Hair Cortisol Concentrations in Opioid-Exposed versus Nonexposed Mother-Infant Dyads.

OBJECTIVE: To pilot measurement of hair cortisol concentration (HCC) in pregnant women with opioid use disorder and their infants over time and study the potential utility of hair cortisol as a biomarker of chronic stress in this population. STUDY DESIGN: In this pilot prospective cohort study of mother-infant dyads with and without prenatal opioid exposure, we obtained mother-infant HCCs at delivery and again within 1 to 3 months' postpartum. HCCs were compared between the opioid and control groups and between the two time points. RESULTS: There were no significant differences between opioid and control group maternal or infant HCCs at either time point. However, within the opioid-exposed group, there was a significant increase in infant HCCs across the two time points. CONCLUSION: This pilot study describes our experience with the measurement of HCCs in opioid-exposed mother-infant dyads. KEY POINTS: · Maternal stress impacts fetal and child health.. · Many stressors in pregnant women with opioid use disorder.. · Hair cortisol may be a useful stress biomarker..

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.

Maternal Immune Activation and Enriched Environments Impact B2 SINE Expression in Stress Sensitive Brain Regions of Rodent Offspring.

Early life stress (ELS) can have wide-spread neurodevelopmental effects with support accumulating for the idea that genomic mechanisms may induce lasting physiological and behavioral changes following stress exposure. Previous work found that a sub-family of transposable elements, SINEs, are repressed epigenetically after acute stress. This gives support to the concept that the mammalian genome may be regulating retrotransposon RNA expression allowing for adaptation in response to environmental challenges, such as maternal immune activation (MIA). Transposon (TE) RNAs are now thought to work at the epigenetic level and to have an adaptive response to environmental stressors. Abnormal expression of TEs has been linked to neuropsychiatric disorders like schizophrenia, which is also linked to maternal immune activation. Environmental enrichment (EE), a clinically utilized intervention, is understood to protect the brain, enhance cognitive performance, and attenuate responses to stress. This study examines the effects of MIA on offspring B2 SINE expression and further, the impact that EE, experienced throughout gestation and early life, may have in conjunction with MIA during development. Utilizing RT-PCR to quantify the expression of B2 SINE RNA in the juvenile brain of MIA exposed rat offspring, we found dysregulation of B2 SINE expression associated with MIA in the prefrontal cortex. For offspring experiencing EE, the prefrontal cortex exhibited an attenuation of the MIA response observed in standard housed animals. Here, the adaptive nature of B2 is observed and thought to be aiding in the animal's adaptation to stress. The present changes indicate a wide-spread stress-response system adaptation that impacts not only changes at the genomic level but potentially observable behavioral impacts throughout the lifespan, with possible translational relevance to psychotic disorders.

B2 SINE RNA as a novel regulator of glucocorticoid receptor transcriptional activity.

Glucocorticoids are a key component to the cellular response to stress. Glucocorticoids act via glucocorticoid receptors found ubiquitously in the brain and body. Glucocorticoid receptors can bind to response elements throughout the genome to elicit changes in transcription, an adaptation observed at the cellular level. Yet, the transcriptional changes as a consequence of glucocorticoid receptor activation are variable across brain regions, stress conditions and recurrent bouts of glucocorticoid exposure. Here we describe a non-coding RNA, B2 SINE, which is regulated by glucocorticoids and can in turn regulate glucocorticoid receptor transcriptional activity. We show that activated glucocorticoid receptors interact directly with B2 SINE RNA via a decoy response element contained within the transcript sequence and alter receptor binding to response elements in the genome and, subsequently, changes in loci expression.

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.

Regulatory Effects of Maternal Immune Activation and Environmental Enrichment on Glucocorticoid Receptor and FKBP5 Expression in Stress-sensitive Regions of the Offspring Brain.

A mother's exposure to immune challenge during pregnancy is well known to be a detrimental factor to the development of the offspring's brain and an impetus for neuropsychiatric disorders. Previous studies have shown that these adverse events can dysregulate the stress response machinery. Two crucial components of the stress axis considered to be affected have been targets in these studies: the glucocorticoid receptor (GR), and FKBP5 which regulates GR activity. The implementation of interventions such as Environmental Enrichment (EE) have shown positive results in protecting the brain against the consequences associated with gestational insults. In light of this, we investigated the transcriptional regulation of GR and FKBP5 from six stress-sensitive brain regions of the offspring using a rat model of maternal immune activation (MIA). Furthermore, we analyzed the effect of an enriched environment on their expression. We found an increase in FKBP5 in MIA rats in five brain regions. RT-qPCR analysis of MIA's effect on GR yielded insignificant results. However, we found that EE increased GR expression in the medial preoptic area which could be indicative of a positive regulation by EE. This study provides evidence of the impact of both gestational insult and EE on the regulation of stress responsive genes in the developing brain.

