Got milk? Maternal immune activation during the mid-lactational period affects nutritional milk quality and adolescent offspring sensory processing in male and female rats.

Previous studies have underscored the importance of breastfeeding and parental care on offspring development and behavior. However, their contribution as dynamic variables in animal models of early life stress are often overlooked. In the present study, we investigated how lipopolysaccharide (LPS)-induced maternal immune activation (MIA) on postnatal day (P)10 affects maternal care, milk, and offspring development. MIA was associated with elevated milk corticosterone concentrations on P10, which recovered by P11. In contrast, both milk triglyceride and percent creamatocrit values demonstrated a prolonged decrease following inflammatory challenge. Adolescent MIA offspring were heavier, which is often suggestive of poor early life nutrition. While MIA did not decrease maternal care quality, there was a significant compensatory increase in maternal licking and grooming the day following inflammatory challenge. However, this did not protect against disrupted neonatal huddling or later-life alterations in sensorimotor gating, conditioned fear, mechanical allodynia, or reductions in hippocampal parvalbumin expression in MIA offspring. MIA-associated changes in brain and behavior were likely driven by differences in milk nutritional values and not by direct exposure to LPS or inflammatory molecules as neither LPS binding protein nor interleukin-6 milk levels differed between groups. These findings reflected comparable microbiome and transcriptomic patterns at the genome-wide level. Animal models of early life stress can impact both parents and their offspring. One mechanism that can mediate the effects of such stressors is changes to maternal lactation quality which our data show can confer multifaceted and compounding effects on offspring physiology and behavior.

The contributions of parental lactation on offspring development: It's not udder nonsense!

The Developmental Origins of Health and Disease (DOHaD) hypothesis describes how maternal stress exposures experienced during critical periods of perinatal life are linked to altered developmental trajectories in offspring. Perinatal stress also induces changes in lactogenesis, milk volume, maternal care, and the nutritive and non-nutritive components of milk, affecting short and long-term developmental outcomes in offspring. For instance, selective early life stressors shape the contents of milk, including macro/micronutrients, immune components, microbiota, enzymes, hormones, milk-derived extracellular vesicles, and milk microRNAs. In this review, we highlight the contributions of parental lactation to offspring development by examining changes in the composition of breast milk in response to three well-characterized maternal stressors: nutritive stress, immune stress, and psychological stress. We discuss recent findings in human, animal, and in vitro models, their clinical relevance, study limitations, and potential therapeutic significance to improving human health and infant survival. We also discuss the benefits of enrichment methods and support tools that can be used to improve milk quality and volume as well as related developmental outcomes in offspring. Lastly, we use evidence-based primary literature to convey that even though select maternal stressors may modulate lactation biology (by influencing milk composition) depending on the severity and length of exposure, exclusive and/or prolonged milk feeding may attenuate the negative in utero effects of early life stressors and promote healthy developmental trajectories. Overall, scientific evidence supports lactation to be protective against nutritive and immune stressors, but the benefits of lactation in response to psychological stressors need further investigation.

Maternal immune activation accelerates puberty initiation and alters mechanical allodynia in male and female C57BL6/J mice.

The mechanisms that link maternal immune activation (MIA) with the onset of neurodevelopmental disorders remain largely unclear. Accelerated puberty is also associated with a heightened risk for psychopathology in later life, but there is a dearth of evidence on the impacts of maternal infection on pubertal timing. We examined the effects of MIA on reproductive development, mechanical allodynia, and sensorimotor gating in juvenile, adolescent, and adult male and female mice. Moreover, we investigated hypothalamic neural markers associated with the reproductive and stress axes. Finally, we tested the mitigating effects of environmental enrichment (EE), which has clinical relevancy in human rehabilitation settings. Our results show that administration of polyinosinic-polycytidylic acid (poly(I:C)) on gestational day 12.5 led to early preputial separation, vaginal openings, and age of first estrus in offspring. MIA exposure altered pain sensitivity across development and modestly altered prepulse inhibition. The downregulation of Nr3c1 and Oprk mRNA in the hypothalamus of juvenile mice suggests that MIA's effects may be mediated through disruption of hypothalamic-pituitary-adrenal axis activity. In contrast, life-long housing with EE rescued many of these MIA-induced consequences. Overall, our findings suggest that accelerated puberty may be associated with the deleterious effects of infection during pregnancy and the onset of psychopathology.

