[Show abstract][Hide abstract] ABSTRACT: Bisphenol A (BPA) is a widespread environmental contaminant, and humans are routinely exposed to BPA. We investigated whether prenatal exposure to BPA influences behavioral development in juvenile cynomolgus monkeys (Macaca fascicularis). Pregnant cynomolgus monkeys were implanted with subcutaneous pumps and exposed to 10μg/kg/day BPA or vehicle (control) from gestational day 20 to 132. Both BPA-exposed and control juvenile monkeys (aged 1-2years) were assessed using the peer-encounter test that was conducted to evaluate behaviors in social interaction with a same-sex, same-treatment peer. In the encounter test, prenatal BPA exposure significantly reduced environmental exploration and presenting, a gesture related to sexual reproduction, and increased visual exploration, but only in males; furthermore, it significantly reduced the typical sexual dimorphism of the aforementioned behaviors normally observed between male and female juvenile cynomolgus monkeys. This study demonstrates that prenatal BPA exposure affects behavioral development during adolescence and results in the demasculinization of key sexually dimorphic behaviors in male juvenile monkeys.
No preview · Article · May 2014 · Neurotoxicology and Teratology
[Show abstract][Hide abstract] ABSTRACT: Acetamiprid (ACE) and imidacloprid (IMI) belong to a new, widely used class of pesticide, the neonicotinoids. With similar chemical structures to nicotine, neonicotinoids also share agonist activity at nicotinic acetylcholine receptors (nAChRs). Although their toxicities against insects are well established, their precise effects on mammalian nAChRs remain to be elucidated. Because of the importance of nAChRs for mammalian brain function, especially brain development, detailed investigation of the neonicotinoids is needed to protect the health of human children. We aimed to determine the effects of neonicotinoids on the nAChRs of developing mammalian neurons and compare their effects with nicotine, a neurotoxin of brain development.
[Show abstract][Hide abstract] ABSTRACT: Polychlorinated biphenyls (PCBs) are endocrine disrupting chemicals that disturb normal development of embryonic brains. In the present study, we evaluated the relationship between maternal plasma PCB concentration and infant behavioral characteristics in mother-infant interactions. We grouped 20 pregnant cynomolgus monkeys (Macaca fascicularis) into higher and lower PCB exposure groups; monkeys in the higher PCB group had PCB concentrations above 15 pg/g, which is representative of natural exposure levels. Maternal PCB concentration correlated negatively with infant behaviors (approach, look, proximity, locomotion) at the age of 6 months (p < .05), when an increase in these behaviors should normally occur. These results suggest that maternal PCB exposure may affect the development of infant social behavior in cynomolgus monkeys. Furthermore, this study provides primate evidence to support observations of associations between behavioral and learning disabilities and prenatal exposure to PCBs in humans.
No preview · Article · Jan 2011 · Developmental Psychobiology
[Show abstract][Hide abstract] ABSTRACT: Bisphenol A (BPA) is an environmental chemical with physiological potencies that cause adverse effects, even at environmentally relevant exposures, on the basis of a number of studies in experimental rodents. Thus, there is an increasing concern about environmental exposure of humans to BPA. In the present study, we used experimentally controlled cynomolgus monkeys (Macaca fascicularis) to assess the influence of prenatal exposure to BPA (10 microg/(kg day)) via subcutaneously implanted pumps and examined social behaviors between infants and their mothers during the suckling period. Mother-infant interactions in cynomolgus monkeys had behavioral sexual dimorphism associated with sex of infant from early suckling period. Prenatal exposure to BPA altered the behaviors of male infants significantly; BPA-exposed male infants behaved as female infants. And it also affected some of female infant behaviors. Consequently, gestational BPA exposure altered some behaviors of their mothers, mainly in male-nursing mothers. These results suggest that BPA exposure affects behavioral sexual differentiation in male monkeys, which promotes the understanding of risk of BPA exposure in human.
