Effects of limited bedding and nesting on postpartum mood state in rats.

This study examined the effect of limited bedding and nesting (LBN) stress on postpartum anhedonia, maternal behaviors, anxiety-like behaviors, and neuroendocrine and neuroimmune function as a potential model of postpartum depression. Dams underwent sucrose preference tests prior to breeding, during gestation and again postpartum, to examine the potential onset of anhedonia. On embryonic day 19, dams were placed into either a LBN or control housing condition. Contrary to our predictions, LBN stress had no effect on postpartum sucrose preference. We also found no effect of LBN condition on fecal estradiol or corticosterone levels, both of which increased at birth and decreased postpartum. Regardless of housing conditions, approximately 40% of new mothers exhibited a decrease in sucrose preference, while others show no change, suggesting an individual susceptibility to postpartum anhedonia. In a separate cohort of LBN and control dams, we measured pup retrieval, hoarding behavior, elevated plus maze (EPM), and marble burying. LBN dams exhibited increased anxiety, associated with decreased time spent in the open arms of the EPM. We also measured a significant increase in IL-6 expression in the dorsal hippocampus and medial prefrontal cortex of postpartum dams compared to nonpregnant dams. These findings suggest that while LBN stress has effects on anxiety and maternal care, it does not induce postpartum anhedonia. Rather, there are inherent differences in susceptibility to anhedonia in individual dams, and future studies should be conducted to better understand individual vulnerability and resilience to postpartum anhedonia.

Maternal immune activation as an epidemiological risk factor for neurodevelopmental disorders: Considerations of timing, severity, individual differences, and sex in human and rodent studies.

Epidemiological evidence suggests that one's risk of being diagnosed with a neurodevelopmental disorder (NDD)-such as autism, ADHD, or schizophrenia-increases significantly if their mother had a viral or bacterial infection during the first or second trimester of pregnancy. Despite this well-known data, little is known about how developing neural systems are perturbed by events such as early-life immune activation. One theory is that the maternal immune response disrupts neural processes important for typical fetal and postnatal development, which can subsequently result in specific and overlapping behavioral phenotypes in offspring, characteristic of NDDs. As such, rodent models of maternal immune activation (MIA) have been useful in elucidating neural mechanisms that may become dysregulated by MIA. This review will start with an up-to-date and in-depth, critical summary of epidemiological data in humans, examining the association between different types of MIA and NDD outcomes in offspring. Thereafter, we will summarize common rodent models of MIA and discuss their relevance to the human epidemiological data. Finally, we will highlight other factors that may interact with or impact MIA and its associated risk for NDDs, and emphasize the importance for researchers to consider these when designing future human and rodent studies. These points to consider include: the sex of the offspring, the developmental timing of the immune challenge, and other factors that may contribute to individual variability in neural and behavioral responses to MIA, such as genetics, parental age, the gut microbiome, prenatal stress, and placental buffering.

A method for the selective depletion of microglia in the dorsal hippocampus in the juvenile rat brain.

BACKGROUND: To understand the role of microglia in brain function and development, methods have emerged to deplete microglia throughout the brain. Liposome-encapsulated clodronate (LEC) can be infused into the brain to deplete microglia in a brain-region and time-specific manner. NEW METHOD: This study validates methodology to deplete microglia in the rat dorsal hippocampus (dHP) during a specific period of juvenile development. Stereotaxic surgery was performed to infuse LEC at postnatal day (P) 16 or 19 into dHP. Rat brains were harvested at various ages to determine specificity of infusion and duration of depletion. RESULTS: P19 infusion of LEC into dHP with a 27G syringe depleted microglia in dHP subregions CA1, dentate gyrus (DG), and CA3 from P24-P30. There was also evidence of depletion in parietal cortex above the infusion site. P16 infusion of LEC with a 32 G syringe depleted microglia only in dHP subregions CA1 and DG from P21-P40. COMPARISON WITH EXISTING METHOD(S): Previous methods have infused LEC intra-hippocampally in adult rats or intra-cerebroventricularly in neonatal rats. This study is the first to publish methodology to deplete microglia in a brain-region specific manner during juvenile rat development. CONCLUSIONS: The timing of LEC infusion during the juvenile period can be adjusted to achieve maximal microglia depletion by a specific postnatal day. A 27G needle results in LEC backflow during the infusion, but also allows LEC to reach all subregions of dHP. Infusion with a 32 G needle prevents backflow during infusion, but results in a more local spread of LEC within dHP.