The Role of Transposable Elements in Sexual Development.

Up to 50% of most mammalian genomes are made up of transposable elements (TEs) that have the potential to mobilize around the genome. Despite this prevalence, research on TEs is only beginning to gain traction within the field of neuroscience. While TEs have long been regarded as "junk" or parasitic DNA, it has become evident that they are adaptive DNA and RNA regulatory elements. In addition to their vital role in normal development, TEs can also interact with steroid receptors, which are key elements to sexual development. In this review, we provide an overview of the involvement of TEs in processes related to sexual development- from TE activity in the germline to TE accumulation in sex chromosomes. Moreover, we highlight sex differences in TE activity and their regulation of genes related to sexual development. Finally, we speculate on the epigenetic mechanisms that may govern TEs' role in sexual development. In this context, we emphasize the need to further the understanding of sexual development through the lens of TEs including in a variety of organs at different developmental stages, their molecular networks, and evolution.

Bridging the Gap Between Environmental Adversity and Neuropsychiatric Disorders: The Role of Transposable Elements.

Long regarded as "junk DNA," transposable elements (TEs) have recently garnered much attention for their role in promoting genetic diversity and plasticity. While many processes involved in mammalian development require TE activity, deleterious TE insertions are a hallmark of several psychiatric disorders. Moreover, stressful events including exposure to gestational infection and trauma, are major risk factors for developing psychiatric illnesses. Here, we will provide evidence demonstrating the intersection of stressful events, atypical TE expression, and their epigenetic regulation, which may explain how neuropsychiatric phenotypes manifest. In this way, TEs may be the "bridge" between environmental perturbations and psychopathology.

Pipette Olympics: An Engaging Exercise for Undergraduate Laboratory Training.

Pipetting is an important technique used in almost every molecular neuroscience method including but not limited to, PCR, reverse transcription, immunohistochemistry, chromatin immunoprecipitation, and cell culture. The COVID-19 pandemic has robbed the undergraduate population of time to practice in person laboratory techniques. In response, we have devised a standardized, quick, and fun way to instruct students on the fundamentals of pipetting, serial dilutions, and basic statistical analysis. Here, we offer a standardized protocol for instructors to use to teach undergraduates valuable skills while providing friendly competition. We also offer an example of an undergraduate performing the steps of this protocol with example results and the results from three separate undergrads' first two attempts. This exercise provides laboratories with a method to reintroduce undergraduates to lab basics while standardizing the training thereby saving time lost to the pandemic.

Bag-1 mediates glucocorticoid receptor trafficking to mitochondria after corticosterone stimulation: Potential role in regulating affective resilience.

Molecular abnormalities within the Glucocorticoid Receptor (GR) stress signaling pathway involved in dysfunction of mitochondria and confer vulnerability to stress-related psychiatric disorders. Bcl-2 associated athanogene (Bag-1) is a target for the actions of mood stabilizers. Bag-1 interacts with GR, thereby regulating glucocorticoid function. In this study, we investigate the potential role of Bag-1 in regulating GR translocation into mitochondria. Corticosterone (CORT) treatment significantly enhanced Bag-1/GR complex formation and GR mitochondrial translocation in cultured rat cortical neurons after treatment for 30 min and 24 hr. By contrast, after stimulation with CORT for 3 days, localization of the Bag-1/GR complex and mitochondrial GR were reduced. Similar results were obtained in mice, in which administrated CORT in drinking water for 21 days significantly impaired the GR levels in the mitochondria, while Bag-1 over-expression rescued this reduction. Furthermore, chronic CORT exposure led to anhedonia-like and depression-like behaviors in the sucrose-consumption test and forced swimming test, and these behaviors were rescued by Bag-1 over-expression. These results suggest that Bag-1 mediates GR trafficking to mitochondria and regulates affective resilience in response to a CORT increase and provide potential insight into the mechanisms by which Bag-1 and GR could contribute to the physiology and pathogenesis of psychiatric disorders in response to the change of stress hormone.

Early experience alters developmental trajectory of central oxytocin systems involved in hypothalamic-pituitary-adrenal axis regulation in Long-Evans rats.

