Gestational exposure to FireMaster® 550 (FM 550) disrupts the placenta-brain axis in a socially monogamous rodent species, the prairie vole (Microtus ochrogaster).

Gestational flame retardant (FR) exposure has been linked to heightened risk of neurodevelopmental disorders, but the mechanisms remain largely unknown. Historically, toxicologists have relied on traditional, inbred rodent models, yet those do not always best model human vulnerability or biological systems, especially social systems. Here we used prairie voles (Microtus ochrogaster), a monogamous and bi-parental rodent, leveraged for decades to decipher the underpinnings of social behaviors, to examine the impact of fetal FR exposure on gene targets in the mid-gestational placenta and fetal brain. We previously established gestational exposure to the commercial mixture Firemaster 550 (FM 550) impairs sociality, particularly in males. FM 550 exposure disrupted placental monoamine production, particularly serotonin, and genes required for axon guidance and cellular respiration in the fetal brains. Effects were dose and sex specific. These data provide insights on the mechanisms by which FRs impair neurodevelopment and later in life social behaviors.

Maternal organophosphate flame retardant exposure alters the developing mesencephalic dopamine system in fetal rat.

Organophosphate flame retardants (OPFRs) have become the predominant substitution for legacy brominated flame retardants but there is concern about their potential developmental neurotoxicity (DNT). OPFRs readily dissociate from the fireproofed substrate to the environment, and they (or their metabolites) have been detected in diverse matrices including air, water, soil, and biota, including human urine and breastmilk. Given this ubiquitous contamination, it becomes increasingly important to understand the potential effects of OPFRs on the developing nervous system. We have previously shown that maternal exposure to OPFRs results in neuroendocrine disruption, alterations to developmental metabolism of serotonin (5-HT) and axonal extension in male fetal rats, and potentiates adult anxiety-like behaviors. The development of the serotonin and dopamine systems occur in parallel and interact, therefore, we first sought to enhance our prior 5-HT work by first examining the ascending 5-HT system on embryonic day 14 using whole mount clearing of fetal heads and 3-dimensional (3D) brain imaging. We also investigated the effects of maternal OPFR exposure on the development of the mesocortical dopamine system in the same animals through 2-dimensional and 3D analysis following immunohistochemistry for tyrosine hydroxylase (TH). Maternal OPFR exposure induced morphological changes to the putative ventral tegmental area and substantia nigra in both sexes and reduced the overall volume of this structure in males, whereas 5-HT nuclei were unchanged. Additionally, dopaminergic axogenesis was disrupted in OPFR exposed animals, as the dorsoventral spread of ventral telencephalic TH afferents were greater at embryonic day 14, while sparing 5-HT fibers. These results indicate maternal exposure to OPFRs alters the development trajectory of the embryonic dopaminergic system and adds to growing evidence of OPFR DNT.

Combining Micropunch Histology and Multidimensional Lipidomic Measurements for In-Depth Tissue Mapping.

While decades of technical and analytical advancements have been utilized to discover novel lipid species, increase speciation, and evaluate localized lipid dysregulation at subtissue, cellular, and subcellular levels, many challenges still exist. One limitation is that the acquisition of both in-depth spatial information and comprehensive lipid speciation is extremely difficult, especially when biological material is limited or lipids are at low abundance. In neuroscience, for example, it is often desired to focus on only one brain region or subregion, which can be well under a square millimeter for rodents. Herein, we evaluate a micropunch histology method where cortical brain tissue at 2.0, 1.25, 1.0, 0.75, 0.5, and 0.25 mm diameter sizes and 1 mm depth was collected and analyzed with multidimensional liquid chromatography, ion mobility spectrometry, collision induced dissociation, and mass spectrometry (LC-IMS-CID-MS) measurements. Lipid extraction was optimized for the small sample sizes, and assessment of lipidome coverage for the 2.0 to 0.25 mm diameter sizes showed a decline from 304 to 198 lipid identifications as validated by all 4 analysis dimensions (~35% loss in coverage for ~88% less tissue). While losses were observed, the ~200 lipids and estimated 4630 neurons contained within the 0.25 punch still provided in-depth characterization of the small tissue region. Furthermore, while localization routinely achieved by mass spectrometry imaging (MSI) and single cell analyses is greater, this diameter is sufficiently small to isolate subcortical, hypothalamic, and other brain regions in adult rats, thereby increasing the coverage of lipids within relevant spatial windows without sacrificing speciation. Therefore, micropunch histology enables in-depth, region-specific lipid evaluations, an approach that will prove beneficial to a variety of lipidomic applications.

