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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.

Large numbers of senile plaques are thought to be characteristic of Alzheimer’s disease (AD), but these deposits are also a by-product of normal senescence. In AD and normal brains, senile plaques are primarily composed of amyloid-s peptides (As P) with aluminum (Al). Evidence suggests the oligomerization of As P is part of the molecular mechanism of AD pathogenesis by forming neurotoxic amyloid channels. However, the relationship between Al and AD has been a subject of scientific debate for many years. The complex nature of Al bioavailability has made it difficult to evaluate its toxicity to the human brain. In 2004, Al concentration in CSF of AD patients analyzed to be 1.6 ± 0.4 times higher than normal people. Importantly, AD patients with more Al in CSF showed less MMSE score, indicating Al may decrease cognitive ability. Recently, Al accumulations in sporadic AD and familial AD brains were reported to be much higher than in normal control brains. Above its neurotoxicity, Al3+ has a crucial role as a cross-linker in s-amyloid oligomerization. Therefore, I propose a hypothesis that s-amyloid oligomerizes with Al, forming non-specific cation amyloid channels in cell membranes, which allows calcium to enter cells and finally causes neuronal death by together with Al’s own neurotoxicity.

Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of longterm (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

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

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.

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.

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.

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.

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. Key words: behavior, bisphenol A, fear, learning, monoamine, nonylphenol.

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.

We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys ( Macaca fascicularis ). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N -methyl- d -aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage–colony stimulating factor (M–CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.

1. Thyroid hormones play important roles in the development of the brain. Increasing evidence suggests that the deprivation of thyroid hormones in the early developmental stage causes structural and functional deficits in the CNS, but the precise mechanism underlying this remains elusive. In this study, we investigated the effects of thyroid hormones on synapse formation between cultured rat cortical neurons, using a system to estimate functional synapse formation in vitro. 2. Exposure to 10(-9) M thyroid hormones, 3,5,3'-triiodothyronine or thyroxine, caused an increase in the frequency of spontaneous synchronous oscillatory changes in intracellular calcium concentration, which correlated with the number of synapses formed. 3. The detection of synaptic vesicle-associated protein synapsin I by immunocytochemical and immunoblot analysis also confirmed that exposure to thyroxine facilitated synapse formation. 4. The presence of amiodarone, an inhibitor of 5'-deiodinase, or amitrole, a herbicide, inhibited the synapse formation in the presence of thyroxine. 5. In conclusion, we established a useful in vitro assay system for screening of miscellaneous chemicals that might interfere with synapse formation in the developing CNS by disrupting the thyroid system.

1. Although microtubule-associated protein (MAP) 1B and its phosphorylation have been suggested to be important for synapse formation among cortical neurons, the localization of MAP1B in synapses has not yet been confirmed. In this report, we examine the localization of MAP1B in synaptic regions. 2. The localization of MAP1B was observed by immunohistochemical and electron microscopic techniques using specific antibodies against MAP1B. 3. MAP1B immunoreactivities were widely distributed in the cerebral cortex and were observed in the postsynaptic area but not in presynaptic terminals. 4. These synapses were classified as the asymmetrical type. 5. Only some synapses exhibited MAP1B immunoreactivities. MAP1B-immunopositive synapses accounted for about half of the total synapses. 6. Such a localization suggests MAP1B's important roles in synaptic functions.

The objective of this investigation is to evaluate whether perinatal maternal exposure to bisphenol A (BPA) at 4, 40, and 400 mg/kg per day affects the behavior of offspring in F344 rats. Perinatal BPA exposure inhibited the body weight increases of male and female offspring in a dose-dependent manner, which continued after weaning. Spontaneous activity analyses revealed that BPA elongated immobile time during the dark phase in female offspring. At 4 weeks of age, male offspring exposed to BPA at 40 and 400 mg/kg per day performed avoidance responses significantly higher in the shuttlebox avoidance test. At 8 weeks of age, however, male offspring only at 4 mg/kg per day showed significantly lower responses. In the open-field behavior test at 8 weeks of age, male offspring exposed to BPA only at 4 mg/kg per day showed a higher percent of grooming than the control male offspring. In conclusion, perinatal exposure to BPA caused the behavioral alterations in the offspring.