Oxytocin is important for postnatal developmental experiences for mothers, infants, and transactions between them. Oxytocin is also implicated in adult affiliative behaviors, including social buffering of stress. There is evidence for connections between early life experience and adult oxytocin system functioning, but effects of early experience on behavioral, endocrine, and neurophysiological outcomes related to adult social buffering are not well explored. We use a limited bedding and nesting (LBN) material paradigm as an environmental disruption of early experiences and assessed central oxytocin systems in brain regions related to hypothalamic-pituitary-adrenal (HPA) axis regulation (paraventricular nucleus of the hypothalamus, amygdala, hippocampus). We also assessed developmentally-appropriate social behaviors and HPA reactivity during social buffering testing in adulthood. LBN litters had larger huddles and more pups visible compared to control litters during the first two weeks of life. LBN also altered the developmental trajectory of oxytocin-expressing cells and oxytocin receptor cells, with increases in oxytocin receptor cells at P15 in LBN pups. By adulthood, LBN females had more and LBN males had fewer oxytocin and oxytocin receptor cells in these areas compared to sex-matched controls. Adult LBN females, but not LBN males, had behavioral changes during social interaction and social buffering testing. The sex-specific effects of early experience on central oxytocin systems and social behavior may contribute to female resilience to early life adversity.

Stress, Adaptation, and the Deep Genome: Why Transposons Matter.

Stress is a common, if often unpredictable life event. It can be defined from an evolutionary perspective as a force an organism perceives it must adapt to. Thus stress is a useful tool to study adaptation and the adaptive capacity of organisms. The deep genome, long neglected as a pile of "junk" has emerged as a source of regulatory DNA and RNA as well as a potential stockpile of adaptive capacity at the organismal and species levels. Recent work on the regulation of transposable elements (TEs), the principle constituents of the deep genome, by stress has shown that these elements are responsive to host stress and other environmental cues. Further, we have shown that some are likely directly regulated by the glucocorticoid receptor (GR), one of the two major vertebrate stress steroid receptors in a fashion that appears adaptive. On the basis of this and other emerging evidence I argue that the deep genome may represent an adaptive toolkit for organisms to respond to their environments at both individual and evolutionary scales. This argues that genomes may be adapted for what Waddington called "trait adaptability" rather than being purely passive objects of natural selection and single nucleotide level mutation.

Maternal hair cortisol levels as a novel predictor of neonatal abstinence syndrome severity: A pilot feasibility study.

Neonatal abstinence syndrome (NAS) after in-utero opioid exposure remains a poorly understood condition with multiple factors contributing to severity. Exposure to maternal stress may be one contributing factor. Hair cortisol measurement represents a novel technique for assessing prenatal stress. In this pilot study, the association between maternal hair cortisol levels and NAS severity was examined in 70 postpartum women with opioid use disorder within 72 hr of delivery. Infants were monitored for NAS and treated according to institutional protocol. Forty-four (63%) of the infants were pharmacologically treated for NAS, with a mean length of hospital stay (LOS) for all infants of 14.2 (SD 9.0) days. The mean cortisol level in the mothers was 131.8 pg/mg (SD 124.7). In bivariate analysis, higher maternal hair cortisol levels were associated with shorter infant LOS (R = -.26, p = .03) and fewer infant opioid treatment days (R = -.28, p = .02). Results were no longer statistically significant in regression models after adjusting for maternal opioid and smoking. In conclusion, we demonstrated the feasibility of hair cortisol assaying within the first few days after delivery in mothers with opioid use disorder as a novel marker for NAS. The findings suggest that maternal stress may impact the severity of infant opioid withdrawal.

In search of optimal resilience ratios: Differential influences of neurobehavioral factors contributing to stress-resilience spectra.

The ability to adapt to stressful circumstances, known as emotional resilience, is a key factor in the maintenance of mental health. Several individual biomarkers of the stress response (e.g., corticosterone) that influence an animal's position along the continuum that ranges from adaptive allostasis to maladaptive allostatic load have been identified. Extending beyond specific biomarkers of stress responses, however, it is also important to consider stress-related responses relative to other relevant responses for a thorough understanding of the underpinnings of adaptive allostasis. In this review, behavioral, neurobiological, developmental and genomic variables are considered in the context of emotional resilience [e.g., explore/exploit behavioral tendencies; DHEA/CORT ratios and relative proportions of protein-coding/nonprotein-coding (transposable) genomic elements]. As complex and multifaceted relationships between pertinent allostasis biomediators are identified, translational applications for optimal resilience are more likely to emerge as effective therapeutic strategies.

Epigenetic Mechanisms of the Glucocorticoid Receptor.

The glucocorticoid receptor (GR) has been shown to be important for mediating cellular responses to stress and circulating glucocorticoids. Ligand-dependent transcriptional changes induced by GR are observed across numerous tissues. However, the mechanisms by which GR achieves cell and tissue-specific effects are less clear. Epigenetic mechanisms have been proposed to explain some of these differences as well as some of the lasting, even transgenerational, effects of stress and glucocorticoid action. GR functions in tandem with epigenetic cellular machinery to coordinate transcription and shape chromatin structure. Here, we describe GR interactions with these effectors and how GR acts to reshape the epigenetic landscape in response to the environment.

Chromatin Immunoprecipitation Techniques in Neuropsychiatric Research.

