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Dysregulation of the oxytocinergic system and excitation/inhibition (E/I) balance in synaptic transmission and neural circuits are common hallmarks of various neurodevelopmental disorders. Several experimental and epidemiological studies have shown that perinatal exposure to endocrine-disrupting chemicals bisphenol A (BPA) and bisphenol S (BPS) may contribute to a range of childhood neurodevelopmental disorders. However, the effects of BPA and BPS on social-cognitive development and the associated mechanisms remain largely unknown. In this study, we explored the impacts of early developmental exposure (2hpf-5dpf) to environmentally relevant concentrations of BPA, and its analog BPS (0.001, 0.01, and 0.1 μM), on anxiety, social behaviors, and memory performance in 21 dpf zebrafish larvae. Our results revealed that early-life exposure to low concentrations of BPA and BPS elevated anxiety-like behavior, while fish exposed to higher concentrations of these chemicals displayed social deficits and impaired object recognition memory. Additionally, we found that co-exposure with an aromatase inhibitor antagonized BPA- and BPS-induced effects on anxiety levels and social behaviors, while the co-exposure to an estrogen receptor antagonist restored recognition memory in zebrafish larvae. These results indicate that BPA and BPS may affect social-cognitive function through distinct mechanisms. On the other hand, exposure to low BPA/BPS concentrations increased both the mRNA and protein levels of isotocin (zebrafish oxytocin) in the zebrafish brain, whereas a reduction in its mRNA level was observed at higher concentrations. Further, alterations in the transcript abundance of chloride transporters, and molecular markers of gamma-aminobutyric acid (GABA) and glutamatergic systems, were observed in the zebrafish brain, suggesting possible E/I imbalance following BPA or BPS exposure. Collectively, the results of this study demonstrate that early-life exposure to low concentrations of the environmental contaminants BPA and BPS can interfere with the isotocinergic signaling pathway and disrupts the establishment of E/I balance in the developing brain, subsequently leading to the onset of a suite of behavioral deficits and neurodevelopmental disorders.

Phthalates are a class of endocrine disruptors found in a variety of consumer goods, and offspring can be exposed to these compounds during gestation and lactation. Our laboratory has found that perinatal exposure to an environmentally relevant mixture of phthalates resulted in a decrease in cognitive flexibility and in neuron number in the adult rat medial prefrontal cortex (mPFC). Here, we examine effects of phthalate treatment on prenatal cellular proliferation and perinatal apoptosis in the mPFC. To examine the phthalate effects on cellular proliferation, dams consumed 0, 1, or 5 mg/kg of the phthalate mixture daily from embryonic day 2 (E2) through the day of birth (P0), and on E16 and E17, they were injected with BrdU. The mPFC of offspring was analyzed on P5 and showed a decrease in labelled cells in the phthalate exposed groups. To examine whether changes in BrdU density observed on P5 were due to altered cell survival, cell death was measured on E18, P0, and P5 using a TUNEL assay in a separate cohort of prenatally exposed offspring. There was an increase in TUNEL labelled cells at E18 in the phthalate exposed groups. In the final experiment, dams consumed the phthalate mixture from E2 through P10, at which time mPFC tissue was stained with TUNEL. Phthalate treated subjects showed a higher density of apoptotic cells at P10. These results indicate both pre- and postnatal phthalate exposure increases apoptosis in the male and female rat mPFC. While the impact of phthalates on proliferation cannot be ruled out, these data do not allow for definitive conclusions.

Exposure to fine ambient particulates (PM(2.5)) during gestation or neonatally has potent neurotoxic effects. While biological and behavioral data indicate a vulnerability to environmental pollutants across distinct neurodevelopmental windows, the behavioral consequences following exposure across the entire developmental period remain unknown. Moreover, several epidemiological studies support a link between developmental exposure to air pollution and an increased risk of later receiving a diagnosis of autism spectrum disorders (ASD), a neurodevelopmental disorder that persists throughout life. In the current study we sought to determine whether perinatal exposure to PM(2.5) would reduce sociability and increase repetitive deficits in mice, two hallmark characteristics of ASD. Pregnant female B(6)C(3) mice were exposed daily to concentrated ambient PM(2.5) (CAPs) (135.8 μg/m(3)) or filtered air (3.1 μg/m(3)) throughout gestation followed by additional exposures to both dams and their litters from days 2-10 postpartum. Adult offspring were subsequently assessed for social and repetitive behaviors at 20 weeks of age. Daily perinatal exposure to CAPs significantly decreased sociability in male and female mice as measured by the social approach task; however, reductions in reciprocal social interaction and increased grooming behavior were only present in male offspring exposed to CAPs. These findings demonstrate that exposure to particulate air pollutants throughout early neurodevelopment induces long lasting behavioral deficits in a sex-dependent manner and may be an underlying cause of neurodevelopmental disorders such as ASD.

