Brain sexual differentiation in rodents results from the perinatal testicular androgen surge. In the preoptic area (POA), estradiol aromatized from testosterone upregulates the production of the proinflammatory molecule, prostaglandin E2 (PGE2) to produce sex-specific brain development. PGE2 produces a two-fold greater density of dendritic spines in males than in females and masculinizes adult copulatory behavior. One neonatal dose of PGE2 masculinizes the POA and behavior, and simultaneous treatment with an inhibitor of additional prostaglandin synthesis prevents this masculinization, indicating a positive feed-forward process that leads to sustained increases in PGE2. The mechanisms underlying this feed-forward process were unknown. Microglia, the primary immunocompetent cells in the brain, are active neonatally, contribute to normal brain development, and both produce and respond to prostaglandins. We investigated whether there are sex differences in microglia in the POA and whether they influence developmental masculinization. Neonatal males had twice as many ameboid microglia as females and a more activated morphological profile, and both estradiol and PGE2 masculinized microglial number and morphology in females. Microglial inhibition during the critical period for sexual differentiation prevented sex differences in microglia, estradiol-induced masculinization of dendritic spine density, and adult copulatory behavior. Microglial inhibition also prevented the estradiol-induced upregulation of PGE2, indicating that microglia are essential to the feed-forward process through which estradiol upregulates prostaglandin production. These studies demonstrate that immune cells in the brain interact with the nervous and endocrine systems during development, and are crucial for sexual differentiation of brain and behavior.

Innate immune cells play a well-documented role in the etiology and disease course of many brain-based conditions, including multiple sclerosis, Alzheimer’s disease, traumatic brain and spinal cord injury, and brain cancers. In contrast, it is only recently becoming clear that innate immune cells, primarily brain resident macrophages called microglia, are also key regulators of brain development. This review summarizes the current state of knowledge regarding microglia in brain development, with particular emphasis on how microglia during development are distinct from microglia later in life. We also summarize the effects of early life perturbations on microglia function in the developing brain, the role that biological sex plays in microglia function, and the potential role that microglia may play in developmental brain disorders. Finally, given how new the field of developmental neuroimmunology is, we highlight what has yet to be learned about how innate immune cells shape the development of brain and behavior.

The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually-dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in females. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation.

Microglia, the resident innate immune cells in the brain, have long been understood to be crucial to maintenance in the nervous system, by clearing debris, monitoring for infiltration of infectious agents, and mediating the brain’s inflammatory and repair response to traumatic injury, stroke, or neurodegeneration. A wave of new research has shown that microglia are also active players in many basic processes in the healthy brain, including cell proliferation, synaptic connectivity, and physiology. Microglia, both in their capacity as phagocytic cells and via secretion of many neuroactive molecules, including cytokines and growth factors, play a central role in early brain development, including sexual differentiation of the brain. In this review, we present the vast roles microglia play in normal brain development and how perturbations in the normal neuroimmune environment during development may contribute to the etiology of brain-based disorders. There are notable differences between microglia and neuroimmune signaling in the male and female brain throughout the life span, and these differences may contribute to the vast differences in the incidence of neuropsychiatric and neurological disorders between males and females.

The study of sex differences in the brain is a topic of neuroscientific study that has broad reaching implications for culture, society and biomedical science. Recent research in rodent models has led to dramatic shifts in our views of the mechanisms underlying the sexual differentiation of the brain. These include the surprising discoveries of a role for immune cells and inflammatory mediators in brain masculinization and a role for epigenetic suppression in brain feminization. How and to what degree these findings will translate to human brain development will be questions of central importance in future research in this field.

Microglia regulate brain development through many processes, such as promoting neurogenesis, supporting cell survival, and phagocytizing progenitor, newly-born, and dying cells. Many of these same developmental processes show robust sex differences, yet very few studies have assessed sex differences in microglia function during development. Hormonally-induced sexual differentiation of the brain occurs during the perinatal period, thus we examined sex differences in microglial morphology, phagocytosis, and proliferation in the hippocampus during the early postnatal period. We found that the neonatal female hippocampus had significantly more microglia with phagocytic cups than the male hippocampus. We subsequently found that female microglia phagocytized more neural progenitor cells and healthy cells compared to males, but there were no sex differences in the number of newly-born or dying cells targeted by microglial phagocytosis. We found that the number of phagocytic microglia in females was reduced to male-typical levels by treatment with estradiol, the hormone responsible for masculinizing the rodent brain. Females also had higher expression of several phagocytic pathway genes in the hippocampus compared to males. In contrast to robust sex differences in phagocytic microglia, we found no sex differences in the number of microglia with amoeboid, transitioning, or ramified morphologies or differences in three-dimensional reconstructions of microglial morphology. While we did not find a baseline sex difference in microglial proliferation during or following the prenatal gonadal hormone surge in males, we found that estradiol treatment increased microglia proliferation in females. Overall, these data show that there are important sex differences in microglia function in the hippocampus during the early neonatal period.

Steroid hormones of gonadal origin act on the neonatal brain to produce sex differences that underlie adult reproductive physiology and behavior. Neuronal sex differences occur on a variety of levels, including differences in regional volume and/or cell number, morphology, physiology, molecular signaling, and gene expression. In the rodent, many of these sex differences are determined by steroid hormones, particularly estradiol, and are established by diverse downstream effects. One brain region that is potently organized by estradiol is the preoptic area (POA), a region critically involved in many behaviors that show sex differences, including copulatory and maternal behaviors. This review focuses on the POA as a case study exemplifying the depth and breadth of our knowledge as well as the gaps in understanding the mechanisms through which gonadal hormones produce lasting neural and behavioral sex differences. In the POA, multiple cell types, including neurons, astrocytes, and microglia are masculinized by estradiol. Multiple downstream molecular mediators are involved, including prostaglandins, various glutamate receptors, protein kinase A, and several immune signaling molecules. Moreover, emerging evidence indicates epigenetic mechanisms maintain sex differences in the POA that are organized perinatally and thereby produce permanent behavioral changes. We also review emerging strategies to better elucidate the mechanisms through which genetics and epigenetics contribute to brain and behavioral sex differences.

Microglia, the innate immune cells of the central nervous system, regulate brain development by promoting cell genesis, pruning synapses, and removing dying, newly-born or progenitor cells. However, the role of microglia in the early life programming of behavior under normal conditions is not well characterized. We used central infusion of liposomal clodronate to selectively deplete microglia from the neonatal rat brain and subsequently assessed the impact of microglial depletion on programming of juvenile and adult motivated behaviors. Liposomal clodronate treatment on postnatal days one and four led to greater than 70% loss of forebrain microglia by postnatal day 6 that lasted for approximately ten days. Neonatal microglia depletion led to reduced juvenile and adult anxiety behavior on the elevated plus maze and open field test, and increased locomotor activity. On a test of juvenile social play, microglial depletion led to decreased chase behaviors relative to control animals. There was no change in active social behavior in adults on a reciprocal social interaction test, but there was decreased passive interaction time and an increased number of social avoidance behaviors in clodronate treated rats relative to controls. There was an overall decrease in behavioral despair on the forced swim test in adult rats treated neonatally with clodronate. Females, but not males, treated neonatally with clodronate showed a blunted corticosterone response after acute stress in adulthood. These results show that microglia are important for the early life programming of juvenile and adult motivated behavior.