The medial preoptic area and acute cocaine's stimulant effects in rats: Potential influences of estradiol and biological sex.

The medial preoptic area (mPOA) in the hypothalamus is an important integrator of neuroendocrine signaling and a key regulator of both natural and drug-induced reward. Although the mPOA modulates sex differences in other behaviors, whether it also modulates sex differences in cocaine response remains unclear. To help us better understand the mPOA's role in sex differences associated with cocaine response, we examined cocaine-induced changes in locomotion and neural activity in the mPOA of male and female rats. In addition, neural activity in the striatum, a brain area known to be involved in cocaine response, was examined for comparison purposes. Fos, the protein product of the immediate early gene c-fos, was used as the marker of neural activity. Locomotion chambers were used to measure behavior, radioimmunoassays and vaginal lavages were used to determine hormonal status, and immunohistochemical assays were used to quantify Fos. To account for the effects of gonadal hormones, rats were left gonadally intact and categorized as either 'low-estradiol' or 'high-estradiol' based on their hormonal status on test day. Results indicate that high-estradiol females experienced greater cocaine-induced mPOA Fos-immunoreactivity (Fos-ir) and displayed greater cocaine-induced locomotion than low estradiol females. Conversely, high-estradiol males experienced less cocaine-induced mPOA Fos-ir and displayed less cocaine-induced locomotion than low-estradiol males. Cocaine-induced Fos-ir in the mPOA also correlated with cocaine-induced Fos-ir in areas of the striatum already associated with cocaine response. These findings further support the mPOA's role in the endocrine-mediated response to cocaine. It also identifies the mPOA as a contributor to sex differences in cocaine response and potential differences in vulnerability to developing cocaine use disorders.

Hormonal contraceptives alter amphetamine place preference and responsivity in the intact female rat.

Hormonal contraceptives (HCs) containing synthetic ovarian hormones are commonly used among reproductive aged women; HCs alter the physiological state of the user by interfering with endogenous hormone concentrations and their actions on the reproductive tract. As ovarian hormones modulate the incidence of substance abuse disorders in women, this experiment explores how modulating female rat ovarian hormonal states with an HC containing the synthetic progestin levonorgestrel influences measures of drug preference and responsivity. First, rats underwent food-light Pavlovian conditioning to measure conditioned orienting, a known predictor of amphetamine (AMP) place preference. Then, rats were conditioned and tested for AMP place preference with either an HC implant or during estrous cycle stages associated with opposing ovarian hormone levels, that is, proestrus (P) or metestrus/diestrus (M/D), while recording ultrasonic vocalizations (USVs) as an index of he donic drug responsivity. Because of dopamine's (DA's) role in reward learning and memory, DA cell number and activity were examined using tyrosine hydroxylase and FOS immunohistochemistry after a final AMP challenge. Conditioned orienting did not differ between cycling and HC-implanted rats. HC rats emitted fewer USVs in response to AMP, showed marginally less AMP place preference, and had lower DA cell activity in the substantia nigra after AMP compared to P rats. M/D rats showed a similar behavioral profile and neural response as HC rats. This experiment suggests ovarian hormones affect drug preference and responsivity, while providing novel insight into how hormone-altering contraceptives may reduce these measures. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Two Hits of EDCs Three Generations Apart: Effects on Social Behaviors in Rats, and Analysis by Machine Learning.

All individuals are directly exposed to extant environmental endocrine-disrupting chemicals (EDCs), and indirectly exposed through transgenerational inheritance from our ancestors. Although direct and ancestral exposures can each lead to deficits in behaviors, their interactions are not known. Here we focused on social behaviors based on evidence of their vulnerability to direct or ancestral exposures, together with their importance in reproduction and survival of a species. Using a novel "two hits, three generations apart" experimental rat model, we investigated interactions of two classes of EDCs across six generations. PCBs (a weakly estrogenic mixture Aroclor 1221, 1 mg/kg), Vinclozolin (antiandrogenic, 1 mg/kg) or vehicle (6% DMSO in sesame oil) were administered to pregnant rat dams (F0) to directly expose the F1 generation, with subsequent breeding through paternal or maternal lines. A second EDC hit was given to F3 dams, thereby exposing the F4 generation, with breeding through the F6 generation. Approximately 1200 male and female rats from F1, F3, F4 and F6 generations were run through tests of sociability and social novelty as indices of social preference. We leveraged machine learning using DeepLabCut to analyze nuanced social behaviors such as nose touching with accuracy similar to a human scorer. Surprisingly, social behaviors were affected in ancestrally exposed but not directly exposed individuals, particularly females from a paternally exposed breeding lineage. Effects varied by EDC: Vinclozolin affected aspects of behavior in the F3 generation while PCBs affected both the F3 and F6 generations. Taken together, our data suggest that specific aspects of behavior are particularly vulnerable to heritable ancestral exposure of EDC contamination, that there are sex differences, and that lineage is a key factor in transgenerational outcomes.