Previously, we established a culture system of the accessory olfactory bulb in order to investigate the functional role of each accessory olfactory bulb neurons in pheromonal signal processing. In the present study, we developed a co-culture system of cultured accessory olfactory bulb neurons with partially dissociated cells of the vomeronasal organ. The dissociated cells of the vomeronasal organ form spherical structures surrounding a central cavity in culture, referred to as the vomeronasal pockets. The projection and activity of olfactory receptor neurons affect the differentiation and maturation of main olfactory bulb neurons. It was also reported induction of tyrosine hydroxylase expression in main olfactory bulb neurons when they were co-cultured with explants of the olfactory epithelium. Thus, we investigated the effects of co-culture with vomeronasal pockets on the differentiation and/or maturation of cultured accessory olfactory bulb neurons in relation to tyrosine hydroxylase expression. The number of tyrosine hydroxylase-containing neurons developmentally increased over time in the accessory olfactory bulb culture. This increase was significantly enhanced by coculture with vomeronasal pockets. Interestingly, a significant change in tyrosine hydroxylase expression was not observed when main olfactory bulb neurons were co-cultured with vomeronasal pockets. Moreover, significant changes in tyrosine hydroxylase expression were not observed when accessory olfactory bulb neurons were co-cultured with olfactory epithelium explants, as was previously observed in co-culture of main olfactory bulb neurons and olfactory epithelium explants. These results suggest that the differentiation and/or maturation of accessory olfactory bulb neurons is modified by vomeronasal organ neurons via specific interactions between the sensory organ and its target.

We established the procedures for cryopreservation and primary culture of fetal cerebral neurons of cynomolgus monkeys (Macaca fascicularis). Three developmental stages of fetuses (80, 93, and 102 days of gestation) were compared to determine the optimal stage of cerebrum development for primary culture. Among the three fetuses, the 80-day-old fetus produced the most process-rich neurons with the highest survival. The number of total recovery cells from the cryopreserved 80-day-old fetus corresponded to 83.4% of that from fresh tissue. Besides, synchronous oscillations of intracellular calcium were first seen in primate cerebral neurons, which suggested the formation of synapse-networks. Cultured neurons expressed synaptophysin protein. Successful cryopreservation and subsequent cell culture of primate neurons would be useful tools for neuroscience research with species specificity.

Using a well-defined medium with insulin, transferrin and selenium but without serum and albumin, we quantitatively determined the effect of thyroid hormones on the development of Purkinje cells in mouse cerebellar monolayer cultures. Addition of a thyroid hormone, T3 or T4, to the serum-free medium resulted in a highly elaborate dendritic development of Purkinje cells. The cultured Purkinje cells in the presence of T4 even showed similarities in shape and in synapse formation to normal Purkinje cells in vivo. Such effect of T4 on the dendritic arborization of Purkinje cells was dose dependent and significantly sensitive to a low dose of T4 even at 50 pM. The effect of T4 was confirmed by an inhibition experiment using amiodarone, which was reported to induce thyroid dysfunction. Furthermore, T4 affected not only Purkinje cell development but also the shape of other neural cells such as small interneurons (mainly granule cells) and astrocytes in cerebellar cultures. T4 induced development of both interneurons and astrocytes having long processes. These results indicate that thyroid hormones play a pivotal role in the development of mouse Purkinje cell dendrites acting on Purkinje cells directly and/or indirectly via the close interaction with interneurons and astrocytes.