Poly (I:C)-induced maternal immune activation modifies ventral hippocampal regulation of stress reactivity: prevention by environmental enrichment.

Environmental enrichment (EE) has been successfully implemented in human rehabilitation settings. However, the mechanisms underlying its success are not understood. Incorporating components of EE protocols into our animal models allows for the exploration of these mechanisms and their role in mitigation. Using a mouse model of maternal immune activation (MIA), the present study explored disruptions in social behavior and associated hypothalamic pituitary adrenal (HPA) axis functioning, and whether a supportive environment could prevent these effects. We show that prenatal immune activation of toll-like receptor 3, by the viral mimetic polyinosinic-polycytidylic acid (poly(I:C)), led to disrupted maternal care in that dams built poorer quality nests, an effect corrected by EE housing. Standard housed male and female MIA mice engaged in higher rates of repetitive rearing and had lower levels of social interaction, alongside sex-specific expression of several ventral hippocampal neural stress markers. Moreover, MIA males had delayed recovery of plasma corticosterone in response to a novel social encounter. Enrichment housing, likely mediated by improved maternal care, protected against these MIA-induced effects. We also evaluated c-Fos immunoreactivity associated with the novel social experience and found MIA to decrease neural activation in the dentate gyrus. Activation in the hypothalamus was blunted in EE housed animals, suggesting that the putative circuits modulating social behaviors may be different between standard and complex housing environments. These data demonstrate that augmentation of the environment supports parental care and offspring safety/security, which can offset effects of early health adversity by buffering HPA axis dysregulation. Our findings provide further evidence for the viability of EE interventions in maternal and pediatric settings.

Interactive effects of compounding multidimensional stressors on maternal and male and female rat offspring outcomes.

Exposure to adverse childhood experiences (ACEs) is a risk factor for the development of psychiatric disorders in addition to cardiovascular associated diseases. This risk is elevated when the cumulative burden of ACEs is increased. Laboratory animals can be used to model the changes (as well as the underlying mechanisms) that result in response to adverse events. In this study, using male and female Sprague Dawley rats, we examined the impact of increasing stress burden, utilizing both two adverse early life experiences (parental/offspring high fat diet + limited bedding exposure) and three adverse early life experiences (parental/offspring high fat diet + limited bedding exposure + neonatal inflammation), on maternal care quality and offspring behavior. Additionally, we measured hormones and hippocampal gene expression related to stress. We found that the adverse perinatal environment led to a compensatory increase in maternal care. Moreover, these dams had reduced maternal expression of oxytocin receptor, compared to standard housed dams, in response to acute stress on postnatal day (P)22. In offspring, the two-hit and three-hit models resulted in a hyperlocomotor phenotype and increased body weights. Plasma leptin and hippocampal gene expression of corticotropin releasing hormone (Chrh)1 and Crhr2 were elevated (males) while expression of oxytocin was reduced (females) following acute stress. On some measures (e.g., hyperlocomotion, leptin), the magnitude of change was lower in the three-hit compared to the two-hit model. This suggests that multiple early adverse events can have interactive, and often unpredictable, impacts, highlighting the importance of modeling complex interactions amongst stressors during development.

Environmental influences on placental programming and offspring outcomes following maternal immune activation.

Adverse experiences during pregnancy induce placental programming, affecting the fetus and its developmental trajectory. However, the influence of 'positive' maternal experiences on the placenta and fetus remain unclear. In animal models of early life stress, environmental enrichment (EE) has ameliorated and even prevented associated impairments in brain and behavior. Here, using a maternal immune activation (MIA) model in rats, we test whether EE attenuates maternal, placental and/or fetal responses to an inflammatory challenge, thereby offering a mechanism by which fetal programming may be prevented. Moreover, we evaluate life-long EE exposure on offspring development and examine a constellation of genes and epigenetic writers that may protect against MIA challenges. In our model, maternal plasma corticosterone and interleukin-1ß were elevated 3 h after MIA, validating the maternal inflammatory response. Evidence for developmental programming was demonstrated by a simultaneous decrease in the placental enzymes Hsd11b2 and Hsd11b2/Hsd11b1, suggesting disturbances in glucocorticoid metabolism. Reductions of Hsd11b2 in response to challenge is thought to result in excess glucocorticoid exposure to the fetus and altered glucocorticoid receptor expression, increasing susceptibility to behavioral impairments later in life. The placental, but not maternal, glucocorticoid implications of MIA were attenuated by EE. There were also sustained changes in epigenetic writers in both placenta and fetal brain as a consequence of environmental experience and sex. Following MIA, both male and female juvenile animals were impaired in social discrimination ability. Life-long EE mitigated these impairments, in addition to the sex specific MIA associated disruptions in central Fkbp5 and Oprm1. These data provide the first evidence that EE protects placental functioning during stressor exposure, underscoring the importance of addressing maternal health and well-being throughout pregnancy. Future work must evaluate critical periods of EE use to determine if postnatal EE experience is necessary, or if prenatal exposure alone is sufficient to confer protection.

