Pediatric traumatic brain injury (TBI) increases the risk for long-term physical, cognitive, and psychosocial changes, many of which can lead to challenges in daily life functioning. Our understanding of how pediatric TBI interferes with brain development to lead to long-term behavioral outcomes is still incomplete. Despite emerging evidence for sex-specific outcomes in people, few rodent studies have examined sex differences after pediatric TBI. Here, we investigated the impact of pediatric lateral fluid percussion injury on acute neuroimmune and chronic neurobehavioral outcomes in male and female Sprague Dawley rats. At 3 days post injury (DPI), both sham and TBI increased microglia immunoreactivity relative to naive controls and had sex-specific effects on astrocytes. TBI increased brain mast cells relative to naive controls at 3 DPI in both sexes, and this increase persisted in females at 7 DPI. Sham and TBI procedures increased juvenile social play behavior in males and rearing behavior in both sexes relative to naive controls. Pediatric TBI reduced adult social interaction and had sex-differential effects on social avoidance, with increased avoidance in TBI females and decreased avoidance in TBI males. Injury condition did not affect perineuronal nets or oxytocin immunoreactivity at 7 DPI or oxytocin in adulthood. These studies show that immune cells in the male and female brain are differentially sensitive to the acute and sub-acute neuroinflammatory consequences of pediatric head trauma, and that pediatric head trauma in rats has age- and sex-dependent consequences for social behavior.

Traumatic brain injury (TBI) is one of the leading causes of emergency room visits in children under 10. Children are potentially more vulnerable to the adverse effects of TBI, given that their brains are still developing at the time of injury. Indeed, early life TBI has been linked to cognitive, social, and mood-related impairments later in life. The neuroimmune system has been implicated in adult TBI mechanisms and plays numerous key roles in brain development, making it an interesting candidate for linking pediatric TBI and prolonged behavioral alterations. Here we establish a rat model of mild pediatric TBI to investigate the relationship between early life TBI, acute responses of neuroimmune cells, and chronic behavioral dysregulation. At postnatal day 15, which is roughly equivalent to toddler age, male and female rat pups received a TBI via lateral fluid percussion injury. At 3 days post injury, TBI increased microglia and astrocyte coverage locally in the Perilesional Cortex but not in more distant corticolimbic regions. However, the hippocampus and prefrontal cortex did exhibit increased expression of the phagocytic marker CD68 in microglia, suggesting widespread glial activation even in the absence of gross coverage change. TBI also impacted mast cells, early-response innate immune cells, increasing their number and degranulation in multiple regions. In the juvenile and early adult periods, TBI impaired cognitive function, reduced sociability, and increased avoidance, with no change in anxiety-like behavior. Later in adulthood, TBI continued to impact cognitive behavior, increasing risky decision-making and impairing optimization months after injury. Together, these results suggest that pediatric TBI causes lasting cognitive and social dysregulation, possibly via acute neuroimmune alterations following injury at a critical period of brain development.

7 % of pregnant people use opioids. Opioid use during pregnancy can negatively impact maternal and offspring health. Medications for opioid use disorder (MOUD), commonly buprenorphine, are the recommended treatment for opioid use disorder during pregnancy to prevent cycles of withdrawal and relapse. In addition to effects on opioid receptors, opioids have strong binding affinity to toll-like receptor (TLR) 4, an immune cell receptor, and thereby impact neuroinflammatory signaling. We have previously shown that neuroimmune alterations are important for the display of maternal behavior. Here, we used a rodent model to assess the impact of chronic peripartum opioid exposure or MOUD on maternal caregiving and neuroinflammation in the postpartum brain. Female rats were exposed to vehicle (VEH), buprenorphine (BUP) to model MOUD, or oxycodone (OXY), to model peripartum drug use, before, during, and after pregnancy. Opioid exposure reduced gestation length and maternal weight gain. Postpartum maternal caretaking behaviors, including pup retrieval, huddling and nursing, and pup-directed sniffing and licking, were reduced in opioid-exposed mothers. Following behavioral testing, tissue was collected from brain regions important for maternal caretaking, including the prefrontal cortex (PFC), nucleus accumbens (NAc), preoptic area (POA), amygdala (AMY), and periaqueductal grey (PAG). Immunofluorescent labeling showed that BUP increased astrocyte labeling, while OXY increased microglia labeling in the PAG, but not other regions. Gene expression analysis also showed regional and treatment differences in immune transcripts. BUP and OXY increased TLR4 in the PFC. BUP increased TNF in the NAc but decreased IL1 beta in the POA. OXY increased CD68 in the POA, and IL1 beta, TNF, and TLR4 in the PAG. Together, these results provide novel evidence of peripartum neuroimmune alterations following chronic opioid exposure that could be mediating maternal care deficits. This work provides a foundation to explore the extent to which modulation of neuroimmune activation may be a potential intervention for caregiving deficits in mothers exposed to opioids during pregnancy.