The impact of sub-lethal doses of herbicides on human health and the environment is a matter of controversy. Due to the fact that evidence particularly of the effects of glyphosate on the central nervous system of rat offspring by in utero exposure is scarce, the purpose of the present study was to assess the neurobehavioral effects of chronic exposure to a glyphosate-containing herbicide during pregnancy and lactation. To this end, pregnant Wistar rats were exposed through drinking water to 0.2% or 0.4% of a commercial formulation of glyphosate (corresponding to a concentration of 0.65 or 1.30 g/L of glyphosate, respectively) during pregnancy and lactation and neurobehavioral alterations in offspring were analyzed. The postnatal day on which each pup acquired neonatal reflexes (righting, cliff aversion and negative geotaxis) and that on which eyes and auditory canals were fully opened were recorded for the assessment of sensorimotor development. Locomotor activity and anxiety levels were monitored via open field test and plus maze test, respectively, in 45- and 90-day-old offspring. Pups exposed to a glyphosate-based herbicide showed early onset of cliff aversion reflex and early auditory canal opening. A decrease in locomotor activity and in anxiety levels was also observed in the groups exposed to a glyphosate-containing herbicide. Findings from the present study reveal that early exposure to a glyphosate-based herbicide affects the central nervous system in rat offspring probably by altering mechanisms or neurotransmitter systems that regulate locomotor activity and anxiety. Fil: Gallegos, Cristina Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina Fil: Bartos, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina Fil: Bras, Cristina Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina Fil: Gumilar, Fernanda Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina Fil: Antonelli, Marta Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina Fil: Minetti, Silvia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina

Early life exposure to endocrine disruptors is considered to disturb normal development of hormone sensitive parameters and contribute to advanced puberty and reduced fecundity in humans. Kisspeptin is a positive regulator of the hypothalamic-pituitary-gonadal axis, and plays a key role in the initiation of puberty. In the adult, Kiss1 gene expression occurs in two hypothalamic nuclei, namely the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), which are differentially regulated by peripheral sex steroid hormones. In this study we determined the effects on puberty onset and Kiss1 mRNA levels in each of the two nuclei after long-term perinatal exposure of rats to ethinyl oestradiol (EE2) or to five different pesticides, individually and in a mixture. Rat dams were per orally administered with three doses of EE2 (5, 15 or 50 μg/kg/day) or with the pesticides epoxiconazole, mancozeb, prochloraz, tebuconazole, and procymidone, alone or in a mixture of the five pesticides at three different doses. Kiss1 mRNA expression was determined in the AVPV and in the ARC of the adult male and female pups in the EE2 experiment, and in the adult female pups in the pesticide experiment. We find that perinatal EE2 exposure did not affect Kiss1 mRNA expression in this study designed to model human exposure to estrogenic compounds, and we find only minor effects on puberty onset. Further, the Kiss1 system does not exhibit persistent changes and puberty onset is not affected after perinatal exposure to a pesticide mixture in this experimental setting. However, we find that the pesticide mancozeb tends to increase Kiss1 expression in the ARC, presumably through neurotoxic mechanisms rather than via classical endocrine disruption, calling for increased awareness that Kiss1 expression can be affected by environmental pollutants through multiple mechanisms.

Bisphenol A (BPA) is widely used in polymer products in food and beverage containers, baby bottles, dental sealants and fillings, adhesives, protective coatings, flame retardants, water supply pipes, and compact discs, and is found in the environment and in placental tissue, fetuses and breast milk. We have recently reported that a single neonatal exposure to bisphenol A can induce persistent aberrations in spontaneous behavior, in a dose-dependent manner, and affect the adult response to the cholinergic agent nicotine. Furthermore, other recent reports indicate that pre- and perinatal exposure to bisphenol A can induce neurotoxic effects. The present study indicates that a single neonatal exposure to bisphenol A, on postnatal day 10, during the peak of the brain growth spurt, can alter the adult levels of proteins important for normal brain development (CaMKII and synaptophysin). These alterations are induced in both male and female mice and effects are seen in both hippocampus and cerebral cortex. These results further support our recent study showing that neonatal exposure to bisphenol A can act as a developmental neurotoxicant and the effects are similar to effects seen after a single postnatal exposure to other POPs, such as PBDEs, PCBs and PFCs.