Access to a high resource environment protects against accelerated maturation following early life stress: A translational animal model of high, medium and low security settings.

Early life exposure to a low security setting, characterized by a scarcity of resources and limited food access, increases the risk for psychiatric illness and metabolic dysfunction. We utilized a translational rat model to mimic a low security environment and determined how this manipulation affected offspring behavior, metabolism, and puberty. Because food insecurity in humans is associated with reduced access to healthy food options the "low security" rat manipulation combined a Western diet with exposure to a limited bedding and nesting manipulation (WD-LB). In this setting, dams were provided with limited nesting materials during the pups' early life (P2-P10). This manipulation was contrasted with standard rodent caging (SD) and environmental enrichment (EE), to model "medium security" and "high security" environments, respectively. To determine if transitioning from a low to high security environment improved outcomes, some juvenile WD-LB offspring were exposed to EE. Maternal care was impacted by these environments such that EE dams engaged in high quality care when on the nest, but spent less time on the nest than SD dams. Although WD-LB dams excessively chased their tails, they were very attentive to their pups, perhaps to compensate for limited resources. Offspring exposed to WD-LB only displayed subtle changes in behavior. However, WD-LB exposure resulted in significant metabolic dysfunction characterized by increased body weight, precocious puberty and alterations in the hypothalamic kisspeptin system. These negative effects of WD-LB on puberty and weight regulation were mitigated by EE exposure. Collectively, these studies suggest that both compensatory maternal care and juvenile enrichment can reduce the impact of a low security environment. Moreover, they highlight how utilizing diverse models of resource (in)stability can reveal mechanisms that confer vulnerability and resilience to early life stress.

Resilience priming: Translational models for understanding resiliency and adaptation to early life adversity.

Despite the increasing attention to early life adversity and its long-term consequences on health, behavior, and the etiology of neurodevelopmental disorders, our understanding of the adaptations and interventions that promote resiliency and rescue against such insults are underexplored. Specifically, investigations of the perinatal period often focus on negative events/outcomes. In contrast, positive experiences (i.e. enrichment/parental care//healthy nutrition) favorably influence development of the nervous and endocrine systems. Moreover, some stressors result in adaptations and demonstrations of later-life resiliency. This review explores the underlying mechanisms of neuroplasticity that follow some of these early life experiences and translates them into ideas for interventions in pediatric settings. The emerging role of the gut microbiome in mediating stress susceptibility is also discussed. Since many negative outcomes of early experiences are known, it is time to identify mechanisms and mediators that promote resiliency against them. These range from enrichment, quality parental care, dietary interventions and those that target the gut microbiota.

Between mind and heart: Sex-based cognitive bias in cardiovascular disease treatment.

Given that both men and women experience cardiovascular disease (CVD), a common misconception is that they have similar risk factors and clinical presentation, receive comparable treatment, and have equivalent clinical outcomes; in reality differences are observed between men and women for each of these endpoints. Moreover, these differences occur as a function of both gender and sex. A review of the literature reveals widespread bias in the selection of research subjects based on these factors, in addition to implicit patient and provider biases that impede the access of women to recommended primary and secondary CVD management. In this perspective, we identify strategies to eliminate such biases and improve women's access to CVD treatments to ensure their care is consistent with current guidelines.

Mechanical allodynia corresponds to Oprm1 downregulation within the descending pain network of male and female rats exposed to neonatal immune challenge.