Adolescence is a period of protracted neurodevelopment, during which the prefrontal cortex (PFC) undergoes significant remodeling. Microglia are integral to neurodevelopment and are sensitive to gonadal hormones, which increase during adolescence. Microglia and gonadal hormones can interact to influence adolescent development of the PFC (or medial prefrontal cortex [mPFC] in rodents). In females, gonadal hormones can be perturbed by using hormonal contraceptives (HCs). We predicted that HC administration over adolescence could affect microglia, other immunocompetent cells, and the neuroimmune environment of the developing mPFC. We also assessed HC effects on neuroimmune measures in the hippocampus, as the hippocampus also matures throughout adolescence and is sensitive to ovarian hormones. Intact post-pubertal female Sprague-Dawley rats received daily subcutaneous injections of vehicle or 10 ug ethinyl estradiol + 20 ug levonorgestrel (HCs) throughout adolescence from postnatal day (PND) 35-56. On PND 57 or 58, brains were collected for immunohistochemistry and qPCR. In the mPFC, HC-treated rats showed less Iba1 (microglia) immunolabeling and fewer Iba1+ cells. HC treatment also altered microglia morphology and reduced the spacing between microglia in the mPFC. In the hippocampus, HC-treated rats had reduced Iba1 immunolabeling in the dorsal CA1 and reductions in microglial cell complexity in dorsal CA1, ventral CA1, and ventral CA3. There were no effects of HCs on GFAP (astrocyte) immunolabeling in the mPFC or on astrocytes in any hippocampal subregion analyzed, except an increase in astrocyte number in the dorsal dentate gyrus. mPFC expression of genes related to phagocytosis (Cd68, Trem2) and neuroimmune signaling (Cx3cr1, Cx3cl1) were reduced in rats treated with HCs, but no gene expression changes were seen in the hippocampus. These data provide the first evidence that HCs given during the critical developmental period of adolescence can affect microglia properties in limbic brain regions.

Millions of people take hormonal contraceptives (HCs), often starting during adolescence when ovarian hormones influence brain and behavioral maturation. However, there is a fundamental lack of information about the neurobehavioral consequences of hormonal alterations via adolescent HC use. To begin addressing this gap, we validated a rodent model of adolescent HC administration and characterized its impact on endocrine, transcriptional, and behavioral endpoints. Cohorts of intact post-pubertal female Sprague-Dawley rats received daily subcutaneous injections of either vehicle or HC [10 μg ethinyl estradiol (EE) + 20 μg levonorgestrel (LNG)] for the duration of adolescence from postnatal day (PND) 35 to PND56. Blood and brain tissue was collected at PND57. Other cohorts received daily injections of vehicle or HC from PND35 until behavioral assays were completed on PND57-64. HC treatment was effective, as vaginal lavage indicated disrupted estrous cycling and ELISA indicated suppressed serum luteinizing hormone in HC-treated rats. Liquid chromatography-mass spectrometry analysis showed EE and LNG in serum and brain as well as diminished serum and brain levels of allopregnanolone and testosterone in HC-treated rats. NanoString nCounter analysis indicated that adolescent HC administration impacted expression of genes related to synapses, white matter, neuroimmune, monoamine, and hormone signaling in the hypothalamus and medial prefrontal cortex. While no effects of HCs were seen on sociability in the social preference test or stress coping behavior in the forced swim test, adolescent HC administration diminished risk-assessment behaviors in the novelty-induced hypophagia paradigm and altered anxiety-like behavior in the open field test and elevated plus maze. Overall, these data suggest that exposure to contraceptive hormones during the critical developmental period of adolescence may shape the brain and behavior.