No preview · Article · May 2009 · Psychoneuroendocrinology
[Show abstract][Hide abstract] ABSTRACT: Polychlorinated biphenyls (PCBs), major environmental hormonally active agents, are metabolized into hydroxylated PCBs in the liver to facilitate excretion. Some of hydroxylated PCBs also have potencies disturbing endogenous hormonal activities at least in vitro. Hormonal activities of hydroxylated PCBs raise a possibility of their interfering with normal brain development which is strictly regulated by endogenous hormones. We investigated whether and how prenatal exposure to a congener of hydroxylated PCBs (4-OH-2',3,3',4',5'-penta CB; 4-OH-PCB106) having activities to disrupt thyroid hormone-dependent signals in vitro could perturb normal gene expression in the developing brain in vivo. Pregnant rats were exposed to 4-OH-PCB106 subcutaneously at the dose of 1.0mg/(kgday) from day 7 of gestation to postnatal day 1. Then three brain regions (cerebral cortex, hippocampus and striatum) were obtained from offspring on postnatal day 1 and subjected to further gene expression analyses. Comprehensive analyses of mRNA expression by oligo DNA microarrays and subsequent validations by quantitative RT-PCR revealed that prenatal exposure to 4-OH-PCB106 affected mRNA expression of glutamate receptors as well as that of thyroid hormone-responsive genes in region-specific manners. Concomitantly 4-OH-PCB106 exposure increased mRNA expression of genes related to exocytosis in the three brain regions. These results raise the possibility that prenatal exposure to some hydroxylated PCBs with thyroid hormone-disrupting potencies leads to abnormal brain development via perturbations on the expression of genes involved in glutamatergic neurotransmission.
[Show abstract][Hide abstract] ABSTRACT: Background: Deficits of function in autism spectrum disorder are explained by abnormal neural connectivity as a consequence of alternations in synapse formation during development. Thyroid hormones (THs) are essential for functional brain development through TH-dependent gene expressions which are disrupted by polychlorinated biphenyls (PCBs) at pM order concentrations (Miyazaki et al.J.B.C.279:18195,2004). PCBs and their metabolites hydroxy-PCBs (OH-PCBs), which have similar chemical structure to THs, have accumulated in almost all human blood examined, even in their brain by global environmental contamination. 4- OH-PCB 187 is found as the major OH-PCB in human cerebrospinal fluid (Takasuga et al.Org.Halogen Comp.66:2529,2004). These toxic chemicals pass easily through placenta to fetal brain where the blood-brain barrier has not yet developed. We reported two other OH-PCB congeners inhibited TH-dependent dendrite arborization of Purkinje cells in culture (Kimura-Kuroda et al.Dev.Brain Res.154:259,2005). Monkey experiments showed maternal PCBs exposure caused some difficulties in the development of social communications of their offspring (Nakagami et al., 2007). Objectives: Effects of OH-PCB congeners on synapse formation and dendrite extension were investigated using cultured Purkinje cells of fetal mice. Methods: Dissociated cerebellar culture was prepared as previously described (Kimura-Kuroda et al.,Dev.Brain Res.137:55,2002).After OH-PCB treatments, Purkinje cells and synapses were immunostained , observed by confocal laser microscope and quantified using CCD - MetaMorph imaging system. Results: Addition of 4-OH-PCB 187 significantly inhibited synapse formation on Purkinje cells at pM order concentrations. 4-OH-PCB 187 also caused abnormal dendrite extension in the Purkinje cells. Conclusions: These data further support the hypothesis that interaction with genetic backgrounds, PCBs (and probably other neurotoxic chemicals) contaminated in perinatal brain cause abnormal synaptic connections during development, result in heterogeneous symptoms of autism spectrum disorders and/or other comorbid disorders like LD, ADHD, depending on spatio-temporal difference in sensitive synaptogenesis and in chemical exposure doses (Kuroda, Environ. Sci.,10:23,2003).
