Extinction training suppresses activity of fear memory ensembles across the hippocampus and alters transcriptomes of fear-encoding cells.

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells-the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.

Extinction Training Suppresses Activity of Fear Memory Ensembles Across the Hippocampus and Alters Transcriptomes of Fear-Encoding Cells.

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells - the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.

A 2b-RAD parentage analysis pipeline for complex and mixed DNA samples.

Next-generation sequencing technology has revolutionized genotyping in many fields of study, yet parentage analysis often still relies on microsatellite markers that are costly to generate and are currently available only for a limited number of species. 2b-RAD sequencing (2b-RAD) is a DNA sequencing technique developed for ecological population genomics that utilizes type IIB restriction enzymes to generate consistent, uniform fragments across samples. This technology is inexpensive, effective with low DNA inputs, and robust to DNA degradation. Here, we developed a probabilistic genotyping-by-sequencing genetic testing pipeline for parentage analysis by using 2b-RAD for inferring familial relationships from mixed DNA samples and populations. Our approach to partial paternity assignment utilizes a novel weighted outlier paternity index (WOPI) adapted for next-generation sequencing data and an identity-by-state (IBS) matrix-based clustering method for pedigree reconstruction. The combination of these two parentage assignment methods overcomes two major obstacles faced by other genetic testing methods: 1) It allows detection of parentage when closely related or inbred individuals are in the alleged parent population (e.g., in laboratory strains); and 2) it resolves mixed DNA samples. We successfully demonstrate this novel approach by correctly inferring paternity for samples pooled from multiple offspring (i.e., entire clutches) in a highly inbred population of an East African cichlid fish. The unique advantages of 2b-RAD in combination with our bioinformatics pipeline enable straightforward and cost-effective parentage analysis in any species regardless of genomic resources available.

Early rearing history influences oxytocin receptor epigenetic regulation in rhesus macaques.

Adaptations to stress can occur through epigenetic processes and may be a conduit for informing offspring of environmental challenge. We employed ChIP-sequencing for H3K4me3 to examine effects of early maternal deprivation (peer-rearing, PR) in archived rhesus macaque hippocampal samples (male, n = 13). Focusing on genes with roles in stress response and behavior, we assessed the effects of rearing on H3K4me3 binding by ANOVA. We found decreased H3K4me3 binding at genes critical to behavioral stress response, the most robust being the oxytocin receptor gene OXTR, for which we observed a corresponding decrease in RNA expression. Based on this finding, we performed behavioral analyses to determine whether a gain-of-function nonsynonymous OXTR SNP interacted with early stress to influence relevant behavioral stress reactivity phenotypes (n = 194), revealing that this SNP partially rescued the PR phenotype. PR infants exhibited higher levels of separation anxiety and arousal in response to social separation, but infants carrying the alternative OXTR allele did not exhibit as great a separation response. These data indicate that the oxytocin system is involved in social-separation response and suggest that epigenetic down-modulation of OXTR could contribute to behavioral differences observed in PR animals. Epigenetic changes at OXTR may represent predictive adaptive responses that could impart readiness to respond to environmental challenge or maintain proximity to a caregiver but also contribute to behavioral pathology. Our data also demonstrate that OXTR polymorphism can permit animals to partially overcome the detrimental effects of early maternal deprivation, which could have translational implications for human psychiatric disorders.

Distinct actions of ancestral vinclozolin and juvenile stress on neural gene expression in the male rat.

Exposure to the endocrine disrupting chemical vinclozolin during gestation of an F0 generation and/or chronic restraint stress during adolescence of the F3 descendants affects behavior, physiology, and gene expression in the brain. Genes related to the networks of growth factors, signaling peptides, and receptors, steroid hormone receptors and enzymes, and epigenetic related factors were measured using quantitative polymerase chain reaction via Taqman low density arrays targeting 48 genes in the central amygdaloid nucleus, medial amygdaloid nucleus, medial preoptic area (mPOA), lateral hypothalamus (LH), and the ventromedial nucleus of the hypothalamus. We found that growth factors are particularly vulnerable to ancestral exposure in the central and medial amygdala; restraint stress during adolescence affected neural growth factors in the medial amygdala. Signaling peptides were affected by both ancestral exposure and stress during adolescence primarily in hypothalamic nuclei. Steroid hormone receptors and enzymes were strongly affected by restraint stress in the mPOA. Epigenetic related genes were affected by stress in the ventromedial nucleus and by both ancestral exposure and stress during adolescence independently in the central amygdala. It is noteworthy that the LH showed no effects of either manipulation. Gene expression is discussed in the context of behavioral and physiological measures previously published.

Nature, nurture and epigenetics.

Real life by definition combines heritability (e.g., the legacy of exposures) and experience (e.g. stress during sensitive or 'critical' periods), but how to study or even model this interaction has proven difficult. The hoary concept of evaluating traits according to nature versus nurture continues to persist despite repeated demonstrations that it retards, rather than advances, our understanding of biological processes. Behavioral genetics has proven the obvious, that genes influence behavior and, vice versa, that behavior influences genes. The concept of Genes X Environment (G X E) and its modern variants was viewed as an improvement on nature-nurture but has proven that, except in rare instances, it is not possible to fractionate phenotypes into these constituent elements. The entanglement inherent in terms such as nature-nurture or G X E is a Gordian knot that cannot be dissected or even split. Given that the world today is not what it was less than a century ago, yet the arbitrator (differential survival and reproduction) has stayed constant, de novo principles and practices are needed to better predict what the future holds. Put simply, the transformation that is now occurring within and between individuals as a product of global endocrine disruption is quite independent of what has been regarded as evolution by selection. This new perspective should focus on how epigenetic modifications might revise approaches to understand how the phenotype and, in particular its components, is shaped. In this review we summarize the literature in this developing area, focusing on our research on the fungicide vinclozolin.

Sexually dimorphic effects of ancestral exposure to vinclozolin on stress reactivity in rats.

How an individual responds to the environment depends upon both personal life history as well as inherited genetic and epigenetic factors from ancestors. Using a 2-hit, 3 generations apart model, we tested how F3 descendants of rats given in utero exposure to the environmental endocrine-disrupting chemical (EDC) vinclozolin reacted to stress during adolescence in their own lives, focusing on sexually dimorphic phenotypic outcomes. In adulthood, male and female F3 vinclozolin- or vehicle-lineage rats, stressed or nonstressed, were behaviorally characterized on a battery of tests and then euthanized. Serum was used for hormone assays, and brains were used for quantitative PCR and transcriptome analyses. Results showed that the effects of ancestral exposure to vinclozolin converged with stress experienced during adolescence in a sexually dimorphic manner. Debilitating effects were seen at all levels of the phenotype, including physiology, behavior, brain metabolism, gene expression, and genome-wide transcriptome modifications in specific brain nuclei. Additionally, females were significantly more vulnerable than males to transgenerational effects of vinclozolin on anxiety but not sociality tests. This fundamental transformation occurs in a manner not predicted by the ancestral exposure or the proximate effects of stress during adolescence, an interaction we refer to as synchronicity.