Postpartum depression (PPD) affects 20 % of new mothers. Little is known about what contributes to this prevalence, though one of the strongest risk factors for developing PPD is stress during pregnancy. Pregnancy and the postpartum period are accompanied by dramatic changes in the endocrine, nervous, and immune systems, which may confer heightened vulnerability to maternal mental illness. Microglia, the brain's main immune cells, are important for displaying maternal behaviors. Environmental insults to microglia, such as stress, lead to hormonal dysregulation and aberrant synaptic pruning, both of which have been implicated in the pathophysiology of mood disorders like depression. Given these links, we hypothesized that stress during pregnancy would lead to dysregulated neuroimmune and endocrine profiles and produce synaptic changes in the maternal brain. We used a 2-week variable stress model in pregnancy and assessed neuroimmune changes in the nucleus accumbens (NAc), a brain region that regulates functions impaired in PDD including mood and maternal caregiving. Peripheral hormones and cytokines were also measured. While peripheral cytokine levels were not affected by stress exposure, gestational stress induced central immune changes in the NAc of maternal rats. This included increased microglia immunolabeling and changes in pro- and anti-inflammatory transcripts measured by a Nanostring nCounter panel. Astrocytes also increased in the NAc following gestational stress exposure. Peripheral estradiol and progesterone concentrations were reduced in late pregnancy of stressed mothers, and at the transcript level, hormone receptor and synthesis molecules were altered in the NAc. Gestational stress also altered transcripts associated with synapses and synaptic plasticity during late pregnancy and the postpartum period. There was no impact of stress on engulfment of pre- and postsynaptic proteins by microglia. However, microglia engulfed more of the postsynaptic marker, PSD95, in late pregnancy relative to postpartum, indicating a potential role for microglia in remodeling synapses in the NAc. Overall, these studies provide novel evidence that gestational stress impacts endocrine, synaptic and neuroimmune factors in the maternal NAc, suggesting possible mechanisms by which stress during pregnancy contributes to peripartum mood disorders.

Microglia play numerous important roles in brain development. From early embryonic stages through adolescence, these immune cells influence neuronal genesis and maturation, guide connectivity, and shape brain circuits. They also interact with other glial cells and structures, influencing the brain's supportive microenvironment. While this central role makes microglia essential, it means that early life perturbations to microglia can have widespread effects on brain development, potentially resulting in long-lasting behavioral impairments. Here, we will focus on the effects of early life psychosocial versus physiological stressors in rodent models. Psychosocial stress refers to perceived threats that lead to stress axes activation, including prenatal stress, or chronic postnatal stress, including maternal separation and resource scarcity. Physiological stress refers to physical threats, including maternal immune activation, postnatal infection, and traumatic brain injury. Differing sources of early life stress have varied impacts on microglia, and these effects are moderated by factors such as developmental age, brain region, and sex. Overall, these stressors appear to either 1) upregulate basal microglia numbers and activity throughout the lifespan, while possibly blunting their responsivity to subsequent stressors, or 2) shift the developmental curve of microglia, resulting in differential timing and function, impacting the critical periods they govern. Either could contribute to behavioral dysfunctions that occur after the resolution of early life stress. Exploring how different stressors impact microglia, as well as how multiple stressors interact to alter microglia's developmental functions, could deepen our understanding of how early life stress changes the brain's developmental trajectory. This article is part of the Special Issue on "Microglia".

20% of new mothers experience Postpartum Depression (PPD). The mechanisms contributing to PPD aren't well understood, but stress during pregnancy increases risk. Microglia, the brain’s innate immune cells, undergo peripartum-related changes within regions (e.g., medial prefrontal cortex, mPFC) regulating behaviors disrupted in PPD including mood and maternal care. Pregnancy also induces widespread neuroplasticity, and because microglia have been linked to synaptic remodeling, aberrant microglia-mediated synaptic remodeling may contribute to PPD.