Neuropsychiatric disorders are highly prevalent (e.g., affecting children 2-8 years old at a rate of 14%). Many of these disorders are highly heritable such as major depressive disorder and schizophrenia. Despite this, genome-wide association has failed to identify gene(s) significantly associated with diagnostic status suggesting a strong role for environmental factors and the epigenome. From a molecular standpoint, the study of DNA-protein interactions yields fruitful information regarding the regulation of cellular processes above the level of the nucleotide sequence. Understanding chromatin dynamics may continue to explain individual variation to environmental perturbation and subsequent behavioral response. Chromatin immunoprecipitation (ChIP) techniques have allowed for probing of epigenetic effectors at specific regions of the genome. The following article reviews the current techniques and considerations when incorporation ChIP into neuropsychiatric models.

Seeing a Face in a Crowd of Emotional Voices: Changes in Perception and Cortisol in Response to Emotional Information across the Senses.

One source of information we glean from everyday experience, which guides social interaction, is assessing the emotional state of others. Emotional state can be expressed through several modalities: body posture or movements, body odor, touch, facial expression, or the intonation in a voice. Much research has examined emotional processing within one sensory modality or the transfer of emotional processing from one modality to another. Yet, less is known regarding interactions across different modalities when perceiving emotions, despite our common experience of seeing emotion in a face while hearing the corresponding emotion in a voice. Our study examined if visual and auditory emotions of matched valence (congruent) conferred stronger perceptual and physiological effects compared to visual and auditory emotions of unmatched valence (incongruent). We quantified how exposure to emotional faces and/or voices altered perception using psychophysics and how it altered a physiological proxy for stress or arousal using salivary cortisol. While we found no significant advantage of congruent over incongruent emotions, we found that changes in cortisol were associated with perceptual changes. Following exposure to negative emotional content, larger decreases in cortisol, indicative of less stress, correlated with more positive perceptual after-effects, indicative of stronger biases to see neutral faces as happier.

Early life exposures, neurodevelopmental disorders, and transposable elements.

Transposable elements make up a much larger portion of the genome than protein-coding genes, yet we know relatively little about their function in the human genome. However, we are beginning to more fully understand their role in brain development, neuroinflammation, and adaptation to environmental insults such as stress. For instance, glucocorticoid receptor activation regulates transposable elements in the brain following acute stress. Early life is a period of substantial brain development during which transposable elements play a role. Environmental exposures and experiences during early life that promote abnormal regulation of transposable elements may lead to a cascade of events that ultimately increase susceptibility to disorders later in life. Recent attention to transposable elements in psychiatric illness has begun to clarify associations indicative of dysregulation of different classes of transposable elements in stress-related and neurodevelopmental illness. Though individual susceptibility or resiliency to psychiatric illness has not been explained by traditional genetic studies, the wide inter-individual variability in transposable element composition in the human genome make TEs attractive candidates to elucidate this differential susceptibility. In this review, we discuss evidence that regulation of transposable elements in the brain are stage-specific, sensitive to environmental factors, and may be impacted by early life perturbations. We further present evidence of associations with stress-related and neurodevelopmental psychiatric illness from a developmental perspective.

Toxic stress history and hypothalamic-pituitary-adrenal axis function in a social stress task: Genetic and epigenetic factors.

Histories of early life stress (ELS) or social discrimination can reach levels of severity characterized as toxic to mental and physical health. Such toxic social stress during development has been linked to altered acute hypothalamic-pituitary-adrenal (HPA) response to social stress in adulthood. However, there are important individual differences in the size and direction of these effects. We explored developmental, genetic, epigenetic, and contextual sources of individual differences in the relationship between ELS, discrimination, and adult responses to acute social stress in a standard laboratory test. Additional measures included perceived status, social support, background activity of HPA axis, and genetic variants in aspects of the stress response system. Participants (n = 90) answered questions about historical and ongoing stress, provided a DNA sample to examine genetic polymorphisms and epigenetic marks, and underwent the Trier Social Stress Test (TSST) during which three saliva samples were collected to assess HPA function. Individuals who reported high levels of childhood adversity had a blunted salivary cortisol response to the TSST. Childhood adversity, discrimination experiences, and FKBP5 genotype were found to predict pretest cortisol levels. Following up on recent observations that the glucocorticoid receptor directly interacts with the mitochondrial genome, particularly the NADH dehydrogenase 6 (MT-ND6) gene, individuals who reported high childhood adversity were also found to have higher percent methylation across six CpG sites upstream of MT-ND6. These findings suggest multiple contributions across psychological, genetic, epigenetic, and social domains to vulnerability and resilience in hypothalamic-pituitary-adrenal axis regulation. Further study to examine how these multiple contributors affect developmental endpoints through integrated or independent pathways will be of use.