Regional Differences in Various Risk Factors for Postpartum Depression: Applying Mixed Models to the PRAMS Dataset.

Purpose: The purpose of this study was to assess the association between various risk factors with postpartum depression severity using a large dataset that included variables such as previous mental health status, social factors, societal factors, health care access, and other state-wide or region-specific variables. Methods: We obtained the most recently available (2016-2017) dataset from the Pregnancy Risk Assessment Monitoring System (PRAMS), which is a dataset compiled by the Centers for Disease Control (CDC) that collects state-specific, population-based data on maternal attitudes and experiences before, during, and shortly after pregnancy from over 73,000 women in 39 states. We utilized a hierarchical linear model to analyze the data across various levels, with a symptom severity scale (0-8) as the dependent variable. Results: Of the 21 variables included in the final model, nine variables were statistically significant predictors of symptom severity. Statistically significant predictors of increased postpartum depression symptom severity included previous depression diagnosis and depression symptoms during pregnancy, baby not residing with mother, unintentional pregnancy, women with less than a high school degree and more than a college degree, Women Infants Children (WIC) enrollment, and married women. In contrast to these other factors, attendance at a postpartum follow up appointment was associated with significantly increased symptom severity. Age revealed an inverted curve in predicting postpartum symptom severity. Conclusions: There was no significant difference in symptom severity scores across the 39 participating states. Most notably, postpartum depression symptom severity was associated with previous depression diagnosis and previous symptom severity, but our results also reveal novel social and education factors that contribute to the support and well-being of the mother and child.

A Rat Model of Prenatal Zika Virus Infection and Associated Long-Term Outcomes.

Zika virus (ZIKV) is a mosquito-borne flavivirus that became widely recognized due to the epidemic in Brazil in 2015. Since then, there has been nearly a 20-fold increase in the incidence of microcephaly and birth defects seen among women giving birth in Brazil, leading the Centers for Disease Control and Prevention (CDC) to officially declare a causal link between prenatal ZIKV infection and the serious brain abnormalities seen in affected infants. Here, we used a unique rat model of prenatal ZIKV infection to study three possible long-term outcomes of congenital ZIKV infection: (1) behavior, (2) cell proliferation, survival, and differentiation in the brain, and (3) immune responses later in life. Adult offspring that were prenatally infected with ZIKV exhibited motor deficits in a sex-specific manner, and failed to mount a normal interferon response to a viral immune challenge later in life. Despite undetectable levels of ZIKV in the brain and serum in these offspring at P2, P24, or P60, these results suggest that prenatal exposure to ZIKV results in lasting consequences that could significantly impact the health of the offspring. To help individuals already exposed to ZIKV, as well as be prepared for future outbreaks, we need to understand the full spectrum of neurological and immunological consequences that could arise following prenatal ZIKV infection.

Examining the impact of neuroimmune dysregulation on social behavior of male and female juvenile rats.

Many individuals diagnosed with neuropsychiatric disorders, such as autism, attention-deficit/hyperactivity disorder, schizophrenia, and social anxiety disorder, all share a common dimension of aberrant social behavior. Epidemiological data indicate that adverse environmental factors contribute to the risk for neurodevelopmental disorders, including those associated with aberrant social behavior. Early-life exposure to infectious pathogens is one of those adverse environmental factors, suggesting that activation of the immune system during early development may contribute to disease pathology associated with altered social behavior. In the current project, we examined the impact of neonatal infection, with or without juvenile immune activation, on the expression of juvenile social behavior and on the expression of inflammatory cytokines and microglial signaling molecules in the juvenile rat brain. The outcomes of these experiments revealed that neonatal infection significantly decreased juvenile social interaction, but significantly increased juvenile play behavior in male and female rats. Moreover, neonatal infection alone, juvenile immune activation alone, and neonatal infection plus juvenile immune activation all significantly impaired social recognition in juvenile male rats. Juvenile female rats (including controls) did not demonstrate social recognition as measured in our three-chamber social recognition test. Taken together, the behavioral and molecular data presented here support the sensitivity of the developing brain to immune activation, particularly in the expression of age-appropriate social behaviors. These data warrant the design of additional studies to examine the mechanistic relationship between early-life immune activation and aberrant social behavior to develop novel as well as modify existing therapeutic targets and preventative measures to help those who display aberrant social behavior.