Individual and Combined Effects of Paternal Deprivation and Developmental Exposure to Firemaster 550 on Socio-Emotional Behavior in Prairie Voles.

The prevalence of neurodevelopmental disorders (NDDs) is rapidly rising, suggesting a confluence of environmental factors that are likely contributing, including developmental exposure to environmental contaminants. Unfortunately, chemical exposures and social stressors frequently occur simultaneously in many communities, yet very few studies have sought to establish the combined effects on neurodevelopment or behavior. Social deficits are common to many NDDs, and we and others have shown that exposure to the chemical flame retardant mixture, Firemaster 550 (FM 550), or paternal deprivation impairs social behavior and neural function. Here, we used a spontaneously prosocial animal model, the prairie vole (Microtus ochrogaster), to explore the effects of perinatal chemical (FM 550) exposure alone or in combination with an early life stressor (paternal absence) on prosocial behavior. Dams were exposed to vehicle (sesame oil) or 1000 µg FM 550 orally via food treats from conception through weaning and the paternal absence groups were generated by removing the sires the day after birth. Adult offspring of both sexes were then subjected to open-field, sociability, and a partner preference test. Paternal deprivation (PD)-related effects included increased anxiety, decreased sociability, and impaired pair-bonding in both sexes. FM 550 effects include heightened anxiety and partner preference in females but reduced partner preference in males. The combination of FM 550 exposure and PD did not exacerbate any behaviors in either sex except for distance traveled by females in the partner preference test and, to a lesser extent, time spent with, and the number of visits to the non-social stimulus by males in the sociability test. FM 550 ameliorated the impacts of parental deprivation on partner preference behaviors in both sexes. This study is significant because it provides evidence that chemical and social stressors can have unique behavioral effects that differ by sex but may not produce worse outcomes in combination.

Effects of developmental exposure to FireMaster® 550 (FM 550) on microglia density, reactivity and morphology in a prosocial animal model.

Microglia are known to shape brain sex differences critical for social and reproductive behaviors. Chemical exposures can disrupt brain sexual differentiation but there is limited data regarding how they may impact microglia distribution and function. We focused on the prevalent flame retardant mixture Firemaster 550 (FM 550) which is used in foam-based furniture and infant products including strollers and nursing pillows because it disrupts sexually dimorphic behaviors. We hypothesized early life FM 550 exposure would disrupt microglial distribution and reactivity in brain regions known to be highly sexually dimorphic or associated with social disorders in humans. We used prairie voles (Microtus ochrogaster) because they display spontaneous prosocial behaviors not seen in rats or mice and are thus a powerful model for studying chemical exposure-related impacts on social behaviors and their underlying neural systems. We have previously demonstrated that perinatal FM 550 exposure sex-specifically impacts socioemotional behaviors in prairie voles. We first established that, unlike in rats, the postnatal colonization of the prairie vole brain is not sexually dimorphic. Vole dams were then exposed to FM 550 (0, 500, 1000, 2000 µg/day) via subcutaneous injections through gestation, and pups were directly exposed beginning the day after birth until weaning. Adult offspring's brains were assessed for number and type (ramified, intermediate, ameboid) of microglia in the medial prefrontal cortex (mPFC), cerebellum (lobules VI-VII) and amygdala. Effects were sex- and dose-specific in the regions of interests. Overall, FM 550 exposure resulted in reduced numbers of microglia in most regions examined, with the 1000 µg FM 550 exposed males particularly affected. To further quantify differences in microglia morphology in the 1000 µg FM 550 group, Sholl and skeleton analysis were carried out on individual microglia. Microglia from control females had a more ramified phenotype compared to control males while 1000 µg FM 550-exposed males had decreased branching and ramification compared to same-sex controls. Future studies will examine the impact on the exposure to FM 550 on microglia during development given the critical role of these cells in shaping neural circuits.

