Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine disrupting chemical commonly used as a plasticizer in medical equipment, food packaging, flooring, and children’s toys. DEHP exposure during early development has been associated with adverse neurobehavioral outcomes in children. In animal models, early exposure to DEHP results in abnormal development of the reproductive system as well as altered behavior and neurodevelopment. Based on these data, we hypothesized that developmental exposure to DEHP would decrease social interactions and increase anxiety-like behaviors in mice in a dose-dependent manner, and that the effects would persist over generations. C57BL/6J mice consumed one of three DEHP doses (0, 5, 40, and 400 μg/kg body weight) throughout pregnancy and during the first ten days of lactation. The two higher doses yielded detectable levels of DEHP metabolites in serum. Pairs of mice from control, low, and high DEHP doses were bred to create three dose lineages in the third generation (F3). Average anogenital index (AGI: anogenital distance/body weight) was decreased in F1 males exposed to the low dose of DEHP and in F1 females exposed to the highest dose. In F1 mice, juvenile pairs from the two highest DEHP dose groups displayed fewer socially investigative behaviors and more exploratory behaviors as compared with control mice. The effect of DEHP on these behaviors was reversed in F3 mice as compared with F1 mice. F1 mice exposed to low and medium DEHP doses spent more time in the closed arms of the elevated plus maze than controls, indicating increased anxiety-like behavior. The generation-dependent effects on behavior and AGI suggest complex mechanisms by which DEHP directly impacts reproductive and neurobehavioral development and influences germline-inherited traits.

Bisphenol A (BPA) is a ubiquitous man-made endocrine disrupting compound (EDC). Developmental exposure to BPA changes behavioral and reproductive phenotypes, and these effects can last for generations. We exposed embryos to BPA, producing two lineages: controls and BPA exposed. In the third filial generation (F3), brain tissues containing the preoptic area, the bed nucleus of the stria terminalis, and the anterior hypothalamus were collected. RNA sequencing (RNA-seq) and subsequent data analyses revealed 50 differentially regulated genes in the brains of F3 juveniles from BPA vs control lineages. BPA exposure can lead to loss of imprinting, and one of the two imprinted genes in our data set, maternally expressed gene 3 (Meg3), has been associated with EDCs and neurobehavioral phenotypes. We used quantitative polymerase chain reaction to examine the two imprinted genes in our data set, Meg3 and microRNA-containing gene Mirg (residing in the same loci). Confirming the RNA-seq, Meg3 messenger RNA was higher in F3 brains from the BPA lineage than in control brains. This was true in brains from mice produced with two different BPA paradigms. Next, we used pyrosequencing to probe differentially methylated regions of Meg3. We found transgenerational effects of BPA on imprinted genes in brain. Given these results, and data on Meg3 methylation in humans, we suggest this gene may be a biomarker indicative of early life environmental perturbation.

Evidence suggests that when presented with novel acute stress, animals previously exposed to chronic homotypic or heterotypic stressors exhibit normal or enhanced hypothalamic-pituitary-adrenal (HPA) response compared with animals exposed solely to that acute stressor. The molecular mechanisms involved in this effect remain unknown. The extracellular signal-regulated kinase (ERK) is one of the key pathways regulated in the hippocampus in both acute and chronic stress. The aim of this study was to examine the interaction of prior chronic stress, using the chronic variable stress model (CVS), with exposure to a novel acute stressor (2,5-dihydro-2,4,5-trimethyl thiazoline; TMT) on ERK activation, expression of the downstream protein BCL-2, and the glucocorticoid receptor co-chaperone BAG-1 in control and chronically stressed male rats. TMT exposure after chronic stress resulted in a significant interaction of chronic and acute stress in all 3 hippocampus subregions on ERK activation and BCL-2 expression. Significantly, acute stress increased ERK activation, BCL-2 and BAG-1 protein expression in the dentate gyrus (DG) of CVS-treated rats compared with control, CVS-treated alone, and TMT-only animals. Furthermore, CVS significantly increased ERK activation in medial prefrontal cortex, but acute stress had no significant effect. Inhibition of corticosterone synthesis with metyrapone had no significant effect on ERK activation in the hippocampus; therefore, glucocorticoids alone do not mediate the molecular effects. Finally, because post-translational modifications of histones are believed to play an important role in the stress response, we examined changes in histone acetylation. We found that, in general, chronic stress decreased K12H4 acetylation, whereas acute stress increased acetylation. These results indicate a molecular mechanism by which chronic stress-induced HPA axis plasticity can lead to neurochemical alterations in the hippocampus that influence reactivity to subsequent stress exposure. This may represent an important site of dysfunction that contributes to stress-induced pathology such as depression, anxiety disorders, and posttraumatic stress disorder.

Calbindin-D(28K) (Calb1), a high-affinity calcium buffer/sensor, shows abundant expression in neurons and has been associated with a number of neurobehavioral diseases, many of which are sexually dimorphic in incidence. Behavioral and physiological end points are affected by experimental manipulations of calbindin levels, including disruption of spatial learning, hippocampal long-term potentiation, and circadian rhythms. In this study, we investigated novel aspects of calbindin function on social behavior, anxiety-like behavior, and fear conditioning in adult mice of both sexes by comparing wild-type to littermate Calb1 KO mice. Because Calb1 mRNA and protein are sexually dimorphic in some areas of the brain, we hypothesized that sex differences in behavioral responses of these behaviors would be eliminated or revealed in Calb1 KO mice. We also examined gene expression in the amygdala and prefrontal cortex, two areas of the brain intimately connected with limbic system control of the behaviors tested, in response to sex and genotype. Our results demonstrate that fear memory and social behavior are altered in male knockout mice, and Calb1 KO mice of both sexes show less anxiety. Moreover, gene expression studies of the amygdala and prefrontal cortex revealed several significant genotype and sex effects in genes related to brain-derived neurotrophic factor signaling, hormone receptors, histone deacetylases, and γ-aminobutyric acid signaling. Our findings are the first to directly link calbindin with affective and social behaviors in rodents; moreover, the results suggest that sex differences in calbindin protein influence behavior.

Bisphenol A (BPA) is an endocrine disrupting chemical used in the production of polycarbonate plastics and resins. Exposure to BPA during gestation has been proposed as a risk factor for the development of neurobehavioral disorders, such as autism spectrum disorder. To address the behavioral impact of developmental exposure to BPA, we tested offspring of mice exposed to a daily low dose of BPA during pregnancy. We also asked if preconception exposure of the sire affected behaviors in offspring. Sires that consumed BPA for 50days prior to mating weighed less than controls, but no effects on any reproductive measures were noted. Juvenile offspring exposed to BPA maternally, but not paternally, spent less time in the open arms of the elevated plus maze than controls, indicating increased anxiety-like behavior. However, neither parental exposure group differed significantly from controls in the social recognition task. We also assessed the behaviors of maternally exposed offspring in two novel tasks: ultrasonic vocalizations (USVs) in pups and operant reversal learning in adults. Maternal BPA exposure increased the duration and median frequency of USVs emitted by pups during maternal separation. In the reversal learning task, females responded more accurately and earned more rewards than males. Additionally, control females received more rewards than BPA females during the acquisition phase of the task. These are among the first studies conducted to ask if BPA exposure via the sire affects offspring behavior and the first study to report effects of gestational BPA exposure on pup USVs and adult operant responding.