[Show abstract][Hide abstract] ABSTRACT: Polychlorinated biphenyls (PCBs) and hydroxy-PCB (OH-PCB) metabolites are widely distributed bioaccumulative environmental chemicals and have similar chemical structures to those of thyroid hormones (THs). Previously, we reported that THs are essential for neuronal development and the low doses of two OH-PCBs, namely, 4-OH-2',3,3',4',5'-pentachlorobiphenyl (4'-OH-PeCB-106) and 4-OH-2',3,3',4',5,5'-hexachlorobiphenyl (4'-OH-HxCB-159), inhibited the TH-dependent dendritic development of Purkinje cells in mouse cerebellar cultures using serum-free defined medium. To determine which type of OH-PCBs affect neuronal development, we further examined several OH-PCBs and other estrogenic chemicals using this simple and sensitive assay system. Two-way ANOVA was used to assess the effects of OH-PCBs and other chemicals on both factors of their concentrations and with/without T4 in the assay of TH-dependent dendritic development of Purkinje cells. Aside from the two OH-PCBs, 4-OH-2',3,4',5,6'-pentachlorobiphenyl (4'-OH-PeCB-121) and bisphenol A significantly inhibited the TH-dependent dendritic development of Purkinje cells, whereas 4-OH-2',3,3',5',6'-pentachlorobiphenyl (4'-OH-PeCB-112), 4-OH-2',3,3',5,5',6'-hexachlorobiphenyl (4'-OH-HxCB-165), 4-OH-2,2',3,4',5,5',6-heptachlorobiphenyl (4-OH-HpCB-187), progesterone and nonylphenol did not induce any inhibition, but significantly promoted the dendritic extension of Purkinje cells in the absence of THs. Other estrogenic chemicals, including beta-estradiol, diethyl stilbestrol and p-octylphenol did not show significant inhibitory or promoting effects. From these results, it is suggested that exposure to OH-PCBs and other environmental chemicals may disrupt normal neuronal development and cause some developmental brain disorders, such as LD, ADHD, and autism.
[Show abstract][Hide abstract] ABSTRACT: Pregnant rhesus monkeys (Macaca mulatta) were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 30 and 300 ng/kg by subcutaneous injection at gestational day 20, followed by additional injections of TCDD (1.5 and 15 ng/kg, respectively) every 30 days till 90 days after parturition. The offspring delivered from these experimentally TCDD-exposed mothers were subjected to a series of behavioral tests after the weaning at 12-14 months old (MO): a finger maze learning task (12-15 MO), encounter tests between two monkeys (at 12-15 and 24-27 MO), and an eye-contact test (23-26 MO) to estimate learning ability, social interaction with a peer subject, and interest or hostility to a human observer, respectively. TCDD exposure had no significant effect on learning ability or interest/hostility to an observer. It did, however, significantly affect behavioral characteristics in the encounter tests. In the first encounter test, monkeys exposed to TCDD showed more visual exploration and mutual proximity but less stereotypy behavior compared to control monkeys. In the second encounter test, these differences seemed to disappear, suggesting that the behavioral effects of TCDD exposure in the encounter tests might disappear as the monkey develops. This study produced evidence of the behavioral toxicity of TCDD in social interactions using non-human primates.
No preview · Article · Feb 2006 · Toxicology Letters
[Show abstract][Hide abstract] ABSTRACT: Use of DNA microarrays in neuroscience have been limited to rough screening purposes, mainly because the reliability and sensitivity of available arrays are not high enough. Because only a few hundred to one thousand genes are usually found to change expression levels in most experiments, we attempted to develop a more quantitative array by the following strategies: 1) limit the genes to those relevant to the experimental system, 2) design oligonucleotide probes of specified molecular properties so that more stringent hybridization conditions can be employed, 3) place six spots per probe on one slide and use multiple normalization genes, and 4) use a new type of gold-coated slide with higher S/N ratio. Genes involved in the construction and functioning of the synapse were selected from the literature as well as experimentally by comparing cerebella from hypothyroid and control mice at postnatal day 15 (P15). Although the number of genes covered was not yet large (172 genes), the custom array "Synaptoarray" thus constructed was capable of detecting +/-20% difference in gene expression ratios. Analysis of the postnatal development of the mouse cerebellum by using Synaptoarray demonstrated a general expression pattern with a peak at P7, followed by a decline at P15 and a partial recovery after P21. P10 clearly marked the end of the initial growth stage and a major transcriptional turning point in this system. This result suggests that such a custom array should be useful in monitoring perturbations to the normal developmental program.