FireMaster® 550 (FM 550) exposure during the perinatal period impacts partner preference behavior and nucleus accumbens core medium spiny neuron electrophysiology in adult male and female prairie voles, Microtus ochrogaster.

One of the most widely used flame retardant (FR) mixtures in household products is Firemaster 550 (FM 550). FM 550 leaches from items such as foam-based furniture and infant products, resulting in contamination of the household environment and biota. Previous studies indicate sex-specific behavioral deficits in rodents and zebrafish in response to developmental FM 550 exposure. These deficits include impacts on social and attachment behaviors in a prosocial rodent: the prairie vole (Microtus ochrogaster). The prairie vole is a laboratory-acclimated rodent that exhibits spontaneous attachment behaviors including pair bonding. Here we extend previous work by addressing how developmental exposure to FM 550 impacts pair bonding strength via an extended-time partner preference test, as well as neuron electrophysiological properties in a region implicated in pair bond behavior, the nucleus accumbens (NAcc) core. Dams were exposed to vehicle or 1000 µg of FM 550 via subcutaneous injections throughout gestation, and female and male pups were directly exposed beginning the day after birth until weaning. Pair bond behavior of adult female and male offspring was assessed using a three hour-long partner preference test. Afterwards, acute brain slices of the NAcc core were produced and medium spiny neuron electrophysiological attributes recorded via whole cell patch-clamp. Behavioral impacts were sex-specific. Partner preference behavior was increased in exposed females but decreased in exposed males. Electrophysiological impacts were similar between sexes and specific to attributes related to input resistance. Input resistance was decreased in neurons recorded from both sexes exposed to FM 550 compared to vehicle. This study supports the hypothesis that developmental exposure to FM 550 impacts attachment behaviors and demonstrates a novel FM 550 effect on neural electrophysiology.

Sex-specific Disruption of the Prairie Vole Hypothalamus by Developmental Exposure to a Flame Retardant Mixture.

Prevalence of neurodevelopmental disorders (NDDs) with social deficits is conspicuously rising, particularly in boys. Flame retardants (FRs) have long been associated with increased risk, and prior work by us and others in multiple species has shown that developmental exposure to the common FR mixture Firemaster 550 (FM 550) sex-specifically alters socioemotional behaviors including anxiety and pair bond formation. In rats, FRs have also been shown to impair aspects of osmoregulation. Because vasopressin (AVP) plays a role in both socioemotional behavior and osmotic balance we hypothesized that AVP and its related nonapeptide oxytocin (OT) would be vulnerable to developmental FM 550 exposure. We used the prairie vole (Microtus ochrogaste) to test this because it is spontaneously prosocial. Using siblings of prairie voles used in a prior study that assessed behavioral deficits resulting from developmental FM 550 exposure across 3 doses, here we tested the hypothesis that FM 550 sex-specifically alters AVP and OT neuronal populations in critical nuclei, such as the paraventricular nucleus (PVN), that coordinate those behaviors, as well as related dopaminergic (determined by tyrosine hydroxylase (TH) immunolabeling) populations. Exposed females had fewer AVP neurons in the anterior PVN and more A13 TH neurons in the zona incerta than controls. By contrast, in FM 550 males, A13 TH neuron numbers in the zona incerta were decreased but only in 1 dose group. These results expand on previous work showing evidence of endocrine disruption of OT/AVP pathways, including to subpopulations of PVN AVP neurons that coordinate osmoregulatory functions in the periphery.

Perinatal exposure to FireMaster® 550 (FM550), brominated or organophosphate flame retardants produces sex and compound specific effects on adult Wistar rat socioemotional behavior.