An Examination of the Long-Term Neurodevelopmental Impact of Prenatal Zika Virus Infection in a Rat Model Using a High Resolution, Longitudinal MRI Approach.

Since Zika virus (ZIKV) first emerged as a public health concern in 2015, our ability to identify and track the long-term neurological sequelae of prenatal Zika virus (ZIKV) infection in humans has been limited. Our lab has developed a rat model of maternal ZIKV infection with associated vertical transmission to the fetus that results in significant brain malformations in the neonatal offspring. Here, we use this model in conjunction with longitudinal magnetic resonance imaging (MRI) to expand our understanding of the long-term neurological consequences of prenatal ZIKV infection in order to identify characteristic neurodevelopmental changes and track them across time. We exploited both manual and automated atlas-based segmentation of MR images in order to identify long-term structural changes within the developing rat brain following inoculation. The paradigm involved scanning three cohorts of male and female rats that were prenatally inoculated with 107 PFU ZIKV, 107 UV-inactivated ZIKV (iZIKV), or diluent medium (mock), at 4 different postnatal day (P) age points: P2, P16, P24, and P60. Analysis of tracked brain structures revealed significantly altered development in both the ZIKV and iZIKV rats. Moreover, we demonstrate that prenatal ZIKV infection alters the growth of brain regions throughout the neonatal and juvenile ages. Our findings also suggest that maternal immune activation caused by inactive viral proteins may play a role in altered brain growth throughout development. For the very first time, we introduce manual and automated atlas-based segmentation of neonatal and juvenile rat brains longitudinally. Experimental results demonstrate the effectiveness of our novel approach for detecting significant changes in neurodevelopment in models of early-life infections.

The effects of early-life immune activation on microglia-mediated neuronal remodeling and the associated ontogeny of hippocampal-dependent learning in juvenile rats.

Many neurodevelopmental disorders and associated learning deficits have been linked to early-life immune activation or ongoing immune dysregulation (Laskaris et al., 2016; O'Connor et al., 2014; Frick et al., 2013). Neuroscientists have begun to understand how the maturation of neural circuits allows for the emergence of cognitive and learning behaviors; yet we know very little about how these developing neural circuits are perturbed by certain events, including risk-factors such as early-life immune activation and immune dysregulation. To answer these questions, we examined the impact of early-life immune activation on the emergence of hippocampal-dependent learning in juvenile male and female rats using a well-characterized hippocampal-dependent learning task and we investigated the corresponding, dynamic multicellular interactions in the hippocampus that may contribute to these learning deficits. We found that even low levels of immune activation can result in hippocampal-depedent learning deficits days later, but only when this activation occurs during a sensitive period of development. The initial immune response and associated cytokine production in the hippocampus resolved within 24 h, several days prior to the observed learning deficit, but notably the initial immune response was followed by altered microglial-neuronal communication and synapse remodeling that changed the structure of hippocampal neurons during this period of juvenile brain development. We conclude that immune activation or dysregulation during a sensitive period of hippocampal development can precipitate the emergence of learning deficits via a multi-cellular process that may be initiated by, but not the direct result of the initial cytokine response. SIGNIFICANCE STATEMENT: Many neurodevelopmental disorders have been linked to early-life immune activation or immune dysregulation; however, very little is known about how dynamic changes in neuroimmune cells mediate the transition from normal brain function to the early stages of cognitive disorders, or how changes in immune signaling are subsequently integrated into developing neuronal networks. The current experiments examined the consequences of immune activation on the cellular and molecular changes that accompany the emergence of learning deficits during a sensitive period of hippocampal development. These findings have the potential to significantly advance our understanding of how early-life immune activation or dysregulation can result in the emergence of cognitive and learning deficits that are the largest source of years lived with disability in humans.