Perinatal exposure to methylmercury (MeHg) in rodents has been linked to changes in sensitivity to dopaminergic agents later in life. In an effort to determine the behavioral and neurochemical response to the indirect dopaminergic and serotonergic agonist amphetamine following neonatal exposure to MeHg, male BALB/c mice were administered MeHg during critical periods of neural development and challenged with amphetamine as adults. Mice were observed 15, 30 and 60 min after a single amphetamine injection (7.5 mg/kg i.p.) for presence of stereotypic and self-injurious behaviors, abnormal posture, and hyperthermia. Mice treated with 2 or 4 mg/kg MeHg on alternate days 3-15 of life demonstrated an increase in body temperature and the appearance of stereotypic and self-injurious behaviors not observed when amphetamine was administered to either vehicle-exposed mice or those treated with an equivalent total amount of MeHg administered on postnatal days 13 and 15. Neurochemical analysis of MeHg- and vehicle-exposed mice challenged with amphetamine or saline revealed alterations in dopaminergic and serotonergic activity which corresponded to the sensitized behavioral response to amphetamine. These observations demonstrate a critical window for MeHg exposure affecting the later appearance of amphetamine-induced self-injurious behavior and support the hypothesis that early exposure to environmental neurotoxicants may predispose individuals to engage in aberrant, intrusive behaviors later in life.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. The most toxic PCBs are the non-ortho-substituted (“dioxin-like”) congeners that act through the aryl hydrocarbon receptor (AHR) pathway. In humans, perinatal exposure to dioxin-like PCBs is associated with neurodevelopmental toxicity in children. Yet, the full potential for later-life neurobehavioral effects that result from early-life low level exposure to dioxin-like PCBs is not well understood. The objective of this study was to determine the effects of developmental exposure to low levels of dioxin-like PCBs on early-and later-life behavioral phenotypes using zebrafish as a model system. We exposed zebrafish embryos to either vehicle (DMSO) or low concentrations of PCB126 (0.3, 0.6, 1.2 nM) for 20 hours (4–24 hours post fertilization), and then reared them to adulthood in clean water. Locomotor activity was tested at two larval stages (7 and 14 days post fertilization). Adult fish were tested for anxiety-related behavior using the novel tank and shoaling assays. Adult behavioral assays were repeated several times on the same group of fish and effects on intra-and inter-trial habituation were determined. While there was no effect of PCB126 on larval locomotor activity in response to changes in light conditions, developmental exposure to PCB126 resulted in impaired short-and long-term habituation to a novel environment in adult zebrafish. Cyp1a induction was measured as an indicator for AHR activation. Despite high induction at early stages, cyp1a expression was not induced in the brains of developmentally exposed adult fish that showed altered behavior, suggesting that AHR was not activated at this stage. Our results demonstrate the effectiveness of the zebrafish model in detecting subtle and delayed behavioral effects resulting from developmental exposure to an environmental contaminant.

Dioxin and its related compounds are suspected to cause neurological and nueroendocrinological disruption in human and laboratory animal offspring upon in utero and lactational exposure during growth and development. We tested the hypothesis by utilizing Long-Evans Hooded rats that perinatal exposure to dioxins affects the neocortical function and expression of sexual behavior in adulthood. In the sexual behavior test, perinatal exposure to TCDD significantly reduced the number of mounts and intromissions. The mRNA semi-quantification in in situ hybridization showed that the mating stimulus in control males induced c-fos mRNA expression in the preoptic area (POA) and the brain derived neurotrophic factor (BDNF) mRNA upregulation in the frontal cortex. In contrast, perinatal exposure to TCDD lowered the upregulation of BDNF mRNA in the frontal cortex but not that of c-fos mRNA in the POA. The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was not affected. The results suggest that perinatal TCDD affects the neocortical function independently from the brain sexual differentiation and alters the expression of sexual behavior.

