolismoRESUMO
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.
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Longevidade , Ratos-Toupeira , Animais , Encéfalo , Plasticidade Neuronal , Comportamento SocialRESUMO
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.
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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.
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Hipotálamo/metabolismo , Sistemas Neurossecretores/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Vasopressinas/metabolismo , Animais , Biomarcadores/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osmorregulação/genética , Osmorregulação/fisiologia , Vasopressinas/genéticaRESUMO
Developmental cell death eliminates half of the neurons initially generated in the mammalian brain, and occurs perinatally in many species. It is possible that the timing of neuronal cell death is developmentally programmed, and only coincidentally associated with birth. Alternatively, birth may play a role in shaping cell death. To test these competing hypotheses, we experimentally advanced or delayed birth by 1 d in mice (within the normal range of gestation for the species) and examined effects on the temporal pattern and magnitude (amount) of neuronal cell death, using immunohistochemical detection of activated caspase-3 as a cell death marker. In order to detect effects of subtle changes in birth timing, we focused on brain areas that exhibit sharp postnatal peaks in cell death. We find that advancing birth advances peak cell death, supporting the hypothesis that birth triggers cell death. However, a delay of birth does not delay cell death. Thus, birth can advance cell death, but if postponed, a developmental program governs. Advancing or delaying birth also caused region-specific changes in the overall magnitude of cell death. Our findings shed light on the long-standing question of what controls the timing and magnitude of developmental neuronal cell death, and position birth as an orchestrator of brain development. Because humans across the world now routinely alter birth timing, these findings may have implications for current obstetric practices.
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Encéfalo , Parto , Animais , Apoptose , Morte Celular , Feminino , Camundongos , Neurônios , GravidezRESUMO
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.
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Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética , Feminino , Hipotálamo/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Fatores SexuaisRESUMO
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.
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Encéfalo/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Diferenciação Sexual/efeitos dos fármacos , Testosterona/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/metabolismo , Química Encefálica/efeitos dos fármacos , Calbindinas/metabolismo , Citidina/administração & dosagem , Citidina/análogos & derivados , Citidina/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Kisspeptinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Diferenciação Sexual/fisiologia , Fatores Sexuais , Testosterona/administração & dosagemRESUMO
Developmental neuronal cell death has been characterized as a cell autonomous "suicide" program, but recent findings suggest that microglia play an active role in determining the survival of developing neurons. Results have been contradictory, however, with some studies concluding that microglia promote cell death, while others report that microglia are neuroprotective. Here, we depleted microglia throughout the newborn mouse brain using intracerebroventricular injections of clodronate liposomes, and examined effects on naturally occurring cell death across multiple brain areas. Microglial density varied significantly by brain region, and clodronate liposome treatment at birth reduced the number of microglia in all regions examined. The effect of microglia reduction on cell death, however, varied by region: the number of dying cells was reduced in the medial septum and medial amygdala in clodronate treated animals, but was increased in the oriens layer of the hippocampus, and unchanged in several other brain regions. In most brain regions, the average size of microglia was greater in microglia-depleted than in control animals, suggesting that the remaining microglia compensate to some extent for a reduction in microglial number. The hippocampal oriens was exceptional in this regard, in that microglial size was reduced following treatment with clodronate. Microglia produce cytokines which mediate many of their effects, and we found higher expression of inflammatory cytokines in the hippocampus than in the septum, independent of clodronate treatment. Thus, microglial depletion has opposite effects on cell death in different brain regions of the newborn brain, which may be related to regional heterogeneity in microglia.
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Encéfalo/citologia , Microglia/citologia , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Ácido Clodrônico/farmacologia , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacosRESUMO
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.
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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.
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Encéfalo/embriologia , Cesárea/efeitos adversos , Parto/fisiologia , Animais , Morte Celular/fisiologia , Parto Obstétrico/veterinária , Feminino , Idade Gestacional , Trabalho de Parto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Hipotalâmico Paraventricular/fisiologia , GravidezRESUMO
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 35-40â¯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.
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Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Neurônios Motores/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Caracteres Sexuais , Medula Espinal/efeitos dos fármacos , Testosterona/farmacologia , Animais , Animais Recém-Nascidos , Contagem de Células , Tamanho Celular , Feminino , Macaca mulatta , Masculino , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/psicologia , Medula Espinal/citologia , Medula Espinal/fisiologiaRESUMO
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.
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Encéfalo/crescimento & desenvolvimento , Morte Celular , Encefalite/microbiologia , Microbiota , Microglia/fisiologia , Neurônios/fisiologia , Animais , Encéfalo/microbiologia , Encefalite/metabolismo , Feminino , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Microglia/microbiologia , Neurônios/microbiologia , GravidezRESUMO
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.
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Encéfalo/efeitos dos fármacos , Citidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Caracteres Sexuais , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Citidina/farmacologia , DNA (Citosina-5-)-Metiltransferase 1 , Regulação para Baixo/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/fisiologia , Fenótipo , Processos de Determinação Sexual/efeitos dos fármacos , Processos de Determinação Sexual/genética , Fatores de TempoRESUMO
Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase-3 (AC3) and ionized calcium-binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a > tenfold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex, septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3-P5. Minocycline treatment from P3 to P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose 1 week later (P10-P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial "inhibitor" increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 753-766, 2017.
Assuntos
Antibacterianos/farmacologia , Encéfalo/citologia , Morte Celular/efeitos dos fármacos , Microglia/efeitos dos fármacos , Minociclina/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 3/metabolismo , Contagem de Células , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismoRESUMO
The importance of cell death in brain development has long been appreciated, but many basic questions remain, such as what initiates or terminates the cell death period. One obstacle has been the lack of quantitative data defining exactly when cell death occurs. We recently created a "cell death atlas," using the detection of activated caspase-3 (AC3) to quantify apoptosis in the postnatal mouse ventral forebrain and hypothalamus, and found that the highest rates of cell death were seen at the earliest postnatal ages in most regions. Here we have extended these analyses to prenatal ages and additional brain regions. We quantified cell death in 16 forebrain regions across nine perinatal ages from embryonic day (E) 17 to postnatal day (P) 11 and found that cell death peaks just after birth in most regions. We found greater cell death in several regions in offspring delivered vaginally on the day of parturition compared with those of the same postconception age but still in utero at the time of collection. We also found massive cell death in the oriens layer of the hippocampus on P1 and in regions surrounding the anterior crossing of the corpus callosum on E18 as well as the persistence of large numbers of cells in those regions in adult mice lacking the pro-death Bax gene. Together these findings suggest that birth may be an important trigger of neuronal cell death and identify transient cell groups that may undergo wholesale elimination perinatally. J. Comp. Neurol. 525:47-64, 2017. © 2016 Wiley Periodicals, Inc.