Anxiety in transition: Neuroendocrine mechanisms supporting the development of anxiety pathology in adolescence and young adulthood.

Adolescence marks a key developmental window during which emotion dysregulation increases, along with risk for the onset of anxiety and other affect-related pathologies. Although emotion dysregulation and related pathologies normatively decline during the transition into adulthood, this does not occur for a sizable minority of individuals. Finally, sex differences in anxiety emerge during adolescence, with females developing a 2-fold increase in risk relative to males. Unfortunately, a neurobiological model of the mechanisms that cause these changes during adolescence has yet to be proposed. In the present work, we first provide brief reviews of relevant literature. Next, we outline a dual-mechanism model focused on (i) the influence of pubertal testosterone on key emotion-regulation circuitry (i.e., orbitofrontal cortex-amygdala coupling) and (ii) myelination of the fiber bundles connecting such circuitry (i.e., uncinate fasciculus). The proposed model offers a set of specific, testable hypotheses that will hopefully spur much needed cross-disciplinary research.

Zika virus infection of pregnant rats and associated neurological consequences in the offspring.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with microcephaly and other neurological disorders in infants born to infected mothers. Despite being declared an international emergency by the World Health Organization, very little is known about the mechanisms of ZIKV pathogenesis or the long-term consequences of maternal ZIKV infection in the affected offspring, largely due to the lack of appropriate rodent models. To address this issue, our lab has developed a working model of prenatal ZIKV infection in rats. In this study, we infected immune competent pregnant female rats with 105-107 PFU of ZIKV (PRVABC59, Puerto Rico/Human/Dec 2015) in order to examine its pathogenesis in the dams and pups. We examined the febrile response and sickness behavior in the dams, in addition to neonatal mortality, microglia morphology, cortical organization, apoptosis, and brain region-specific volumes in the offspring. Here, we demonstrate that pregnant and non-pregnant female rats have a distinct febrile response to ZIKV infection. Moreover, prenatal ZIKV infection increased cell death and reduced tissue volume in the hippocampus and cortex in the neonatal offspring. For the first time, we demonstrate the efficacy and validity of an immunocompetent rat model for maternal ZIKV infection that results in significant brain malformations in the neonatal offspring.

An investigation into the immune response of cultured neural rat cells following Zika virus infection.

The most notable effect of prenatal Zika virus (ZIKV) infection is severe microcephaly. ZIKV has a selective tropism for neural progenitor cells; however, it is not clear what role the immune cells of the brain, microglia, may have in mitigating or exacerbating neuronal cell death following ZIKV infection. We cultured hippocampal and cortical neural cells from neonatal rat pups and infected them with ZIKV at various multiplicities of infection (MOI). We found that the neuroimmune response to ZIKV infection is composed of both pro-inflammatory and type I interferon responses and is largely dependent upon the viral dose.

Frank Beach Award Winner - The future of mental health research: Examining the interactions of the immune, endocrine and nervous systems between mother and infant and how they affect mental health.

Pregnancy and the postpartum period are periods of significant change in the immune and endocrine systems. This period of life is also associated with an increased risk of mental health disorders in the mother, and an increased risk of developmental and neuropsychiatric disorders in her infant. The collective data described here supports the idea that peripartum mood disorders in mother and developmental disorders in her infant likely reflects multiple pathogeneses, stemming from various interactions between the immune, endocrine and nervous systems, thereby resulting in various symptom constellations. In this case, testing the mechanisms underlying specific symptoms of these disorders (e.g. deficits in specific types of learning or anhedonia) may provide a better understanding of the various physiological interactions and multiple etiologies that most likely underlie the risk of mental health disorders during this unique time in life. The goal here is to summarize the current understanding of how immune and endocrine factors contribute to maternal mental health, while simultaneously understanding the impact these unique interactions have on the developing brain of her infant.

An IL-6 receptor antagonist attenuates postpartum anhedonia, but has no effect on anhedonia precipitated by subchronic stress in female rats.

