Epigenetic processes associated with neonatal spinal transection.

In early development, the spinal cord in healthy or disease states displays remarkable activity-dependent changes in plasticity, which may be in part due to the increased activity of brain derived neurotrophic factor (BDNF). Indeed, BDNF delivery has been efficacious in partially ameliorating many of the neurobiological and behavioral consequences of spinal cord injury (SCI), making elucidating the role of BDNF in the normative developing and injured spinal cord a critical research focus. Recent work in our laboratory provided evidence for aberrant global and locus-specific epigenetic changes in methylation of the Bdnf gene as a consequence of SCI. In the present study, animals underwent thoracic lesions on P1, with cervical and lumbar tissue being later collected on P7, P14, and P21. Levels of Bdnf expression and methylation (exon IX and exon IV), in addition to global methylation levels were quantified at each timepoint. Results indicated locus-specific reductions of Bdnf expression that was accompanied by a parallel increase in methylation caudal to the injury site, with animals displaying increased Bdnf expression at the P14 timepoint. Together, these findings suggest that epigenetic activity of the Bdnf gene may act as biomarker in the etiology and intervention effort efficacy following SCI.

Developmental administration of valproic acid alters DNA methylation and maternal behavior.

Exposure to adversity in early development has powerful and potentially lasting consequences on behavior. Previous work in our laboratory using female Long-Evans rats has demonstrated that exposure to early-life maltreatment manifests into alterations in dam behavior, including a perpetuation of the maltreatment phenotype. These observed behavioral changes coincide with changes in epigenetic activity in the prefrontal cortex (PFC). Further, treating dams with a chromatin modifying agent (Zebularine) normalizes methylation and maltreatment phenotypes, suggesting a link between epigenetic programming and phenotypic outcomes. Here, we sought to investigate if administration of a chromatin modifying agent concurrent with the experience of maltreatment normalizes epigenetic activity associated with maltreatment and alters behavioral trajectories. Administration of valproic acid (VPA) transiently lowered levels of global DNA methylation in the PFC, regardless of exposure to nurturing care or maltreatment. When VPA-exposed animals reached adulthood, they engaged in more adverse behaviors toward their offspring. These data provide further evidence linking epigenetic changes in the developing brain with effects on behavior.

Early Life Stress Affects Bdnf Regulation: A Role for Exercise Interventions.

Early life stress (ELS) encompasses exposure to aversive experiences during early development, such as neglect or maltreatment. Animal and human studies indicate that ELS has maladaptive effects on brain development, leaving individuals more vulnerable to developing behavioral and neuropsychiatric disorders later in life. This result occurs in part to disruptions in Brain derived neurotrophic factor (Bdnf) gene regulation, which plays a vital role in early neural programming and brain health in adulthood. A potential treatment mechanism to reverse the effects of ELS on Bdnf expression is aerobic exercise due to its neuroprotective properties and positive impact on Bdnf expression. Aerobic exercise opens the door to exciting and novel potential treatment strategies because it is a behavioral intervention readily and freely available to the public. In this review, we discuss the current literature investigating the use of exercise interventions in animal models of ELS to reverse or mitigate ELS-induced changes in Bdnf expression. We also encourage future studies to investigate sensitive periods of exercise exposure, as well as sufficient duration of exposure, on epigenetic and behavioral outcomes to help lead to standardized practices in the exercise intervention field.

Preliminary indications that the Attachment and Biobehavioral Catch-up Intervention alters DNA methylation in maltreated children.