Polybrominated diphenyl ethers (PBDEs), a class of widely used flame retardants, are extensively diffused in the environment as shown by several studies on sentinel animal species, as well as humans. Of particular concern are the reported high levels of PBDEs in human milk, as almost no information is available on their potential effects on developing organisms. We investigated the effects of perinatal PBDE exposure on mouse neurobehavioural development. 2,2′,4,4′,5-pentabromodiphenylether (PBDE 99; 0.6, 6 and 30 mg/kg per day) was administered daily to CD-1 Swiss females by gavage from gestational day (GD) 6 to postnatal day (PND) 21. Aroclor 1254 (A1254; 6 mg/kg per day), a PCB mixture, was administered following the same schedule and served as a positive control. The PBDE 99 medium dose had an effect on litter viability. Sensori-motor development analysis (PNDs 2–20) revealed a delayed appearance of climbing response in the PBDE 99 high-dose group. On PND 11, the homing test revealed a trend for treated animals, particularly the A1254 group, to be more active than controls. This activity level alteration was strongly increased on PNDs 34 and 60 in an open-field arena. On PND 60, treated mice showed also an altered thigmotaxis, spending more time in the centre of the arena than controls. At adulthood, A1254 treated mice were still hyperactive, whereas the PBDE 99 groups tended to be hypoactive. These findings showed that perinatal exposure to PBDE 99 produces several behavioural alterations and that its effects are not always similar to those of A1254. The possibility of exposure of neonates to PBDEs warrants further studies to characterise their developmental neurotoxicity.

Nonylphenols (NP) are endocrine-disruptors known to be widely present in our environment. This study evaluated the effects of 4-n-NP on neurobehavioral development and memory capacity after perinatal exposure on the offspring rats. Dams were gavaged with 4-n-NP (0, 50 and 200 mg/kg/day) from gestational day 5 to postnatal day (PND) 21. Dams exposed to the higher dose lost weight during gestation and had a longer gestational duration. Juvenile female pups of the 200 mg 4-n-NP/kg/day group were lighter. Their thyroid somatic index (TSI) was also affected. For male pups, a decrease of TSI at weaning for the 200 mg 4-n-NP/kg/day group and an increase of GSI for the 50 mg 4-n-NP/kg/day group were observed. Physical maturation (incisives and eyes) were likewise affected. In open field (OF) tests, females were more active than males. In the first OF (PND 36), a treatment effect was observed only for males, particularly for the high dose group, which became as active as females. The second OF (PND 71) showed few differences between groups (treated vs control), the gender difference whatever the dose was not abolished. In the Morris Water Maze test, the study of the first 30 s showed that females (200 mg/kg/day) were mainly affected. Their performances were improved by 4-n-NP. These effects were particularly important for the first short-term memory test and observed to a lesser extent in the second evaluation of the long-term memory (PND 69). These data showed that perinatal 4-n-NP exposure induced behavioral and neuro-developmental impairments from 50 mg/kg/day.

Developmental lead (Pb) exposure is associated with cognitive impairments in humans and rodents alike. In particular, impaired spatial learning and memory, as assessed using the Morris water maze (MWM), has been noted in developmentally Pb –exposed rats. Although sex and rearing environment can influence MWM performance in normal animals, the interactions of sex and rearing environment on the impact of developmental Pb exposure on hippocampal-dependent processes has not been well characterized. The present study examined the effects of perinatal exposure (i.e., gestation through weaning) to different levels of Pb (250, 750 and 1,500 ppm Pb acetate in food) in males and females raised in a non-enriched environment (standard cage with 3 animals and no toys) or an enriched environment (large cage containing a variety of toys that were changed twice weekly). Testing in the MWM began at postnatal day 55. Behavioral outcomes were influenced by sex and rearing environment, with complex interactions with Pb exposure. In non-Pb exposed control animals, beneficial effects of environmental enrichment on spatial learning and memory were observed in males and females, with greater effects in females. Pb exposure in females mitigated at least some of the benefits of enrichment on learning, particularly at the lowest and highest exposure levels. In males, enrichment conferred a modest learning advantage and for the most part, Pb exposure did not affect this. However, in males with the highest Pb exposure, enrichment did help to overcome detrimental effects of Pb on learning. In females, any potential benefit to reference memory contributed by enrichment was muted by exposure to Pb and for the most part, this was not reproduced in males. Thus, there are complex interactions between sex, environment, and Pb exposure on spatial learning and memory. Environmental manipulation is a potential risk modifier of developmental Pb exposure and interacts with other factors including sex and amount of Pb exposure to affect the functional influences of Pb on the brain.