The effects of temperature on adenosine A1 receptor activation were studied both by electrophysiological analysis of synaptically evoked responses in CA1 neurons in guinea pig hippocampal slices, and by measuring the binding of adenosine analogues to adenosine A1 receptors in crude synaptosomes from guinea pig hippocampal neurons. Increasing the temperature of the perfusing medium from 30 degrees C to 45 degrees C attenuated the amplitude of the synaptically and the non-synaptically evoked CA1 population spikes. Bath application of 1 microM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist, did not affect non-synaptically evoked CA1 population spikes, but significantly increased the amplitude of synaptically evoked population spikes in the upper range of hyperthermia (37-43 degrees C). In contrast, application of 5 microM L- N(6)-phenylisopropyladenosine, an adenosine A1 receptor agonist, did not affect non-synaptically evoked CA1 population spikes, but significantly decreased the amplitude of synaptically evoked population spikes in the upper range of hyperthermia. Binding assays using crude hippocampal synaptosomes showed that the affinity of adenosine A1 receptors for a radio-labeled adenosine analogue increased in response to a temperature increase. These results suggest that increased activation of adenosine A1 receptors in response to a temperature increase depresses excitatory synaptic responses in hippocampal CA1 neurons.

Zinc is an essential trace element and present at high concentrations in the central nervous system. Recent studies have revealed that excess amount of extracellular zinc is neurotoxic, and that the disruption of zinc homeostasis may be related to various neurodegenerative diseases. Zinc (25-100 microM) caused significant death of immortalized hypothalamic neuronal cells (GT1-7 cells) in a dose- and time-dependent manner. LD50 was estimated to be 34 microM. The degenerated cells were TUNEL-positive and exhibited apoptosis-like characteristics. Preadministration of sodium pyruvate (1-2 mM), a downstream energy substrate, inhibited the zinc-induced neurotoxicity in GT1-7 cells. GT1-7 cells can be used as a good tool for the investigation of zinc neurotoxicity in the hypothalamus.

1. The elevation of intracellular Ca2+ levels ([Ca2+]i) in immortalized hypothalamic neurons (GT1-7 cells) after exposure to Alzheimer's beta-amyloid protein (AbetaP[25-35]) was investigated using a multisite fluorometry system. 2. The marked rise in [Ca2+]i appeared after exposure to 5-20-microM AbetaP[25-35]. Analysis of the spatiotemporal patterns of [Ca2+]i changes revealed that the magnitude and the latency of the response to AbetaP in each cell were highly heterogeneous. 3. The preadministration of 17beta-estradiol, 17alpha-estradiol, phloretin and cholesterol, which influence the properties of membranes, such as membrane fluidity or membrane potential, significantly decreased the rise in [Ca2+]i. 4. These findings support the idea that disruption of calcium homeostasis by AbetaP channels may be the molecular basis of the neurotoxicity of AbetaP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.

Iron (Fe) and aluminum (Al) have been implicated in the pathogenesis of Alzheimer’s disease (AD). In this study, we examined neuronal and glial cells to clarify which contributes most to metal accumulation after internalization through the transferrin-independent iron uptake (Tf-IU) systems in primary neuronal and glial predominant (NP and GP) cells from rat cerebral cortex, which affect the accumulation of transition metals in a variety of cultured cells. Al more significantly upregulated the Tf-IU activity in GP cells than in NP cells. GP cells were more resistant to Fe and Al exposure than NP cells. However, a chemiluminescence analysis specific for reactive oxygen species (ROS) showed that ROS levels in Fe- or Al-loaded NP cells were twice as high as in Fe- or Al-loaded GP cells. Northern blot analysis and gel retardation assay showed that the Al and Fe exposure taken up by the cells suppress Tf receptor mRNA expression to a greater extent in GP than NP cells, indicating that Al and Fe more markedly accumulate in glial than in neuronal cells. These results suggest that glial cells rather than neuronal cells contribute to the metal accumulation and are more resistant to oxidative stress caused by metals than neuronal cells. The present study may help to explain the pathogenesis of neurodegeneration in AD disorders caused by metal-generated oxidative stress.