Firemaster 550 (FM550) is a flame retardant (FR) mixture that has become one of the most commonly used FRs in household items such as foam-based furniture and baby products. Because this mixture readily leaches from products, contamination of the environment and human tissues is widespread. Prior work by us and others has reported sex-specific behavioral deficits in rodents and zebrafish following early life exposure. In an effort to understand the mechanisms by which these behavioral effects occur, here we explored the effects of its constituents on behavioral outcomes previously shown to be altered by developmental FM550 exposure. The FM550 commercial mixture is composed of two brominated compounds (BFR) and two organophosphate compounds (OPFRs) at almost equivalent proportions. Both the BFR and the OPFR components are differentially metabolized and structurally distinct, but similar to known neurotoxicants. Here we examined adult Wistar rat offspring socioemotional behaviors following perinatal exposure (oral, to the dam) to vehicle, 2000 µg/day FM550, 1000 µg/day BFR or 1000 µg/day OPFR from gestation day 0 to weaning. Beginning on postnatal day 65 offspring from all groups were subjected to a series of behavioral tasks including open field, elevated plus maze, marble burying, social interaction tests, and running wheel. Effects were exposure-, sex- and task-specific, with BFR exposure resulting in the most consistent behavioral deficits. Overall, exposed females showed more deficits compared to males across all dose groups and tasks. These findings help elucidate how different classes of flame retardants, independently and as a mixture, contribute to sex-specific behavioral effects of exposure.

Sex-specific effects of perinatal FireMaster® 550 (FM 550) exposure on socioemotional behavior in prairie voles.

The rapidly rising incidence of neurodevelopmental disorders with social deficits is raising concern that developmental exposure to environmental contaminants may be contributory. Firemaster 550 (FM 550) is one of the most prevalent flame-retardant (FR) mixtures used in foam-based furniture and baby products and contains both brominated and organophosphate components. We and others have published evidence of developmental neurotoxicity and sex specific effects of FM 550 on anxiety-like and exploratory behaviors. Using a prosocial animal model, we investigated the impact of perinatal FM 550 exposure on a range of socioemotional behaviors including anxiety, attachment, and memory. Virtually unknown to toxicologists, but widely used in the behavioral neurosciences, the prairie vole (Microtus ochrogaster) is a uniquely valuable model organism for examining environmental factors on sociality because this species is spontaneously prosocial, biparental, and displays attachment behaviors including pair bonding. Dams were exposed to 0, 500, 1000, or 2000 µg of FM 550 via subcutaneous (sc) injections throughout gestation, and pups were directly exposed beginning the day after birth until weaning. Adult offspring of both sexes were then subjected to multiple tasks including open field, novel object recognition, and partner preference. Effects were dose responsive and sex-specific, with females more greatly affected. Exposure-related outcomes in females included elevated anxiety, decreased social interaction, decreased exploratory motivation, and aversion to novelty. Exposed males also had social deficits, with males in all three dose groups failing to show a partner preference. Our studies demonstrate the utility of the prairie vole for investigating the impact of chemical exposures on social behavior and support the hypothesis that developmental FR exposure impacts the social brain. Future studies will probe the possible mechanisms by which these effects arise.

Simultaneous Voltammetric Measurements of Glucose and Dopamine Demonstrate the Coupling of Glucose Availability with Increased Metabolic Demand in the Rat Striatum.

Cerebral blood flow ensures delivery of nutrients, such as glucose, to brain sites with increased metabolic demand. However, little is known about rapid glucose dynamics at discrete locations during neuronal activation in vivo. Acute exposure to many substances of abuse elicits dopamine release and neuronal activation in the striatum; however, the concomitant changes in striatal glucose remain largely unknown. Recent developments have combined fast-scan cyclic voltammetry with glucose oxidase enzyme modified carbon-fiber microelectrodes to enable the measurement of glucose dynamics with subsecond temporal resolution in the mammalian brain. This work evaluates several waveforms to enable the first simultaneous detection of endogenous glucose and dopamine at single recording sites. These molecules, one electroactive and one nonelectroactive, were found to fluctuate in the dorsal striatum in response to electrical stimulation of the midbrain and systemic infusion of cocaine/raclopride. The data reveal the second-by-second dynamics of these species in a striatal microenvironment, and directly demonstrate the coupling of glucose availability with increased metabolic demand. This work provides a foundation that will enable detailed investigation of local mechanisms that regulate the coupling of cerebral blood flow with metabolic demand under normal conditions, and in animal studies of drug abuse and addiction.

Region specific regulation of NR1 in rhesus monkeys following chronic antipsychotic drug administration.