Exposure to painful procedures and/or stressors during the early neonatal period can reprogram the underlying neurocircuitry involved in nociception and neuropathic pain perception. The reprogramming of these systems can result in an enduring elevation in sensitivity towards mechanical and thermal stimuli. Recent evidence suggests that exposure to mild inflammatory mediators during the neonatal period can induce similar pain responses in both adolescent and adult rats. Therefore, we sought to profile changes in the expression of several genes across brain areas involved in the active modulation of nociception and neuropathic pain using a well-recognized model of neonatal inflammation. In the present study male and female Sprague-Dawley rats were administered either the inflammatory endotoxin lipopolysaccharide (LPS; 0.05mg/kg, i.p.) or saline (equivolume) on postnatal days (PND) 3 and 5. During adolescence, hind paw mechanical withdrawal thresholds were evaluated using an electronic von Frey anesthesiometer. Animals challenged neonatally with LPS (nLPS) had increased pain sensitivity on this measure which was associated with decreased Oprm1 expression in the prefrontal cortex (PFC) and periaqueductal gray (PAG) of both male and female rats. Although a 'second hit' with LPS in adolescence (aLPS) did not confer protection or reveal additional vulnerabilities, aLPS given to animals treated neonatally with saline was associated with increased pain sensitivity, but only in females. Interestingly, adolescent inflammatory challenge decreased Hcrt2 mRNA in the PAG and elevated Trpv1 in the PAG and PFC of both sexes. There was no effect of inflammatory treatment on either anxiety or depressive-like behavior suggesting that affective functioning did not account for differences in mechanical pain sensitivity. Finally, a preliminary investigation demonstrated that administration of a broad spectrum antibiotic cocktail attenuated the mechanical sensitivity that followed nLPS. Together, these data extend upon evidence that inflammation imparts long term changes in quality of life and pain responses via interference within the descending pain network. Moreover, they highlight a potential window of opportunity to target the microbiota-gut-brain axis and reverse pain processing disturbances following perinatal inflammation.

Microglia-dependent alteration of glutamatergic synaptic transmission and plasticity in the hippocampus during peripheral inflammation.

Peripheral inflammatory diseases are often associated with behavioral comorbidities including anxiety, depression, and cognitive dysfunction, but the mechanism for these is not well understood. Changes in the neuronal and synaptic functions associated with neuroinflammation may underlie these behavioral abnormalities. We have used a model of colonic inflammation induced by 2,4,6-trinitrobenzenesulfonic acid in Sprague Dawley rats to identify inflammation-induced changes in hippocampal synaptic transmission. Hippocampal slices obtained 4 d after the induction of inflammation revealed enhanced Schaffer collateral-induced excitatory field potentials in CA1 stratum radiatum. This was associated with larger-amplitude mEPSCs, but unchanged mEPSC frequencies and paired-pulse ratios, suggesting altered postsynaptic effects. Both AMPA- and NMDA-mediated synaptic currents were enhanced, and analysis of AMPA-mediated currents revealed increased contributions of GluR2-lacking receptors. In keeping with this, both transcripts and protein levels of the GluR2 subunit were reduced in hippocampus. Both long-term potentiation (LTP) and depression (LTD) were significantly reduced in hippocampal slices taken from inflamed animals. Chronic administration of the microglial/macrophage activation inhibitor minocycline to the inflamed animals both lowered the level of the cytokine tumor necrosis factor α in the hippocampus and completely abolished the effect of peripheral inflammation on the field potentials and synaptic plasticity (LTP and LTD). Our results reveal profound synaptic changes caused by a mirror microglia-mediated inflammatory response in hippocampus during peripheral organ inflammation. These synaptic changes may underlie the behavioral comorbidities seen in patients.

Environmental enrichment rescues the effects of early life inflammation on markers of synaptic transmission and plasticity.