Arsenic is one of the most common heavy metal contaminants found in the environment, particularly in water. We examined the impact of perinatal exposure to relatively low levels of arsenic (50 parts per billion, ppb) on neuroendocrine markers associated with depression and depressive-like behaviors in affected adult C57BL/6J mouse offspring. Whereas most biomedical research on arsenic has focused on its carcinogenic potential, a few studies suggest that arsenic can adversely affect brain development and neural function. Compared to controls, offspring exposed to 50 parts per billion arsenic during the perinatal period had significantly elevated serum corticosterone levels, reduced whole hippocampal CRFR 1 protein level and elevated dorsal hippocampal serotonin 5HT 1A receptor binding and receptor-effector coupling. 5HT 1A receptor binding and receptor-effector coupling were not different in the ventral hippocampal formation, entorhinal or parietal cortices, or inferior colliculus. Perinatal arsenic exposure also significantly increased learned helplessness and measures of immobility in a forced swim task. Taken together, these results suggest that perinatal arsenic exposure may disrupt the regulatory interactions between the hypothalamic-pituitary-adrenal axis and the serotonergic system in the dorsal hippocampal formation in a manner that predisposes affected offspring to depressive-like behavior. These results are the first to demonstrate that relatively low levels of arsenic exposure during development can have long-lasting adverse effects on behavior and neurobiological markers associated with these behavioral changes.

Perinatal cadmium (Cd) exposure has been shown to alter behaviors and reduce learning ability of offspring. A few studies have shown that Cd reduced serum thyroid hormones (THs), which are important for brain development during the perinatal period. Brain specific genes, neurogranin (RC3) and myelin basic protein (BMP), are known to be regulated by TH through TH receptors (TR). It has been suggested that RC3 may play roles in memory and learning. In addition, Cd has been suggested to have estrogen-like activity. To evaluate the effects of perinatal low-dose exposure to Cd on thyroid hormone-related gene (RC3, TR-beta1, MBP, RAR-beta) and sex hormone receptor gene (ER-alpha, ER-beta and PgR) expressions in the brain and on behaviors of offspring, mice were administered with 10ppm Cd (from gestational day 1 to postnatal day 10) and/or 0.025% methimazole (MMI; anti-thyroid drug) (from gestational day 12 to postnatal day 10) in drinking water. Also, 0.1% MMI was administered as a positive control (high MMI group). RC3 mRNA expression was reduced in the female brain of combined exposure and high MMI groups and was negatively correlated with the activity in the open-field. ER-alpha, ER-beta and PgR mRNA expressions were decreased in male and female Cd, and female Cd+MMI groups, respectively; among these changes the reduced expression of PgR was opposite to estrogenic action. These results suggested that perinatal exposure to Cd disrupted the gene expressions of sex hormone receptors, which could not be considered to be a result of estrogenic action. Our study indicates that alteration in the gene expressions of RC3 and sex hormone receptors in the brain induced by perinatal Cd and MMI exposure might be one mechanism of developmental toxicity of Cd.

Polybrominated diphenyl ethers (PBDEs), a class of widely used flame retardants, are becoming widespread environmental pollutants, as indicated by studies on sentinel animal species, as well as humans. Of particular concern are the reported increasingly high levels of PBDEs in human milk, as should be given that almost no information is available on their potential effects on developing organisms. In order to address this issue, studies have been conducted in mice and rats to assess the potential neurotoxic effects of perinatal exposure to PBDEs (congeners 47, 99, 153 and the penta-BDE mixture DE-71). Characteristic endpoints of PBDE neurotoxicity are, among others, endocrine disruption (e.g. decreased thyroid hormone levels), alteration in cholinergic system activity (behavioral hyporesponsivity to nicotine challenge), as well as alterations of several behavioral parameters. In particular, the main hallmark of PBDE neurotoxicity is a marked hyperactivity at adulthood. Furthermore, a deficit in learning and memory processes has been found at adulthood in neonatally exposed animals. Some of neurotoxic effects of PBDEs are comparable to those of polychlorinated biphenyls (PCBs), though the latter class of compounds seems to exert a stronger toxic effect. Available information on PBDE neurotoxicity obtained from animal studies and the possibility of neonatal exposure to PBDEs via the mother's milk suggest that these compounds may represent a potential risk for neurobehavioral development in humans.