Peritubular Macrophages Are Recruited to the Testis of Peripubertal Rats After Mono-(2-Ethylhexyl) Phthalate Exposure and Is Associated With Increases in the Numbers of Spermatogonia.

Peripubertal exposure of male rodents to the phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) causes testicular inflammation, spermatocyte apoptosis, and disruption of the blood-testis barrier. The MEHP-induced inflammatory response in the testis includes an infiltration of macrophages and neutrophils, although the cause and purpose of this response is unknown. Recently, a population of testicular macrophages known as peritubular macrophages that are phenotypically distinct from those resident in interstitium was described in mice. Peritubular macrophages aggregate near the spermatogonial stem cell niche and are believed to stimulate their differentiation. We hypothesized that if testicular peritubular macrophages do indeed stimulate spermatogonial differentiation, MEHP exposure would result in an increase of peritubular macrophages to stimulate the replacement of lost spermatocytes. Male rats were exposed to 700 mg/kg MEHP or corn oil (vehicle control) via oral gavage at postnatal day 28 and euthanized at 48 h, 1 or 2 weeks later. Seminiferous tubules were stained with immunofluorescent markers for macrophages (major histocompatibility complex class II [MHC-II+]) and undifferentiated spermatogonia (PLZF). Peritubular macrophages were observed in rat testis: MHC-II+ cells on the surface of seminiferous tubules with heterogeneous morphology. Quantification of MHC-II+ cells revealed that, unlike in the mouse, their numbers did not increase through puberty (2-week period). MEHP increased macrophage presence by 6-fold 48 h after exposure and remained elevated by 2-fold 2 weeks after exposure. An increase of differentiating spermatogonia occurred 2 weeks after MEHP exposure. Taken together, our results suggest that peritubular macrophages play a crucial role in the testis response to acute injury and the subsequent recovery of spermatogenesis.

Social and neuromolecular phenotypes are programmed by prenatal exposures to endocrine-disrupting chemicals.

Exposures to endocrine-disrupting chemicals (EDCs) affect the development of hormone-sensitive neural circuits, the proper organization of which are necessary for the manifestation of appropriate adult social and sexual behaviors. We examined whether prenatal exposure to polychlorinated biphenyls (PCBs), a family of ubiquitous industrial contaminants detectable in virtually all humans and wildlife, caused changes in sexually-dimorphic social interactions and communications, and profiled the underlying neuromolecular phenotype. Rats were treated with a PCB commercial mixture, Aroclor 1221 (A1221), estradiol benzoate (EB) as a positive control for estrogenic effects of A1221, or the vehicle (4% DMSO), on embryonic day (E) 16 and 18. In adult F1 offspring, we first conducted tests of ultrasonic vocalization (USV) calls in a sociosexual context as a measure of motivated communications. Numbers of certain USV call types were significantly increased by prenatal treatment with A1221 in males, and decreased by EB in females. In a test of sociosexual preference for a hormone-vs. a non-hormone-primed opposite sex conspecific, male (but not female) nose-touching with opposite-sex rats was significantly diminished by EDCs. Gene expression profiling was conducted in two brain regions that are part of the social decision-making network in the brain: the medial preoptic nucleus (MPN) and the ventromedial nucleus (VMN). In both regions, many more genes were affected by A1221 or EB in females than males. In female MPN, A1221 changed expression of steroid hormone receptor and neuropeptide genes (e.g., Ar, Esr1, Esr2, and Kiss1). In male MPN, only Per2 was affected by A1221. The VMN had a number of genes affected by EB compared to vehicle (females: Kiss1, Kiss1r, Pgr; males: Crh) but not A1221. These differences between EB and A1221 indicate that the mechanism of action of A1221 goes beyond estrogenic pathways. These data show sex-specific effects of prenatal PCBs on adult behaviors and the neuromolecular phenotype.

Passing experiences on to future generations: endocrine disruptors and transgenerational inheritance of epimutations in brain and sperm.

All animals have body burdens of polychlorinated biphenyls (PCBs) despite their ban decades ago. These and modern endocrine-disrupting chemicals (EDCs) such as the fungicide vinclozolin (VIN) perturb hormone signaling and lead to dysfunctions following prenatal exposures. Beyond direct exposures, transgenerational disease phenotypes can persist for multiple generations without subsequent exposure. The mechanisms of action of these EDCs differ: VIN is anti-androgenic while the PCB mixture Aroclor 1221 (A1221) is weakly estrogenic. Based on limited evidence for the inheritance of epimutations in germline, we measured DNA methylation in brain and sperm of rats. Pregnant dams were exposed from day 8-18 of gestation to low dosages of VIN, A1221, or the vehicle. To produce paternal lineages, exposed F1 males were bred with untreated females, creating the F2 and subsequently F3 generations. In adult F1 and F3 males, mature sperm was collected, and brain nuclei involved in anxiety and social behaviors (CA3 of the hippocampus; central amygdala) were selected for assays of epimutations in CpG islands using reduced representation bisulfite sequencing. In F1 sperm, VIN and PCBs induced differential methylation in 215 and 284 CpG islands, respectively, compared to vehicle. The majority of effects were associated with hypermethylation. Fewer epimutations were detected in the brain. A subset of differentially methylated regions were retained from the F1 to the F3 generation, suggesting a common mechanism of EDC and germline epigenome interaction. Thus, EDCs can cause heritable epimutations in the sperm that may embody the future phenotype of brain-behavior disorders caused by direct or transgenerational exposures.