Environmental enrichment (EE) has been successful at rescuing the brain from a variety of early-life psychogenic stressors. However, its ability to reverse the behavioral and neural alterations induced by a prenatal maternal infection model of schizophrenia is less clear. Moreover, the specific interactions between the components (i.e. social enhancement, novelty, physical activity) of EE that lead to its success as a supportive intervention have not been adequately identified. In the current study, standard housed female Sprague-Dawley rats were administered either the inflammatory endotoxin lipopolysaccharide (LPS; 100µg/kg) or pyrogen-free saline (equivolume) on gestational day 15. On postnatal day 50, offspring were randomized into one of three conditions: EE (group housed in a large multi-level cage with novel toys, tubes and ramps), Colony Nesting (CN; socially-housed in a larger style cage), or Standard Care (SC; pair-housed in standard cages). Six weeks later we scored social engagement and performance in the object-in-place task. Afterwards hippocampus and prefrontal cortex (n=7-9) were collected and evaluated for excitatory amino acid transporter (EAAT) 1-3, brain-derived neurotrophic factor (BDNF), and neurotrophic tyrosine kinase, receptor type 2 (TrkB) gene expression (normalized to GAPDH) using qPCR methods. Overall, we show that gestational inflammation downregulates genes critical to synaptic transmission and plasticity, which may underlie the pathogenesis of neurodevelopmental disorders such as schizophrenia and autism. Additionally, we observed disruptions in both social engagement and spatial discrimination. Importantly, behavioral and neurophysiological effects were rescued in an experience dependent manner. Given the evidence that schizophrenia and autism may be associated with infection during pregnancy, these data have compelling implications for the prevention and reversibility of the consequences that follow immune activation in early in life.

Peripubertal antagonism of corticotropin-releasing factor receptor 1 results in sustained, sex-specific changes in behavioral plasticity and the transcriptomic profile of the amygdala.

Peripuberty is a significant period of neurodevelopment with long-lasting effects on the brain and behavior. Blocking type 1 corticotropin-releasing factor receptors (CRFR1) in neonatal and peripubertal rats attenuates detrimental effects of early-life stress on neural plasticity, behavior, and stress hormone action, long after exposure to the drug has ended. CRFR1 antagonism can also impact neural and behavioral development in the absence of stressful stimuli, suggesting sustained alterations under baseline conditions. To investigate this further, we administered a CRFR1 antagonist (CRFR1a), R121919, to young adolescent male and female rats across 4 days. Following each treatment, rats were tested for locomotion, social behavior, mechanical allodynia, or PPI of the acoustic startle reflex. Acute CRFR1 blockade immediately reduced PPI in peripubertal males, but not females. In adulthood, each assay was repeated without CRFR1a exposure to test for long-term effects of the adolescent treatment, with males continuing to experience deficits in PPI, while females displayed altered locomotion, PPI, and social behavior. The amygdala was collected to measure long-term effects on gene expression in pathways related to neural plasticity and neurodevelopmental disorders. Relative expression of cannabinoid type 1 receptors (CB1R), which mediate sensorimotor and HPA function, was also measured. In the adult amygdala, peripubertal CRFR1a induced alterations in pathways related to neural plasticity and stress in males and lower expression of CB1R protein in females. Understanding how acute exposure to neuropharmacological agents can have sustained impacts on brain and behavior, in the absence of further exposures, has important clinical implications for adolescent psychiatric treatment protocols.

Maternal immune activation alters placental histone-3 lysine-9 tri-methylation, offspring sensorimotor processing, and hypothalamic transposable element expression in a sex-specific manner.

Animal models of maternal immune activation (MIA) are central to identifying the biological mechanisms that underly the association between prenatal infection and neuropsychiatric disorder susceptibility. Many studies, however, have limited their scope to protein coding genes and their role in mediating this inherent risk, while much less attention has been directed towards exploring the roles of the epigenome and transposable elements (TEs). In Experiment 1, we demonstrate the ability of MIA to alter the chromatin landscape of the placenta. We induced MIA by injecting 200 µg/kg (i.p.) of lipopolysaccharide (LPS) on gestational day 15 in Sprague-Dawley rats. We found a sex-specific rearrangement of heterochromatin 24-h after exposure to MIA, as evidenced by an increase in histone-3 lysine-9 trimethylation (H3K9me3). In Experiment 2, MIA was associated with long-term sensorimotor processing deficits as indicated by reduced prepulse inhibition (PPI) of the acoustic startle reflex in adult male and female offspring and an increased mechanical allodynia threshold in males. Analyses of gene expression within the hypothalamus-chosen for its involvement in the sex-specific pathogenesis of schizophrenia and the stress response-revealed significantly higher levels of the stress-sensitive genes Gr and Fkbp5. Deleterious TE expression is often a hallmark of neuropsychiatric disease and we found sex-specific increases in the expression of several TEs including IAP, B2 SINE, and LINE-1 ORF1. The data from this study warrant the future consideration of chromatin stability and TEs as part of the mechanism that drives MIA-associated changes in the brain and behavior.