The best understood form of long-term potentiation in hippocampal CA1 neurons is induced by activation of the N-methyl-D-aspartate receptor complex and subsequent activation of the intracellular second messenger systems. In addition to this intracellular mechanism, long-term potentiation can also be induced by an extracellular mechanism involving phosphorylation by ATP-ecto-protein kinase. In the present study, we hypothesize that a putative blocking molecule of the formation of long-term potentiation exists on the synaptic membrane, and examine whether ecto-protein kinases play a role in the block of long-term potentiation by phosphorylating the ecto-domains of this molecule in CA1 neurons of guinea-pig hippocampal slices. Long-term potentiation was induced by theta burst stimulation whether or not the test input was delivered to the CA1 neurons following burst stimulation. However, 5 microM K-252b, an ecto-protein kinase inhibitor, only blocked the induction of long-term potentiation when the test input was delivered during a 30-min period following burst stimulation. The results suggest that the process of formation of long-term potentiation continues independently of test synaptic input, while the block of long-term potentiation results from a combination of an interruption of the ATP-ecto-protein kinase-dependent processes and continued test synaptic input after burst stimulation. This block of long-term potentiation, which should be released by activation of ATP-ecto-protein kinase, is suggested to act as a "safety catch" for synaptic plasticity in hippocampal CA1 neurons.

A train of low-frequency afferent stimuli (LFS, 1 Hz, 1000 pulses), given 60 min prior to a tetanus (100 Hz, 100 pulses), suppresses the induction of long-term potentiation (LTP) in which a short-term potentiation decreases gradually back to the pre-tetanic level within 40–50 min (LTP suppression). We investigated the effects of adenosine A1 or A2 receptor antagonists (8-cyclopentyltheophylline (8-CPT) and CP-66713, respectively) on LTP suppression in CA1 neurons of guinea pig hippocampal slices. When the LFS was delivered in the presence of 8-CPT (1 μM), LTP suppression was not significantly affected. However, when LFS was delivered in the presence of CP-66713 (10 μM), LTP suppression was inhibited, leading to successful LTP induction. These results indicate that endogenous adenosine, acting via A2 receptors, is involved in the mechanism of LTP suppression.

1. Using simultaneous recordings of the field EPSP and the population spike in the CA1 neurons of guinea pig hippocampal slices, we confirmed that delivery of a high-frequency stimulation (tetanus: 100 pulses at 100 Hz) produced robust long-term potentiation of synaptic efficacy (LTP) in two independent components, a synaptic component that increases field excitatory postsynaptic potentials (EPSPs) and a component that results in a larger population spike amplitude for a given EPSP size (E-S potentiation).

1. Long-term potentiation (LTP) of synaptic efficacy comprises two components: a synaptic component consisting of increased field excitatory postsynaptic potentials (EPSPs), and a component consisting of a larger population spike amplitude for a given EPSP size (E-S potentiation). In hippocampal CA1 neurons, delivery of three weak bursts (5 pulses at 100 Hz, 20 min intervals) induced LTP in both the EPSP and E-S components. In the same cells, reversal of LTP (depotentiation, DP) in the field EPSP and the E-S component was achieved by delivering three trains of low-frequency stimuli (LFS; 200 pulses at 1 Hz, 20 min intervals). 2. The effects of adenosine A1 and A2 receptor antagonists on the synaptic and E-S components of LTP and DP in CA1 neurons were studied by perfusing guinea-pig hippocampal slices with either 8-cyclopentyltheophylline (8-CPT) or CP-66713. 3. When bursts or LFS were applied to CA1 inputs in the presence of the A1 receptor antagonist 8-CPT, the field EPSP was enhanced in LTP and attenuated in DP, while the E-S relationship was not significantly affected in either LTP or DP. 4. When similar experiments were performed using the A2 receptor antagonist CP-66713, the field EPSP was blocked in LTP, but facilitated in DP, while E-S potentiation was enhanced during both LTP and DP. 5. The results show that A1 and A2 adenosine receptors modulate both the synaptic and E-S components of the induction and reversal of LTP. Based on these results, we discuss the key issue of the contribution of these receptors to the dynamics of neuronal plasticity modification in hippocampal CA1 neurons.