RATIONALE: Nearly 60-80% of women experience some form of sadness, anxiety, or anhedonia in the weeks following the birth of a child (Patel et al. 23(2):534-42, 2012; Degner 10: 359;j4692, 2017); however, the exact mechanisms that precipitate these changes in mood postpartum are still unknown. It is well-known that the function of the peripheral immune system is significantly altered during pregnancy in order to protect the developing fetus from being rejected by the maternal immune system (Fallon et al. 17(1):7-17, 2002), and we have recently found a dramatic change in the central immune system during and just after pregnancy in female rats (Sherer et al. 66:201-209, 2017). We observed anhedonia in Sprague-Dawley rat dams on the day of birth that is associated with an increase in interleukin (IL)-6 expression in the brain on the day of birth (Posillico and Schwarz 298(Pt B):218-28, 2016). OBJECTIVES: The goal of the current experiments was to determine whether inhibiting the IL-6 receptor could prevent onset of this postpartum anhedonia, or anhedonia precipitated by subchronic stress in non-pregnant females. RESULTS: Treatment with an IL-6 receptor antibody attenuated postpartum anhedonia as characterized by a decrease in sucrose preference. In contrast, this antibody had no effect on the decrease in sucrose preference induced following a week of forced swim stress in non-pregnant female rats. CONCLUSIONS: The results of these studies suggest that the molecular mechanisms that underlie the onset of anhedonia following birth or mild stress in female rats may be distinct.

Programming Effects of Pubertal Lipopolysaccharide Treatment in Male and Female CD-1 Mice.

Puberty is a critical period of development marked by sexual, immune, and neural maturation. Exposure to stress during this period can lead to enduring changes in brain functioning and in behavior; however, the underlying mechanisms and the programming effects of stress during puberty remain unknown. Therefore, the objective of this study was to investigate the programming effects of pubertal immune challenge in response to a homotypic stressor later in life in CD-1 mice. Age and sex differences in the peripheral and central cytokine levels, along with sickness behavior and telemetry data, were analyzed following the secondary treatment. The results showed that pretreatment with LPS attenuated the immune response to a second homotypic challenge. Males pretreated with LPS during puberty and in early adulthood displayed an attenuated hypothermic response following the second LPS treatment compared with saline-pretreated controls, which is consistent with the attenuated peripheral IL-6 and IFN-γ concentrations. Females pretreated with LPS during puberty displayed lower IL-1ß, TNF-α, and IL-6 mRNA expression in the prefrontal cortex following the secondary immune challenge compared with saline controls. The results of this study show that exposure to LPS during puberty programs the peripheral and central immune responses, resulting in an attenuated immune response following a subsequent homotypic stressor. Thus, exposure to an immune challenge during puberty affects immune function later in life, which could permanently affect brain function and have implications on mental health.

Sexual Differentiation and Sex Differences in Neural Development.

Sex determination occurs at the moment of conception, as a result of XX or XY chromosome pairing. From that point, the body undergoes the process of sexual differentiation, inducing the development of physical characteristics that are easily distinguishable between the sexes and are often reflected in one's physical appearance and gender identity. Although less apparent, the brain also undergoes sexual differentiation. Sex differences in the brain are organized during a critical period of neural development and have an instrumental role in determining the physiology and behavior of an individual throughout the lifespan. Understanding the extent of sex differences in neurodevelopment also influences our understanding of the potential risk for a number of neurodevelopmental, neurological, and mental health disorders that exhibit strong sex biases. Advances made in our understanding of sexually dimorphic brain nuclei, sex differences in neural cell communication, and sex differences in the communication between the brain and peripheral organs are all research fields that have provided valuable information related to the physiological and behavioral outcomes of sex differences in brain development. More recently, investigations into the impact of epigenetic mechanisms on sexual differentiation of the brain have indicated that changes in gene expression, via epigenetic modifications, also contribute to sexual differentiation of the developing brain. Still, there are a number of important questions and ideas that have arisen from our current understanding of sex differences in neurodevelopmental processes that necessitate more time and attention in this field.

Sex- and region-specific differences in microglia phenotype and characterization of the peripheral immune response following early-life infection in neonatal male and female rats.