Aging and estradiol effects on gene expression in the medial preoptic area, bed nucleus of the stria terminalis, and posterodorsal medial amygdala of male rats.

Studies on the role of hormones in male reproductive aging have traditionally focused on testosterone, but estradiol (E2) also plays important roles in the control of masculine physiology and behavior. Our goal was to examine the effects of E2 on the expression of genes selected for E2-sensitivity, involvement in behavioral neuroendocrine functions, and impairments with aging. Mature adult (MAT, 5 mo) and aged (AG, 18 mo) Sprague-Dawley male rats were castrated, implanted with either vehicle or E2 subcutaneous capsules, and euthanized one month later. Bilateral punches were taken from the bed nucleus of the stria terminalis (BnST), posterodorsal medial amygdala (MePD) and the preoptic area (POA). RNA was extracted, and expression of 48 genes analyzed by qPCR using Taqman low-density arrays. Results showed that effects of age and E2 were age- and region-specific. In the POA, 5 genes were increased with E2 compared to vehicle, and there were no age effects. By contrast the BnST showed primarily age-related changes, with 6 genes decreasing with age. The MePD had 5 genes that were higher in aged than mature males, and 17 genes with significant interactions between age and E2. Gene families identified in the MePD included nuclear hormone receptors, neurotransmitters and neuropeptides and their receptors. Ten serum hormones were assayed in these same males, with results revealing both age- and E2-effects, in several cases quite profound. These results support the idea that the male brain continues to be highly sensitive to estradiol even with aging, but the nature of the response can be substantially different in mature and aging animals.

Anxiety-like behaviors in adulthood are altered in male but not female rats exposed to low dosages of polychlorinated biphenyls in utero.

Exposure to polychlorinated biphenyls (PCBs), a class of endocrine-disrupting chemicals, can result in altered reproductive behavior in adulthood, especially when exposure occurs during critical periods of brain sexual differentiation in the fetus. Whether PCBs alter other sexually dimorphic behaviors such as those involved in anxiety is poorly understood. To address this, pregnant rat dams were injected twice, on gestational days 16 and 18, with the weakly estrogenic PCB mixture Aroclor 1221 (A1221) at one of two low dosages (0.5mg/kg or 1.0mg/kg, hereafter 1.0 and 0.5), estradiol benzoate (EB; 50µg/kg) as a positive estrogenic control, or the vehicle (3% DMSO in sesame oil). We also conducted a comprehensive assessment of developmental milestones of the F1 male and female offspring. There were no effects of treatment on sex ratio at birth and age at eye opening. Puberty, assessed by vaginal opening in females and preputial separation in males, was not affected in females but was advanced in males treated with A1221 (1.0). Males and females treated with A1221 (both dosages) were heavier in early adulthood relative to controls. The earliest manifestation of this effect developed in males prior to puberty and in females slightly later, during puberty. Anxiety-like behaviors were tested using the light:dark box and elevated plus maze tests in adulthood. In females, anxiety behaviors were unaffected by treatment. Males treated with A1221 (1.0) showed reduced indices of anxiety and increased activity in the light:dark box but not the elevated plus maze. EB failed to replicate the phenotype produced by A1221 for any of the developmental and behavioral endpoints. Collectively, these results indicate that PCBs increase body weight in both sexes, but their effects on anxiety-like behaviors are specific to males. Furthermore, differences between the results of A1221 and EB suggest that the PCBs are likely acting through mechanisms distinct from their estrogenic activity.

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.

Epigenetic transgenerational inheritance of altered stress responses.

Ancestral environmental exposures have previously been shown to promote epigenetic transgenerational inheritance and influence all aspects of an individual's life history. In addition, proximate life events such as chronic stress have documented effects on the development of physiological, neural, and behavioral phenotypes in adulthood. We used a systems biology approach to investigate in male rats the interaction of the ancestral modifications carried transgenerationally in the germ line and the proximate modifications involving chronic restraint stress during adolescence. We find that a single exposure to a common-use fungicide (vinclozolin) three generations removed alters the physiology, behavior, metabolic activity, and transcriptome in discrete brain nuclei in descendant males, causing them to respond differently to chronic restraint stress. This alteration of baseline brain development promotes a change in neural genomic activity that correlates with changes in physiology and behavior, revealing the interaction of genetics, environment, and epigenetic transgenerational inheritance in the shaping of the adult phenotype. This is an important demonstration in an animal that ancestral exposure to an environmental compound modifies how descendants of these progenitor individuals perceive and respond to a stress challenge experienced during their own life history.