The mechanism of adenosine 5'-triphosphate (ATP)-induced long-term potentiation (LTP) was studied pharmacologically using guinea pig hippocampal slices. Application of 10 microM ATP for 10 min transiently depressed, then slowly augmented, synaptic transmission in CA1 neurons, leading to LTP. This ATP-induced LTP was blocked by addition of an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist. Furthermore, co-application of 10 microM ATP and 100 microM L-glutamate for 10 min also induced LTP, and this effect was blocked by the use of Ca2+-free solution during drug application. These results suggest that, in CA1 neurons, a co-operative effect involving extracellular ATP and the activation of NMDA receptors, which increases intracellular Ca2+ levels, is required to trigger the intracellular biological process involved in ATP-induced LTP.

To address the role of brain gangliosides in synaptic activity, the ceramide analogs, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP) and its enantiomer, l-PDMP, were used to inhibit and stimulate ganglioside biosynthesis in cultured cortical neurons. Prolonged treatment with both PDMP isomers exhibited opposite effects on functional synapse formation measured by spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons: suppression by d-PDMP and facilitation by l-PDMP. Up-regulation of synaptic activity by l-PDMP could be correlated with the slow but robust activation of p42 mitogen-activated protein kinase. Treatment with l-PDMP after transient forebrain ischemia in rats ameliorated the deficit of a well-learned spatial memory by an 8-arm maze task, suggesting a new potential therapeutic approach for neurodegenerative disorders.

We have previously shown that the 40-residue peptide termed amyloid beta-protein (A beta P[1–40]) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether A beta P[1–40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A beta P[1–40] leads to the spontaneous formation of cation-selective channels. With Cs+ as the main cation in both the external as well as the internal saline, the amplitude of the A beta P[1–40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50–500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid beta-protein in solution, can block the flow of Cs+ through the A beta P[1–40] channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A beta P[1–40] channel pore. These properties of the A beta P[1–40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid beta-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid beta-protein and neuronal membranes also occur in vivo, lending further support to the idea that A beta P[1–40] channel formation might be a mechanism of amyloid beta-protein neurotoxicity.

The effects of an adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (8-CPT, 1 microM), on the reduction of long-term potentiation were studied in CA1 neurons of guinea pig hippocampal slices. Reduction of long-term potentiation (depotentiation) was achieved by delivering a train of low-frequency afferent stimuli (low-frequency stimulation, 1000 pulses, 1 Hz) 20 min after the tetanus (100 Hz, 100 pulses). In control experiments, low-frequency stimulation reduced the potentiated component of the slope of the field EPSP and the amplitude of the population spike by 68.5 +/- 14.4% and 80.1 +/- 8.8%, respectively (n = 6); these values were significantly reduced to 13.4 +/- 9.7% and 9.0 +/- 10.9% (n = 7) when the low-frequency stimulation was applied during the perfusion with 8-CPT (1 microM). These results indicate that activation of adenosine A1 receptors enhances the depotentiation of long-term potentiation.

To evaluate the role of endogenous gangliosides in synapse formation, the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), was used to deplete glycosphingolipids (GSLs) of cultured rat cerebral cortical neurons. Synapse formation between the neurons was estimated by the synchronous oscillation of synaptic activity monitored by fura-2 calcium imaging. Treatment with D-PDMP resulted in dose- and time-dependent decreases in the frequency of synchronous oscillations. When a series of gangliosides (GM3, GM1, GD3, GD1b, GT1b, and GQ1b) was supplemented to the GSL depleted cells, only GQ1b was able to normalize the decreased frequency by D-PDMP. These results suggest that de novo synthesis of a particular molecular species of the gangliosides, GQ1b, is essential for synapse formation and synaptic activity.