Maltreatment during development is associated with epigenetic changes to the genome. Enhancing caregiving may mitigate these effects. Attachment and Biobehavioral Catch-Up (ABC) is an intervention that has been shown to improve parent-child relationships and a variety of biological and behavioral outcomes among children that are involved in Child Protective Services. This preliminary study, using a small sample size, explored whether children who received ABC exhibit different methylation patterns than those who received a control intervention. The participants included 23 children aged 6-21 months who were randomized to receive ABC (n = 12) or a control intervention (n = 11). While the children displayed similar methylation patterns preintervention, DNA methylation varied between the ABC and control groups at 14,828 sites postintervention. Functional pathway analyses indicated that these differences were associated with gene pathways that are involved in cell signaling, metabolism, and neuronal development. This study is one of the first to explore parenting intervention effects on children's DNA methylation at the whole genome level in infancy. These preliminary findings provide a basis for hypothesis generation in further research with larger-scale studies regarding the malleability of epigenetic states that are associated with maltreatment.

Looking back and moving forward: Evaluating and advancing translation from animal models to human studies of early life stress and DNA methylation.

Advances in epigenetic methodologies have deepened theoretical explanations of mechanisms linking early life stress (ELS) and disease outcomes and suggest promising targets for intervention. To date, however, human studies have not capitalized on the richness of diverse animal models to derive and systematically evaluate specific and testable hypotheses. To promote cross-species dialog and scientific advance, here we provide a classification scheme to systematically evaluate the match between characteristics of human and animal studies of ELS and DNA methylation. Three preclinical models were selected that are highly cited, and that differ in the nature and severity of the ELS manipulation as well as in the affected epigenetic loci (the licking and grooming, maternal separation, and caregiver maltreatment models). We evaluated the degree to which human studies matched these preclinical models with respect to the timing of ELS and of DNA methylation assessment, as well as the type of ELS, whether sex differences were explicitly examined, the tissue sampled, and the targeted loci. Results revealed <50% match (range of 8-83%) between preclinical models and human work on these variables. Immediate and longer-term suggestions to improve translational specificity are offered, with the goal of accelerating scientific advance.

Female pups receive more maltreatment from stressed dams.

The effects of exposure to developmental stress often diverge for males and females. Using the scarcity-adversity model of low nesting resources outside the home cage, our lab has discovered sex differences in both behavioral and epigenetic consequences of repeated exposure to caregiver maltreatment. For the measures we have performed to date, we have found more consequences for females. The reasons underlying this sex disparity are unknown. In the current experiment, we aimed to discern the quality of maternal care received by male and female pups in our model. As we have previously found more behavioral and epigenetic consequences in females, we hypothesized that females receive more adverse care compared to their male littermates. Our hypothesis was supported; in our maltreatment condition, we found that female pups received more adverse care than males. This sex difference in adverse care was not present in our two control conditions (cross-foster and normal maternal care). These data lend support to the notion that one reason females in our model incur more behavioral and epigenetic consequences is a result of greater mistreatment by the dam.

Evidence from clinical and animal model studies of the long-term and transgenerational impact of stress on DNA methylation.

While it is well-known that stress during development and adulthood can confer long-term neurobiological and behavioral consequences, investigators have only recently begun to assess underlying epigenetic modifications. In this review, we highlight clinical research and work from animal models that provide evidence of the impact of stressful experiences either during the perinatal period or adulthood on DNA methylation and behavior. Additionally, we explore the more controversial concept of transgenerational inheritance, including that associated with preconception stress experienced by the mother or father. Finally, we discuss challenges associated with the idea of transgenerational epigenetics and for the field of epigenetics in general.

Chronic early life stress induced by limited bedding and nesting (LBN) material in rodents: critical considerations of methodology, outcomes and translational potential.