BACKGROUND: Altered NMDA receptor subunit protein levels have been reported in various regions of the schizophrenic brain; however, chronic antipsychotic administration in schizophrenic subjects may confound interpretation. METHODS: The effects of chronic antipsychotic drug administration (haloperidol and clozapine) on protein levels of NR1, NR2A and NR2B proteins were evaluated in the nucleus accumbens (NAc), putamen (PUT), dorsolateral prefrontal cortex (DLPFC), superior temporal gyrus (STG), and entorhinal cortex (EC) of rhesus monkeys using Western blot analysis. RESULTS: Haloperidol administration significantly decreased NR1 expression in the DLPFC. In contrast, NR2B expression was not affected by antipsychotic administration in any brain region examined. NR2A was not reliably detected in any of the brain regions. CONCLUSIONS: Results indicate that the NR1 subunit in the DLPFC may be a substrate for antipsychotic action and that glutamatergic hypofunction in the DLPFC commonly associated with cognitive dysfunction in schizophrenia may be associated with haloperidol administration.

Social suppression of cortisol in female marmosets: role of luteinizing hormone/chorionic gonadotropin.

Behaviorally subordinate female common marmosets (Callithrix jacchus) undergo suppression of ovulation and chronic reductions in basal plasma cortisol concentrations. Indirect evidence suggests that hypophyseal chorionic gonadotropin (CG; the major pituitary luteinizing gonadotropin in marmosets) may elevate cortisol concentrations in female marmosets, and therefore that social suppression of CG may contribute to diminution of cortisol in subordinates. To test this hypothesis, we determined whether pharmacological inhibition of pituitary CG release decreases basal and adrenocorticotropin (ACTH)-stimulated cortisol secretion. We characterized cortisol and reproductive hormone concentrations in six ovary-intact and six ovariectomized marmosets during long-term treatment with leuprolide acetate, a gonadotropin-releasing hormone (GnRH) agonist, and vehicle. Leuprolide suppressed basal plasma CG concentrations, abolished the CG response to exogenous GnRH, and, in intact animals, blocked ovarian cyclicity. During treatment with vehicle, plasma cortisol concentrations were elevated during the periovulatory phase in intact females, compared to the follicular phase, the luteal phase, and ovariectomized females. Leuprolide suppressed basal cortisol concentrations of intact females as compared to the periovulatory phase, but did not affect basal cortisol in ovariectomized animals and did not alter responses to exogenous ACTH. These findings suggest that elevations in circulating CG concentrations are associated with elevated cortisol concentrations in female marmosets, and that this relationship requires simultaneous increases in ovarian hormones that occur only during the periovulatory period. Thus, suppression of CG release in anovulatory subordinate females may not play an important role in socially induced diminution of cortisol.

Hypoestrogenism does not mediate social suppression of cortisol in subordinate female marmosets.

Behaviorally subordinate female marmosets undergo social suppression of ovulation and hypoestrogenism, as well as chronic reductions in circulating basal cortisol concentrations. Because estrogen elevates hypothalamic-pituitary-adrenal axis activity and circulating glucocorticoid levels in other species, we tested the hypothesis that socially induced hypoestrogenism contributes to cortisol reductions in subordinate female marmosets. We characterized morning basal plasma cortisol levels, as well as cortisol responses to exogenous adrenocorticotropic hormone (ACTH; 0, 1, or 10 microg/kg), in seven anovulatory subordinate females and six ovariectomized, non-subordinate females under two conditions: during long-term treatment with estradiol (E2) and control. Circulating E2 and cortisol levels were compared to those of six dominant females undergoing ovulatory cycles. Basal cortisol concentrations in the control condition were significantly lower in subordinates than in both dominant and ovariectomized females. E2 treatment elevated circulating E2 levels of subordinate and ovariectomized females into the range seen in dominant females but did not increase either mean basal or ACTH-stimulated cortisol levels. To the contrary, E2 treatment caused a decline in basal cortisol levels over time, especially in ovariectomized animals. These results indicate that treatment with exogenous estrogen does not elevate circulating cortisol levels in previously hypoestrogenemic female marmosets and, correspondingly that socially induced hypoestrogenism does not diminish cortisol levels in subordinate females.