No preview · Article · Jun 2005 · Journal of Neuroscience Research
[Show abstract][Hide abstract] ABSTRACT: Thyroid hormone is essential for the proper development of the mammalian central nervous system (CNS). In the present study, we examined behavioural alterations caused by transient perinatal hypothyroidism induced by an anti-thyroid drug, propylthiouracil (PTU). This drug produces perinatal disruption of the thyroid system and subsequent behavioural changes, which we investigated using a series of behavioural tests and focusing particularly on attention-deficit/hyperactivity disorder (ADHD)-like behaviours. In the open field test, both male and female rats that had experienced perinatal hypothyroidism (HT rats) showed an increased percent of locomotion behaviour and reduced grooming behaviour, suggesting that HT rats may be hyperactive and show fewer anxiety characteristics. Neither male nor female HT rats showed retention in the passive avoidance test. Male HT rats showed a significantly lower rate of correct avoidance responses than control rats in earlier sessions in the active avoidance test. In addition, we observed significant increases in the number of times that rats crossed the partition during inter-trial intervals and the percent of failure of avoidance during 5 s electrical stimuli in HT rats, suggesting that HT rats are restless, have a shortened attention span and panic easily. In measuring spontaneous motor activity during a period of darkness, male HT rats appeared to plunge into active phase with short, quick steps, while male control rats showed only long active phases during a stress-free period of darkness. These abnormal behavioural characteristics in HT rats might coincide with those found in some cases of ADHD.
No preview · Article · May 2005 · Behavioural Brain Research
[Show abstract][Hide abstract] ABSTRACT: Thyroid hormones (THs) are important for brain development, and polychlorinated biphenyl (PCB) accumulation in humans is a serious problem because PCBs may affect TH functions. To determine the effects of hydroxylated metabolites of PCBs (OH-PCBs) on brain development, we performed mouse cerebellar culture assays. 4-OH-2',3,3',4',5'-pentachlorobiphenyl and 4-OH-2',3,3',4',5,5'-hexachlorobiphenyl significantly inhibited the TH-dependent extension of Purkinje cell dendrites even at 5 x 10(-11) M and 5 x 10(-12) M, respectively. OH-PCBs may disturb TH-dependent brain development.
No preview · Article · Mar 2005 · Developmental Brain Research
[Show abstract][Hide abstract] ABSTRACT: We previously established a primary culture system of the accessory olfactory bulb (AOB) to investigate the functional roles of individual types of neuron in pheromonal signal processing. However, the detailed characteristics of cultured AOB neurons were not yet apparent. In the present study, we address the cytological aspects of cultured AOB neurons using immunocytochemical staining methods. Cultured AOB neurons were compared with cultured main olfactory bulb (MOB) neurons in neuronal composition, maturational time course, and cell size. The number of total neurons, measured by microtubule-associated protein 2 (MAP2) immunostaining, progressively decreased, and glutamic acid decarboxylase positive (GAD+) interneurons were scarcely changed in their number in both AOB and MOB cultures over the culture periods. In contrast, the number of tyrosine hydroxylase positive (TH+) neurons in AOB cultures showed a slight, but significant, increase over time in culture, while those in MOB cultures remarkably decreased. The numbers of total neurons and GAD+ neurons were significantly greater in AOB cultures than in MOB cultures at all investigated time points. However, the numbers of TH+ neurons were lower at 7 days in vitro (DIV) and greater at 21 DIV in AOB cultures than in MOB cultures. The somatic sizes of all types of neurons at 14 DIV were significantly larger in AOB cultures than in MOB cultures. Furthermore, the frequency distributions of somatic sizes of total, GAD+, and TH+ neurons were significantly different between AOB and MOB cultures. These subtle differences in vitro may reflect in vivo differences between the AOB and MOB.
Full-text · Article · Jan 2005 · Anatomy and Embryology
[Show abstract][Hide abstract] ABSTRACT: 1. Synchronized spontaneous intracellular Ca2+ spikes in networked neurons are believed to play a major role in the development and plasticity of neural circuits. Glutamate-induced signals through the ionotropic glutamate receptors (iGluRs) are profoundly involved in the generation of synchronized Ca2+ spikes.
1 2. In this study, we examined the involvement of metabotropic glutamate receptors (mGluRs) in cultured mouse cortical neurons. We pharmacologically revealed that glutamate-induced signals through inclusive mGluRs decreased the frequency of Ca2+ spikes. Further experiments indicated that this suppressive effect on the spike frequency was mainly due to the signal through group II mGluR, inactivation of adenylate cyclase-cAMP-PKA signaling pathway. Group I mGluR had little involvement in the spike frequency.