The immediate and long-term effects of exposure to early life stress (ELS) have been documented in humans and animal models. Even relatively brief periods of stress during the first 10 days of life in rodents can impact later behavioral regulation and the vulnerability to develop adult pathologies, in particular an impairment of cognitive functions and neurogenesis, but also modified social, emotional, and conditioned fear responses. The development of preclinical models of ELS exposure allows the examination of mechanisms and testing of therapeutic approaches that are not possible in humans. Here, we describe limited bedding and nesting (LBN) procedures, with models that produce altered maternal behavior ranging from fragmentation of care to maltreatment of infants. The purpose of this paper is to discuss important issues related to the implementation of this chronic ELS procedure and to describe some of the most prominent endpoints and consequences, focusing on areas of convergence between laboratories. Effects on the hypothalamic-pituitary adrenal (HPA) axis, gut axis and metabolism are presented in addition to changes in cognitive and emotional functions. Interestingly, recent data have suggested a strong sex difference in some of the reported consequences of the LBN paradigm, with females being more resilient in general than males. As both the chronic and intermittent variants of the LBN procedure have profound consequences on the offspring with minimal external intervention from the investigator, this model is advantageous ecologically and has a large translational potential. In addition to the direct effect of ELS on neurodevelopmental outcomes, exposure to adverse early environments can also have intergenerational impacts on mental health and function in subsequent generation offspring. Thus, advancing our understanding of the effect of ELS on brain and behavioral development is of critical concern for the health and wellbeing of both the current population, and for generations to come.

Caregiver maltreatment causes altered neuronal DNA methylation in female rodents.

Negative experiences with a caregiver during infancy can result in long-term changes in brain function and behavior, but underlying mechanisms are not well understood. It is our central hypothesis that brain and behavior changes are conferred by early childhood adversity through epigenetic changes involving DNA methylation. Using a rodent model of early-life caregiver maltreatment (involving exposure to an adverse caregiving environment for postnatal days 1-7), we have previously demonstrated abnormal methylation of DNA associated with the brain-derived neurotrophic factor (Bdnf) gene in the medial prefrontal cortex (mPFC) of adult rats. The aim of the current study was to characterize Bdnf DNA methylation in specific cell populations within the mPFC. In the prefrontal cortex, there is approximately twice as many neurons as glia, and studies have recently shown differential and distinctive DNA methylation patterns in neurons versus nonneurons. Here, we extracted nuclei from the mPFC of adult animals that had experienced maltreatment and used fluorescence-activated cell sorting to isolate cell types before performing bisulfite sequencing to estimate methylation of cytosine-guanine sites. Our data indicate that early-life stress induced methylation of DNA associated with Bdnf IV in a cell-type and sex-specific manner. Specifically, females that experienced early-life maltreatment exhibited greater neuronal cytosine-guanine methylation compared to controls, while no changes were detected in Bdnf methylation in males regardless of cell type. These changes localize the specificity of our previous findings to mPFC neurons and highlight the capacity of maltreatment to cause methylation changes that are likely to have functional consequences for neuronal function.

Phenotypic outcomes in adolescence and adulthood in the scarcity-adversity model of low nesting resources outside the home cage.

Early life adversity is known to disrupt behavioral trajectories and many rodent models have been developed to characterize these stress-induced outcomes. One example is the scarcity-adversity model of low nesting resources. This model employs resource scarcity (i.e., low nesting materials) to elicit adverse caregiving conditions (including maltreatment) toward rodent neonates. Our lab utilizes a version of this model wherein caregiving exposures occur outside the home cage during the first postnatal week. The aim of this study was to determine adolescent and adult phenotypic outcomes associated with this model, including assessment of depressive- and anxiety-like behaviors and performance in different cognitive domains. Exposure to adverse caregiving had no effect on adolescent behavioral performance whereas exposure significantly impaired adult behavioral performance. Further, adult behavioral assays revealed substantial differences between sexes. Overall, data demonstrate the ability of repeated exposure to brief bouts of maltreatment outside the home cage in infancy to impact the development of several behavioral domains later in life.

Insight from animal models of environmentally driven epigenetic changes in the developing and adult brain.