Sex differences are prominent defining features of neurodevelopmental disorders. Understanding the sex biases in these disorders can shed light on mechanisms leading to relative risk and resilience for the disorders, as well as more broadly advance our understanding of how sex differences may relate to brain development. The prevalence of neurodevelopmental disorders is increasing, and the two most common neurodevelopmental disorders, Autism Spectrum Disorder (ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) exhibit male-biases in prevalence rates and sex differences in symptomology. While the causes of neurodevelopmental disorders and their sex differences remain to be fully understood, increasing evidence suggests that the immune system plays a critical role in shaping development. In this chapter we discuss sex differences in prevalence and symptomology of ASD and ADHD, review sexual differentiation and immune regulation of neurodevelopment, and discuss findings from human and rodent studies of immune dysregulation and perinatal immune perturbation as they relate to potential mechanisms underlying neurodevelopmental disorders. This chapter will give an overview of how understanding sex differences in neuroimmune function in the context of neurodevelopmental disorders could lend insight into their etiologies and better treatment strategies.

The peripartum period is accompanied by peripheral immune alterations to promote a successful pregnancy. We and others have also demonstrated significant neuroimmune changes that emerge during late pregnancy and persist postpartum, most prominently decreased microglia numbers within limbic brain regions. Here we hypothesized that microglial downregulation is important for the onset and display of maternal behavior. To test this, we recapitulated the peripartum neuroimmune profile by depleting microglia in non-mother (i.e., nulliparous) female rats who are typically not maternal but can be induced to behave maternally towards foster pups after repeated exposure, a process called maternal sensitization. BLZ945, a selective colony-stimulating factor 1 receptor (CSF1R) inhibitor, was administered systemically to nulliparous rats, which led to ~75% decrease in microglia number. BLZ- and vehicle-treated females then underwent maternal sensitization and tissue was stained for ∆fosB to examine activation across maternally relevant brain regions. We found BLZ-treated females with microglial depletion met criteria for displaying maternal behavior significantly sooner than vehicle-treated females and displayed increased pup-directed behaviors. Microglia depletion also reduced threat appraisal behavior in an open field test. Notably, nulliparous females with microglial depletion had decreased numbers of ∆fosB+ cells in the medial amygdala and periaqueductal gray, and increased numbers in the prefrontal cortex and somatosensory cortex, compared to vehicle. Our results demonstrate that microglia regulate maternal behavior in adult females, possibly by shifting patterns of activity in the maternal brain network.

Exposure to inflammatory perturbations, such as infection or allergy, during pregnancy increases the offspring’s risk for neurodevelopmental disorders. Rodent models enable us to investigate the neural mechanisms that may underlie behavioral changes in offspring exposed to inflammation in utero. Our lab has demonstrated that a rat model of allergic maternal immune activation (MIA) induces behavior in offspring resembling that found in neurodevelopmental disorders, including decreased juvenile social play. This study tested whether this fetal programming of altered social behavior may be mediated by reduced innervation of the nonapeptides Oxytocin (OT) and Vasopressin (AVP) in social brain regions such as the Medial Amygdala (MeA) and the Bed Nucleus of the Stria Terminalis (BST). Adult virgin female Sprague Dawley rats (n=7/group) were randomly assigned to be sensitized to ovalbumin with alum adjuvant, or saline. After breeding, pregnant dams were subjected to either OVA challenge or saline control by intranasal infusion on gestational day 15 and were left to give birth undisturbed. On postnatal day 28, 1-2 offspring per sex per litter (n = 40) were sacrificed and brain tissue was collected, fixed, and processed for fluorescence immunohistochemistry of OT and AVP. Fiber fractional area (pixels2) was calculated using Image J software. 2-way ANOVA was used for statistical analysis (alpha=0.05). A significant interaction revealed that Allergic MIA decreased OT fiber density in the BST of female but not male offspring. There was also a trend for reduced AVP fiber density in the medial anterior BST in both sexes, and a main effect such that males had significantly more AVP than females. Allergic MIA did not decrease OT fiber density in the MeA. Allergic MIA may produce sex-specific mechanisms underlying altered social behavior. Future studies will investigate whether pre- or post-natal immunomodulatory treatment may prevent the fetal programming of abnormal social behavior and neuropeptide fiber densities.