リハビリテーションなどによる機能修復は，獲得機能に対するニューロン回路の再構築であり，成熟脳では新しいシナプス形成過程と機能再学習の２つの過程を必要とすることを述べた。機能再学習のためには，繰り返し刺激をはじめとする記憶・学習効果をあげるような方法が有効なことは経験的にも知られている。活動依存性(activity-dependent)の可塑性という概念は，シナプスの関わる記憶をはじめとする脳高次機能の獲得や修復・維持のメカニズムの基本概念といえよう。この活動依存性を担う分子の１例を紹介したが，このような研究が発展すれば，再生・機能修復を促進する薬や新しい手法の開発に役立つことが期待される。

Dissociated hippocampal and cerebral cortical neurons from rat embryonic brain form many synapses in culture. The neuronal networks in culture fire spontaneously in synchronous oscillation and can be maintained for long periods of up to 6 months. To establish the relevance of this long-term culture to aging and neurodegenerative diseases, effects of long-term exposure to aluminum, a risk factor for Alzheimer's disease, on the cultured cortical neurons were investigated. It appeared that aluminium promoted the aggregation of amyloid beta-protein and enhanced its neurotoxicity.

Background 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. Methodology/Principal Findings Primary cultures of cerebellar neurons from neonatal rats allow for examinations of the developmental neurotoxicity of chemicals because the various stages of neurodevelopment—including proliferation, migration, differentiation, and morphological and functional maturation—can be observed in vitro. Using these cultures, an excitatory Ca2+-influx assay was employed as an indicator of neural physiological activity. Significant excitatory Ca2+ influxes were evoked by ACE, IMI, and nicotine at concentrations greater than 1 µM in small neurons in cerebellar cultures that expressed the mRNA of the α3, α4, and α7 nAChR subunits. The firing patterns, proportion of excited neurons, and peak excitatory Ca2+ influxes induced by ACE and IMI showed differences from those induced by nicotine. However, ACE and IMI had greater effects on mammalian neurons than those previously reported in binding assay studies. Furthermore, the effects of the neonicotinoids were significantly inhibited by the nAChR antagonists mecamylamine, α-bungarotoxin, and dihydro-β-erythroidine. Conclusions/Significance This study is the first to show that ACE, IMI, and nicotine exert similar excitatory effects on mammalian nAChRs at concentrations greater than 1 µM. Therefore, the neonicotinoids may adversely affect human health, especially the developing brain.

Glial contribution to in vitro synaptic function was investigated in a neuron-glia co-culture system by monitoring spontaneous oscillations of intracellular Ca 2+ in neurons. Rat cortical neurons, grown stably on a cortical astrocyte monolayer, extended neurites resulting in marked functional synapse formation. Little synapse formation was observed in neuronal co-culture with meaningeal fibroblasts or endothelial cells. Aged astrocytes in vitro (C35) were found to attenuate synaptic development, while young astrocytes (C5) markedly promoted synaptic function. C5 and C35 astrocyte media conditioned yielded no significant synaptogenic effect, indicating diffusible factor(s) are not responsible for our observation. Modulation of astrocytic proliferation and differentiation by gliostatin, a glial growth inhibitor, or dibutyryl cAMP affected neuronal synaptic function on the co-cultures. Site-specific analysis in homologous and heterologous neuron-astrocyte co-cultures among cortex, hippocampus, septum, and striatum revealed that homologous combinations of neurons and astrocytes derived from identical brain regions elicited the largest number of synchronizing neur`ons. Thee results suggest that in vivo neuronal synaptic function essentially requires the participation of adjacent astrocytes, which is site-specific and age-dependent.

The aggregation of amyloid beta-protein has been suggested to enhance its neurotoxicity in cultured hippocampal neurons. We found that aluminum, an epidemiologic risk factor for Alzheimer's disease, promoted the aggregation of synthetic amyloid beta-protein (beta 1-40) using immunoblotting and centrifugation. There were no significant changes by Ca or Mg. Other metals including Zn, Fe caused the small degree of aggregation compared to Al. Furthermore, beta 1-40 which was aggregated by aluminum was applied on cultured rat hippocampal neurons, and the characteristic deposition of amyloid fibrils was observed on cultured neurons. These results suggested that the degeneration of neurons and the deposition of amyloid beta-protein were enhanced by aluminum.