The efforts of many neuroscientists are directed toward understanding the appreciable plasticity of the brain and behavior. In recent years, epigenetics has become a core of this focus as a prime mechanistic candidate for behavioral modifications. Animal models have been instrumental in advancing our understanding of environmentally driven changes to the epigenome in the developing and adult brain. This review focuses mainly on such discoveries driven by adverse environments along with their associated behavioral outcomes. While much of the evidence discussed focuses on epigenetics within the central nervous system, several peripheral studies in humans who have experienced significant adversity are also highlighted. As we continue to unravel the link between epigenetics and phenotype, discerning the complexity and specificity of epigenetic changes induced by environments is an important step toward understanding optimal development and how to prevent or ameliorate behavioral deficits bred by disruptive environments.

Changes in dam and pup behavior following repeated postnatal exposure to a predator odor (TMT): A preliminary investigation in Long-Evans rats.

The present study investigated whether repeated early postnatal exposure to the predator odor 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) alters behavioral responses to the stimulus later in life, at postnatal day (PN30). Long-Evans rat pups with their mothers were exposed for 20 min daily to TMT, water, or a noxious odor, butyric acid (BTA), during the first three weeks of life. Mothers exposed to TMT displayed more crouching and nursing behavior than those exposed to BTA, and TMT exposed pups emitted more ultrasonic vocalizations than BTA exposed pups. At PN30, rats were tested for freezing to TMT, water, or BTA. Rats exposed to TMT during the postnatal period displayed less freezing to TMT than rats exposed postnatally to water or BTA. Our data indicate that early-life experience with a predator cue has a significant impact on later fear responses to that same cue, highlighting the programming capacity of the postnatal environment on the development of behavior.

Long-term effects of early-life caregiving experiences on brain-derived neurotrophic factor histone acetylation in the adult rat mPFC.

Infant-caregiver experiences are major contributing factors to neural and behavioral development. Research indicates that epigenetic mechanisms provide a way in which infant-caregiver experiences affect gene activity and other downstream processes in the brain that influence behavioral development. Our laboratory previously demonstrated in a rodent model that exposure to maltreatment alters methylation of DNA associated with the brain-derived neurotrophic factor (bdnf) and reelin genes as well as mRNA of key epigenetic regulatory genes in the medial prefrontal cortex (mPFC). In the current study, we characterized patterns of histone acetylation at bdnf and reelin gene loci after our caregiver manipulations. Using a within-litter design (n = 8-10/group from eight litters), pups were exposed to adverse (maltreatment condition: exposure to a stressed caregiver) or nurturing (cross-foster condition: exposure to a nurturing caregiver) caregiving environments outside the home cage for 30 min daily during the first postnatal week. Remaining pups in a litter were left with the biological mother during each session (providing normal care controls). We then used chromatin immunoprecipitation (ChIP) and quantitative RT-PCR to measure histone 3 lysine 9/14 acetylation associated with bdnf promoters I and IV and the reelin promoter in the adult mPFC. Maltreated females had decreased acetylation at bdnf IV, while neither males nor females exhibited histone acetylation alterations at bdnf I or reelin. These data demonstrate the ability of maltreatment to have long-term consequences on histone acetylation in the mPFC, and provide further evidence of the epigenetic susceptibility of bdnf IV to the quality of infant-caregiver experiences.

Epigenetic pathways through which experiences become linked with biology.

This article highlights the defining principles, progress, and future directions in epigenetics research in relation to this Special Issue. Exciting studies in the fields of neuroscience, psychology, and psychiatry have provided new insights into the epigenetic factors (e.g., DNA methylation) that are responsive to environmental input and serve as biological pathways in behavioral development. Here we highlight the experimental evidence, mainly from animal models, that factors such as psychosocial stress and environmental adversity can become encoded within epigenetic factors with functional consequences for brain plasticity and behavior. We also highlight evidence that epigenetic marking of genes in one generation can have consequences for future generations (i.e., inherited), and work with humans linking epigenetics, cognitive dysfunction, and psychiatric disorder. Though epigenetics has offered more of a beginning than an answer to the centuries-old nature-nurture debate, continued research is certain to yield substantial information regarding biological determinants of central nervous system changes and behavior with relevance for the study of developmental psychopathology.