3. Taken together, glutamate generates the synchronized Ca2+ spikes through iGluRs and modulates simultaneously their frequency through group II mGluR–adenylate cyclase–cAMP–PKA signaling pathway in the present in vitro neural network. These results provide the evidence of the profound role of group II mGluR in the spontaneous and synchronous neural activities.
No preview · Article · Jan 2005 · Cellular and Molecular Neurobiology
[Show abstract][Hide abstract] ABSTRACT: 1. Synchronous oscillation of intracellular Ca2+ in the central nervous system is essential for neural development. We previously reported that endogenous dopamine was involved with synchronous Ca2+ oscillation of primary cultured midbrain neurons, and that regulation of dopamine in synchronous oscillation was distinctly different through dopamine receptor 1 (D1R) and 2 (D2R): the action of dopamine through D1R or D2R was facilitative or suppressive, respectively, to the Ca2+ influx of synchronous oscillation. 2. In the present study, we confirmed that the suppressive effects of D2R were mediated by the regulation of the L-type voltage-gated Ca2+ channel, not by the regulation of NMDA receptor on the Ca2+ influx in the midbrain neural network showing synchronous oscillation. 3. This evidence promotes better understanding of the regulation of neural activity by endogenous dopamine in networked neurons.
No preview · Article · Jan 2005 · Cellular and Molecular Neurobiology
[Show abstract][Hide abstract] ABSTRACT: The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.
No preview · Article · Oct 2004 · Developmental Brain Research
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to examine whether perinatal exposure to two major environmental endocrine-disrupting chemicals, bisphenol A (BPA; 0.1 mg/kg/day orally) and nonylphenol [NP; 0.1 mg/kg/day (low dose) and 10 mg/kg/day (high dose) orally] daily from gestational day 3 to postnatal day 20 (transplacental and lactational exposures) would lead to behavioral alterations in the male offspring of F344 rats. Neither BPA nor NP exposure affected behavioral characteristics in an open-field test (8 weeks of age), in a measurement of spontaneous motor activity (12 weeks of age), or in an elevated plus-maze test (14 weeks of age). A passive avoidance test (13 weeks of age) showed that both BPA- and NP-treated offspring tended to delay entry into a dark compartment. An active avoidance test at 15 weeks of age revealed that BPA-treated offspring showed significantly fewer avoidance responses and low-dose NP-treated offspring exhibited slightly fewer avoidance responses. Furthermore, BPA-treated offspring significantly increased the number of failures to avoid electrical unconditioned stimuli within 5-sec electrical shock presentation compared with the control offspring. In a monoamine-disruption test using 5 mg/kg (intraperitoneal) tranylcypromine (Tcy), a monoamine oxidase inhibitor, both BPA-treated and low-dose NP-treated offspring at 22–24 weeks of age failed to show a significant increment in locomotion in response to Tcy, whereas control and high-dose NP-treated offspring significantly increased locomotion behavior after Tcy injection. In addition, when only saline was injected during a monoamine-disruption test, low-dose NP-treated offspring showed frequent rearing compared with the control offspring. The present results indicate that perinatal low-dose BPA or NP exposure irreversibly influenced the reception of fear-provoking stimuli (e.g., electrical shock), as well as monoaminergic neural pathways.
Full-text · Article · Sep 2004 · Environmental Health Perspectives
[Show abstract][Hide abstract] ABSTRACT: Synapse plasticity, in particular, formation of new synapses, plays crucial roles in learning and memory. We have developed a convenient assay system for measuring the number of newly formed synapses between cultured rat cerebrocortical neurons using the multisite fluorometry system of intracellular calcium. We found that cultured neurons exhibited spontaneous oscillatory changes in intracellular calcium levels and that the frequency of the oscillation was strongly correlated with synaptic density. Combined with immunohistochemical studies, this assay system enables us to study the molecular mechanism of synapse formation, in particular, the involvement of ecto-protein kinase. Other applications of the assay system are discussed here.
No preview · Article · Aug 2004 · Folia Pharmacologica Japonica