Prenatal exposure to inflammation via maternal infection, allergy, or autoimmunity increases one's risk for developing neurodevelopmental and psychiatric disorders. Many of these disorders are associated with altered social behavior, yet the mechanisms underlying inflammation-induced social impairment remain unknown. We previously found that a rat model of acute allergic maternal immune activation (MIA) produced deficits like those found in MIA-linked disorders, including impairments in juvenile social play behavior. The neuropeptides oxytocin (OT) and arginine vasopressin (AVP) regulate social behavior, including juvenile social play, across mammalian species. OT and AVP are also implicated in neuropsychiatric disorders characterized by social impairment, making them good candidate regulators of social deficits after MIA. We profiled how acute prenatal exposure to allergic MIA changed OT and AVP innervation in several brain regions important for social behavior in juvenile male and female rat offspring. We also assessed whether MIA altered additional behavioral phenotypes related to sociality and anxiety. We found that allergic MIA increased OT and AVP fiber immunoreactivity in the medial amygdala and had sex-specific effects in the nucleus accumbens, bed nucleus of the stria terminalis, and lateral hypothalamic area. We also found that MIA reduced ultrasonic vocalizations in neonates and increased the stereotypical nature of self-grooming behavior. Overall, these findings suggest that there may be sex-specific mechanisms underlying MIA-induced behavioral impairment and underscore OT and AVP as ideal candidates for future mechanistic studies.

The postpartum period is a time associated with high rates of depression and anxiety as well as greater risk for psychosis in some women. A growing number of studies point to aberrations in immune system function as contributing to postpartum mental illness. Here we review evidence from both clinical and animal models suggesting an immune component to postpartum depression, postpartum anxiety, and postpartum psychosis. Thus far, clinical data primarily highlights changes in peripheral cytokine signaling in disease etiology, while animal models have begun to provide insight into the immune environment of the maternal brain and how central inflammation may also be contributing to postpartum mental illnesses. Further research investigating peripheral and central immune function, along with neural and endocrine interactions, will be important in successfully developing novel prevention and treatment strategies for these serious disorders that impact a large portion of new mothers.

Allergic inflammation during pregnancy increases risk for a diagnosis of neurodevelopmental disorders such as Attention Deficit/Hyperactivity Disorder (ADHD) and Autism Spectrum Disorder (ASD) in the offspring. Previously, we found a model of such inflammation, allergy-induced maternal immune activation (MIA), produced symptoms analogous to those associated with neurodevelopmental disorders in rats, including reduced juvenile play behavior, hyperactivity, and cognitive inflexibility. These behaviors were preceded by perinatal changes in microglia colonization and phenotype in multiple relevant brain regions. Given the role that microglia play in synaptic patterning as well as evidence for altered synaptic architecture in neurodevelopmental disorders, we investigated whether allergic MIA altered the dynamics of dendritic spine patterning throughout key regions of the rat forebrain across neurodevelopment. Adult virgin female rats were sensitized to the allergen, ovalbumin, with alum adjuvant, bred, and allergically challenged on gestational day 15. Brain tissue was collected from male and female offspring on postnatal days (P) 5, 15, 30, and 100-120 and processed for Golgi-Cox staining. Mean dendritic spine density was calculated for neurons in brain regions associated with cognition and social behavior, including the medial prefrontal cortex (mPFC), basal ganglia, septum, nucleus accumbens (NAc), and amygdala. Allergic MIA reduced dendritic spine density in the neonatal (P5) and juvenile (P15) mPFC, but these mPFC spine deficits were normalized by P30. Allergic inflammation reduced spine density in the septum of juvenile (P30) rats, with an interaction suggesting increased density in males and reduced density in females. MIA-induced reductions in spine density were also found in the female basal ganglia at P15, as well as in the NAc at P30. Conversely, MIA-induced increases were found in the NAc in adulthood. While amygdala dendritic spine density was generally unaffected throughout development, MIA reduced density in both medial and basolateral subregions in adult offspring. Correlational analyses revealed disruption to amygdala-related networks in the neonatal animals and cortico-striatal related networks in juvenile and adult animals in a sex-specific manner. Collectively, these data suggest that communication within and between these cognitive and social brain regions may be altered dynamically throughout development after prenatal exposure to allergic inflammation. They also provide a basis for future intervention studies targeted at rescuing spine and behavior changes via immunomodulatory treatments.