Early-life infection has been shown to have profound effects on the brain and behavior across the lifespan, a phenomenon termed "early-life programming". Indeed, many neuropsychiatric disorders begin or have their origins early in life and have been linked to early-life immune activation (e.g. autism, ADHD, and schizophrenia). Furthermore, many of these disorders show a robust sex bias, with males having a higher risk of developing early-onset neurodevelopmental disorders. The concept of early-life programming is now well established, however, it is still unclear how such effects are initiated and then maintained across time to produce such a phenomenon. To begin to address this question, we examined changes in microglia, the immune cells of the brain, and peripheral immune cells in the hours immediately following early-life infection in male and female rats. We found that males showed a significant decrease in BDNF expression and females showed a significant increase in IL-6 expression in the cerebellum following E.coli infection on postnatal day 4; however, for most cytokines examined in the brain and in the periphery we were unable to identify any sex differences in the immune response, at least at the time points examined. Instead, neonatal infection with E.coli increased the expression of a number of cytokines in the brain of both males and females similarly including TNF-α, IL-1ß, and CD11b (a marker of microglia activation) in the hippocampus and, in the spleen, TNF-α and IL-1ß. We also found that protein levels of GRO-KC, MIP-1a, MCP1, IP-10, TNF-α, and IL-10 were elevated 8-hours postinfection, but this response was resolved by 24-hours. Lastly, we found that males have more thin microglia than females on P5, however, neonatal infection had no effect on any of the microglia morphologies we examined. These data show that sex differences in the acute immune response to neonatal infection are likely gene, region, and even time dependent. Future research should consider these factors in order to develop a comprehensive understanding of the immune response in males and females as these changes are likely the initiating agents that lead to the long-term, and often sex-specific, effects of early-life infection.

Sex Differences in the Neuroimmune System.

While sex differences in the peripheral immune response have been studied extensively, sex differences in the neuroimmune response, including glial activation and associated cytokine production in the brain, is a recently emerging field. Advances in our understanding of sex differences in the neuroimmune response have important implications for understanding how neural circuits are shaped during early brain development, how activation of the immune system may impact cognitive function and behavior, and how inflammation may be associated with the risk of mental health disorders that have strong sex-biases. The goal of this mini review is to highlight recent work in the field of sex differences in neuroimmune function, with a particular focus on how microglia function is influenced by age and sex hormone exposure.

The psychoneuroimmunology of pregnancy.

Pregnancy is associated with a number of significant changes in maternal physiology. Perhaps one of the more notable changes is the significant alteration in immune function that occurs during pregnancy. This change in immune function is necessary to support a successful pregnancy, but also creates a unique period of life during which a female is susceptible to disease and, as we'll speculate here, may also contribute to mental health disorders associated with pregnancy and the postpartum period. Here, we review the known changes in peripheral immune function that occur during pregnancy and the postpartum period, while highlighting the impact of hormones during these times on immune function, brain or neural function, as well as behavior. We also discuss the known and possible impact of pregnancy-induced immune changes on neural function during this time and briefly discuss how these changes might be a risk factor for perinatal anxiety or mood disorders.

Impact of Prenatal and Subsequent Adult Alcohol Exposure on Pro-Inflammatory Cytokine Expression in Brain Regions Necessary for Simple Recognition Memory.

Microglia, the immune cells of the brain, are important and necessary for appropriate neural development; however, activation of microglia, concomitant with increased levels of secreted immune molecules during brain development, can leave the brain susceptible to certain long-term changes in immune function associated with neurological and developmental disorders. One mechanism by which microglia can be activated is via alcohol exposure. We sought to investigate if low levels of prenatal alcohol exposure can alter the neuroimmune response to a subsequent acute dose of alcohol in adulthood. We also used the novel object location and recognition memory tasks to determine whether there are cognitive deficits associated with low prenatal alcohol exposure and subsequent adulthood alcohol exposure. We found that adult rats exposed to an acute binge-like level of alcohol, regardless of gestational alcohol exposure, have a robust increase in the expression of Interleukin (IL)-6 within the brain, and a significant decrease in the expression of IL-1ß and CD11b. Rats exposed to alcohol during gestation, adulthood, or at both time points exhibited impaired cognitive performance in the cognitive tasks. These results indicate that both low-level prenatal alcohol exposure and even acute alcohol exposure in adulthood can significantly impact neuroimmune and associated cognitive function.