Bdnf DNA methylation modifications in the hippocampus and amygdala of male and female rats exposed to different caregiving environments outside the homecage.

We have previously shown in infant rats that brief and repeated experiences with a stressed dam outside the homecage (maltreatment) alters methylation of DNA associated with the brain-derived neurotrophic factor (bdnf) gene within the developing and adult prefrontal cortex. BDNF is a key mediator of activity-dependent processes that have a profound influence on neural development and plasticity. Here we examined whether maltreatment also alters bdnf DNA methylation in two additional regions known to be prominently affected by diverse forms of early life adversity in humans- the hippocampus and amygdala. We found significant bdnf DNA methylation modifications present within the adult hippocampus (dorsal and ventral) and amygdala (central/basolateral complex). We observed that the nature of change differed between sexes, gene locus (bdnf I vs. IV), and brain region. Furthermore, a manipulation that did not produce any obvious behavior difference in infants (brief and repeated experiences with a nurturing foster dam) also had long-term effects on methylation. These data provide further empirical support of DNA methylation modifications as biological consequences of caregiving environments.

Unlocking the epigenome: Stress and exercise induced Bdnf regulation in the prefrontal cortex.

Aversive caregiving in early life is a risk factor for aberrant brain and behavioral development. This outcome is related to epigenetic dysregulation of the brain-derived neurotrophic factor (Bdnf) gene. The Bdnf gene encodes for BDNF, a neurotrophin involved in early brain development, neural plasticity, learning, and memory. Recent work suggests that exercise may be neuroprotective in part by supporting BDNF protein and gene expression, making it an exciting target for therapeutic interventions. To our knowledge, exercise has never been studied as a therapeutic intervention in preclinical rodent models of caregiver maltreatment. To that end, the current study investigated the effect of an adult voluntary wheel running intervention on Bdnf methylation and expression in the prefrontal cortex of rats who experienced aversive caregiving in infancy. We employed a rodent model (Long Evans rats) wherein rat pups experienced intermittent caregiver-induced stress from postnatal days 1-7 and were given voluntary access to a running wheel (except in the control condition) from postnatal days 70-90 as a young adulthood treatment intervention. Our results indicate that maltreatment and exercise affect Bdnf gene methylation in an exon, CG site, and sex-specific manner. Here we add to a growing body of evidence of the ability for our experiences, including exercise, to permeate the brain. Keywords: Early life stress, Bdnf, exercise, prefrontal cortex.

Differential methylation of genes in the medial prefrontal cortex of developing and adult rats following exposure to maltreatment or nurturing care during infancy.

Quality of maternal care in infancy is an important contributing factor in the development of behavior and psychopathology. One way maternal care could affect behavioral trajectories is through environmentally induced epigenetic alterations within brain regions known to play prominent roles in cognition, emotion regulation, and stress responsivity. Whereas such research has largely focused on the hippocampus or hypothalamus, the prefrontal cortex (PFC) has only just begun to receive attention. The current study was designed to determine whether exposure to maltreatment or nurturing care is associated with differential methylation of candidate gene loci (bdnf and reelin) within the medial PFC (mPFC) of developing and adult rats. Using a within-litter design, infant male and female rats were exposed to an adverse or nurturing caregiving environment outside their home cage for 30 min per day during the first postnatal week. Additional littermates remained with their biological caregiver within the home cage during the manipulations. We observed that infant rats subjected to caregiver maltreatment emitted more audible and ultrasonic vocalizations than littermates subjected to nurturing care either within or outside of the home cage. While we found no maltreatment-induced changes in bdnf DNA methylation present in infancy, sex-specific alterations were present in the mPFC of adolescents and adults that had been exposed to maltreatment. Furthermore, while maltreated females showed differences in reelin DNA methylation that were transient, males exposed to maltreatment and both males and females exposed to nurturing care outside the home cage showed differences in reelin methylation that emerged by adulthood. Our results demonstrate the ability of infant-caregiver interactions to epigenetically mark genes known to play a prominent role in cognition and psychiatric disorders within the mPFC. Furthermore, our data indicate the remarkable complexity of alterations that can occur, with both transient and later emerging DNA methylation differences that could shape developmental trajectories and underlie gender differences in outcomes.