Microglia, the innate immune cells of the brain, regulate brain development through many processes such as synaptic pruning, supporting cell genesis and phagocytosing living and dying cells. There are sex differences in these same developmental processes throughout the brain, thus microglia may contribute to brain sex differences. We examined whether microglia support a known sex difference in neonatal hippocampal neurogenesis and whether juvenile hippocampal neurogenesis was impacted by the loss of neonatal microglia. We used central infusion of liposomal clodronate to selectively deplete microglia and found decreased cell genesis in the male, but not female, dentate gyrus and hippocampus. We found that loss of microglia decreased cell genesis in the cortex and amygdala of both males and females. We assessed the expression of several cytokines and growth factors that have previously been shown to support cell genesis. We found that expression of Il1b and Tnf were decreased in the hippocampus due to microglia depletion however, there were no sex differences in the expression of any immune genes. In adolescence, there was an increase in the number of mitotic cells in the subgranular zone of the dentate gyrus of previously microglia depleted rats however, the number of newly-born neurons was unchanged in the adolescent animals. We also sought to determine whether there was a sex difference in the number of progenitor cells in the dentate gyrus in the neonatal period. We found no sex differences in the number of progenitor cells. Overall, these studies show that microglia are important for regulating region-specific sex differences in cell genesis in the developing brain.

Maternal systemic inflammation increases risk for neurodevelopmental disorders like autism, ADHD, and schizophrenia in offspring. Notably, these disorders are male-biased. Studies have implicated immune system dysfunction in the etiology of these disorders, and rodent models of maternal immune activation provide useful tools to examine mechanisms of sex-dependent effects on brain development, immunity, and behavior. Here, we employed an allergen-induced model of maternal inflammation in rats to characterize levels of mast cells and microglia in the perinatal period in male and female offspring, as well as social, emotional, and cognitive behaviors throughout the lifespan. Adult female rats were sensitized to ovalbumin (OVA), bred, and challenged intranasally on gestational day 15 of pregnancy with OVA or saline. Allergic inflammation upregulated microglia in the fetal brain, increased mast cell number in the hippocampus on the day of birth, and conferred region-, time- and sex- specific changes in microglia measures. Additionally, offspring of OVA-exposed mothers subsequently exhibited abnormal social behavior, hyperlocomotion, and reduced cognitive flexibility. These data demonstrate the long-term effects of maternal allergic challenge on offspring development and provide a basis for understanding neurodevelopmental disorders linked to maternal systemic inflammation in humans.

Sexual differentiation is the early life process by which the brain is prepared for male or female typical behaviors, and is directed by sex chromosomes, hormones and early life experiences. We have recently found that innate immune cells residing in the brain, including microglia and mast cells, are more numerous in the male than female rat brain. Neuroimmune cells are also key participants in the sexual differentiation process, specifically organizing the synaptic development of the preoptic area and leading to male-typical sexual behavior in adulthood. Mast cells are known for their roles in allergic responses, thus in this study we sought to determine if exposure to an allergic response of the pregnant female in utero would alter the sexual differentiation of the preoptic area of offspring and resulting sociosexual behavior in later life. Pregnant rats were sensitized to ovalbumin (OVA), bred, and challenged intranasally with OVA on gestational day 15, which produced robust allergic inflammation, as measured by elevated immunoglobulin E. Offspring of these challenged mother rats were assessed relative to control rats in the early neonatal period for mast cell and microglia activation within their brains, downstream dendritic spine patterning on POA neurons, or grown to adulthood to assess behavior and dendritic spines. In utero exposure to allergic inflammation increased mast cell and microglia activation in the neonatal brain, and led to masculinization of dendritic spine density in the female POA. In adulthood, OVA-exposed females showed an increase in male-typical mounting behavior relative to control females. In contrast, OVA-exposed males showed evidence of dysmasculinization, including reduced microglia activation, reduced neonatal dendritic spine density, decreased male-typical copulatory behavior, and decreased olfactory preference for female-typical cues. Together these studies show that early life allergic events may contribute to natural variations in both male and female sexual behavior, potentially via underlying effects on brain-resident mast cells.