Aluminum is an environmental neurotoxin and a suspected risk factor for Alzheimer's disease. The neurotoxicity of aluminum on cultured neurons of rat cerebral cortex was investigated using an assay system for synapse formation and immunohistochemistry. The frequency of spontaneous oscillations of intracellular Ca2+, which is correlated to the number of synapses, was decreased after exposure to 100 microM of aluminum chloride for 22 days. Long-term application of aluminum (48 days) caused aggregation of cell bodies and fasciculation of processes. Processes and cell bodies were strongly stained by antibody to tau protein, which is one of the main components of Alzheimer's neurofibrillary tangles. It is suggested that the characteristics of the degeneration of cultured neurons induced by aluminum show some similarities to the pathology observed in brains with Alzheimer's disease.

Synchronized Ca2+ transients in cultured hippocampal neurons reflect the pattern of underlying electrical activity. Here we demonstrate a similar synchronization of cerebral cortical neurons in culture, and show that this functional coupling is correlated to the appearance of morphologically identified synapses using electron microscopy. During screening of a series of drugs for inhibition of in vitro synaptogenesis, the continuous presence of a protein kinase inhibitor (K-252b) in the culture medium was found to block the synchronous firing and to decrease significantly the number of morphologically identifiable synapses. Since K-252b does not permeate the cell membrane, the results strongly suggest that phosphorylation of cell surface protein(s) by a K-252b sensitive-protein kinase is an essential process in synapse formation.

In the present study, both potentiation and depression of the synaptic response were induced in hippocampal CA1 neurons by systematically varying the frequency of low frequency afferent stimulation (LFS) between 0.5 and 25 Hz and the pulse number between 40 and 1000. The input-response relationship for CA1 synapses showed that LFS at a higher frequency or with a smaller pulse number increased the magnitude of potentiation of the synaptic response by increasing the contribution of N-methyl-D-aspartate receptors (NMDARs) and metabotropic glutamate receptors (mGluRs) to induction of potentiation. One possible mechanism for this bidirectional plasticity was that specific patterns of LFS differentially activate a uniform receptor population in producing depression or potentiation of synaptic responses. However, a pharmacological study indicated that, despite their opposite effects, both the synaptic depression induced by LFS at 1 Hz and the synaptic potentiation induced by LFS at 10 Hz were triggered by co-activation of NMDARs and mGluRs at CA1 synapses. We suggest that activation of protein kinase C or inositol-1,4,5-trisphosphate receptors, both coupled to group 1 mGluRs, is involved in the bidirectional synaptic plasticity induced in hippocampal CA1 neurons by 1-10 Hz LFS.

We have searched for brain-specific extracellular molecules using a library of monoclonal antibodies against surface antigens of differentiated PC12h cells. One of the monoclonal antibodies, PCH42-14, recognized a 27 26 -kDa protein of 10-week-old rat brain on immunoblotting. PCH42-14 antigen was detected only in brain, especially in cerebrum, olfactory bulb, mesencephalon, hippocampus, medulla oblongata, and spinal cord. On hippocampal neuron culture, PCH42-14 antigen existed extracellularly along with the neuronal extensions.

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.

Summary 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 μg/(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.

Recent studies have focused on the effects of low doses of Bisphenol A (BPA) on the central nervous system, which may prevent sexual dimorphism of the brain in rodents. To assess sensitivity to BPA, mother–infant behaviors in the cynomolgus monkey were studied longitudinally after treating the mothers with low-dose BPA during pregnancy. Mother–infant interaction was observed for 6 months after the birth of the infants. In conclusion, male offspring of BPA-treated females showed female-like behavior patterns. Prenatal BPA exposure altered infant behavior in the early stages of mother–infant interaction, and male infants were affected more seriously than females.

We investigated effects of 17beta-estradiol (E(2)) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E(2), NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E(2) for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 microM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 microM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E(2) but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.

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.

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.