Sex differences in offspring risk and resilience following 11ß-hydroxylase antagonism in a rodent model of maternal immune activation.

Maternal immune activation (MIA) puts offspring at greater risk for neurodevelopmental disorders associated with impaired social behavior. While it is known that immune signaling through maternal, placental, and fetal compartments contributes to these phenotypical changes, it is unknown to what extent the stress response to illness is involved and how it can be harnessed for potential interventions. To this end, on gestational day 15, pregnant rat dams were administered the bacterial mimetic lipopolysaccharide (LPS; to induce MIA) alongside metyrapone, a clinically available 11ß-hydroxylase (11ßHSD) inhibitor used to treat hypercortisolism in pregnant, lactating, and neonatal populations. Maternal, placental, and fetal brain levels of corticosterone and placental 11ßHSD enzymes type 1 and 2 were measured 3-hrs post treatment. Offspring social behaviors were evaluated across critical phases of development. MIA was associated with increased maternal, placental, and fetal brain corticosterone concentrations that were diminished with metyrapone exposure. Metyrapone protected against reductions in placental 11ßHSD2 in males only, suggesting that less corticosterone was inactivated in female placentas. Behaviorally, metyrapone-exposure attenuated MIA-induced social disruptions in juvenile, adolescent, and adult males, while females were unaffected or performed worse. Metyrapone-exposure reversed MIA-induced transcriptional changes in monoamine-, glutamate-, and GABA-related genes in adult male ventral hippocampus, but not in females. Taken together, these findings illustrate that MIA-induced HPA responses act alongside the immune system to produce behavioral deficits. As a clinically available drug, the sex-specific benefits and constraints of metyrapone should be investigated further as a potential means of reducing neurodevelopmental risks due to gestational MIA.

Developmental Manipulation-Induced Changes in Cognitive Functioning.

Schizophrenia is a complex neurodevelopmental disorder with as-yet no identified cause. The use of animals has been critical to teasing apart the potential individual and intersecting roles of genetic and environmental risk factors in the development of schizophrenia. One way to recreate in animals the cognitive impairments seen in people with schizophrenia is to disrupt the prenatal or neonatal environment of laboratory rodent offspring. This approach can result in congruent perturbations in brain physiology, learning, memory, attention, and sensorimotor domains. Experimental designs utilizing such animal models have led to a greatly improved understanding of the biological mechanisms that could underlie the etiology and symptomology of schizophrenia, although there is still more to be discovered. The implementation of the Research and Domain Criterion (RDoC) has been critical in taking a more comprehensive approach to determining neural mechanisms underlying abnormal behavior in people with schizophrenia through its transdiagnostic approach toward targeting mechanisms rather than focusing on symptoms. Here, we describe several neurodevelopmental animal models of schizophrenia using an RDoC perspective approach. The implementation of animal models, combined with an RDoC framework, will bolster schizophrenia research leading to more targeted and likely effective therapeutic interventions resulting in better patient outcomes.

What's wrong with my experiment?: The impact of hidden variables on neuropsychopharmacology research.

The field of neuropsychopharmacology relies on behavioral assays to quantify behavioral processes related to mental illness and substance use disorders. Although these assays have been highly informative, sometimes laboratories have unpublished datasets from experiments that "didn't work". Often this is because expected outcomes were not observed in positive or negative control groups. While this can be due to experimenter error, an important alternative is that under-appreciated environmental factors can have a major impact on results. "Hidden variables" such as circadian cycles, husbandry, and social environments are often omitted in methods sections, even though there is a strong body of literature documenting their impact on physiological and behavioral outcomes. Applying this knowledge in a more critical manner could provide behavioral neuroscientists with tools to develop better testing methods, improve the external validity of behavioral techniques, and make better comparisons of experimental data across institutions. Here we review the potential impact of "hidden variables" that are commonly overlooked such as light-dark cycles, transport stress, cage ventilation, and social housing structure. While some of these conditions may not be under direct control of investigators, it does not diminish the potential impact of these variables on experimental results. We provide recommendations to investigators on which variables to report in publications and how to address "hidden variables" that impact their experimental results.