Epigenetic mechanisms in the development of behavior: advances, challenges, and future promises of a new field.

In the past decade, there have been exciting advances in the field of behavioral epigenetics that have provided new insights into a biological basis of neural and behavioral effects of gene-environment interactions. It is now understood that changes in the activity of genes established through epigenetic alterations occur as a consequence of exposure to environmental adversity, social stress, and traumatic experiences. DNA methylation in particular has thus emerged as a leading candidate biological pathway linking gene-environment interactions to long-term and even multigenerational trajectories in behavioral development, including the vulnerability and resilience to psychopathology. This paper discusses what we have learned from research using animal models and from studies in which the translation of these findings has been made to humans. Studies concerning the significance of DNA methylation alterations in outcomes associated with stress exposure later in life and dysfunction in the form of neuropsychiatric disorders are highlighted, and several avenues of future research are suggested that promise to advance our understanding of epigenetics both as a mechanism by which the environment can contribute to the development of psychiatric disorders and as an avenue for more effective intervention and treatment strategies.

Exercise in Adolescence Enhances Callosal White Matter Refinement in the Female Brain in a Rat Model of Fetal Alcohol Spectrum Disorders.

A total of 1 in 20 infants born annually are exposed to alcohol prenatally, which disrupts neurodevelopment and results in several disorders categorized under the umbrella term Fetal Alcohol Spectrum Disorders (FASD). Children and adolescents affected by FASD exhibit delayed maturation of cerebral white matter, which contributes to deficits in executive function, visuospatial processing, sensory integration, and interhemispheric communication. Research using animal models of FASD have uncovered that oligoglia proliferation, differentiation, and survival are vulnerable to alcohol teratogenesis in the male brain due in part to the activation of the neuroimmune system during gestation and infancy. A comprehensive investigation of prenatal alcohol exposure on white matter development in the female brain is limited. This study demonstrated that the number of mature oligodendrocytes and the production of myelin basic protein were reduced first in the female corpus callosum following alcohol exposure in a rat model of FASD. Analysis of myelin-related genes confirmed that myelination occurs earlier in the female corpus callosum compared to their counterparts, irrespective of postnatal treatment. Moreover, dysregulated oligodendrocyte number and myelin basic protein production was observed in the male and female FASD brain in adolescence. Targeted interventions that support white matter development in FASD-affected youth are nonexistent. The capacity for an adolescent exercise intervention to upregulate corpus callosum myelination was evaluated: we discovered that volunteer exercise increases the number of mature oligodendrocytes in alcohol-exposed female rats. This study provides critical evidence that oligoglia differentiation is difficult but not impossible to induce in the female FASD brain in adolescence following a behavioral intervention.

Epigenetics of neurobiology and behavior during development and adulthood.

Gene-environment interactions have long been recognized for their important role in mediating the development and functions of the central nervous system (CNS). The study of DNA methylation and histone modifications, biochemical processes collectively referred to as epigenetic mechanisms, is helping to elucidate how gene-environmental interactions alter neurobiology and behavior over the course of the lifespan. In this review, landmark and recent studies that highlight the role of epigenetic mechanisms in the sustained effects of early-life experiences on gene activity and behavioral outcome will be discussed. Likewise, studies that implicate epigenetics in CNS and behavioral plasticity in the adult animal will be discussed. As our current understanding of epigenetics in these capacities is still evolving, epigenetic research will continue to be of considerable interest for understanding the molecular mechanisms mediating neurobiology and behavior both within and outside of sensitive periods of development.