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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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

In CA1 neurons of guinea pig hippocampal slices, long-term depression (LTD) was induced in the field EPSP response in the absence of test synaptic inputs (one stimulus every 20 s) by application of the metabotropic glutamate receptor (mGluR) agonist, aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD). This effect was blocked and long-term potentiation (LTP) was induced by co-application of N-methyl-D-aspartate (NMDA) during ACPD perfusion (ACPD/NMDA-induced LTD). These results indicate that the state of NMDA receptor activation during ACPD perfusion determines whether LTP or LTD is induced in hippocampal CA1 neurons. Co-application of an inositol 1, 4, 5-trisphosphate (IP3) receptor inhibitor, 2-aminotheoxydiphenyl borate, during ACPD application had no effect on the ACPD/NMDA-induced LTP, but increased the magnitude of the ACPD-induced LTD, suggesting that the ACPD/NMDA-induced LTP involves NMDA receptors, but not IP3 receptors, whereas the converse applies to the ACPD-induced LTD.

We examined whether bisphenol A (BPA) and 4-nonylphenol (NP) influenced staurosporine-induced neuronal cell death in primary cultured rat hippocampal and cortical neurons. In hippocampal neurons, 17beta-estradiol (E2) (1 nM and 10 microM) and BPA (10 microM) significantly inhibited the staurosporine-induced release of lactate dehydrogenase (LDH). In cortical neurons, BPA significantly inhibited the LDH release, while E2 did not. In hippocampal neurons, E2 and BPA significantly inhibited the staurosporine-induced increase in caspase-3 activity. In cortical neurons, BPA and NP significantly inhibited the increase in caspase-3 activity, while E2 did not. Furthermore, low-dose BPA (10 nM) also significantly inhibited the increase in caspase-3 activity in both hippocampal and cortical neurons. BPA and NP might impede normal brain development by inhibiting even desirable neuronal cell death, interfering with caspase-3 activation.

To investigate the roles of the GABAergic inhibitory system of accessory olfactory bulb (AOB) in pheromonal memory formation, we have developed a primary culture system of AOB neurons, which had numerous excitatory and inhibitory synapses. Using this culture system of AOB neurons, we examined the correlation in rats between neuronal excitation and synaptic morphology by bicuculline-induced disinhibition of cultured AOB neurons. The exposure to bicuculline induced long-lasting oscillatory changes in the intracellular calcium level ([Ca2+]in) of cultured non-GABAergic multipolar neurons, which were identified as mitral/tufted cells (MT cells). These MT cells exhibited the appearance of dendritic filopodia structures after a 10-min treatment with bicuculline. By labelling presynaptic terminals with FM4-64, the appearance of new presynaptic terminals was clearly observed on newly formed filopodia after 120 min treatment with bicuculline. These results suggest that bicuculline-induced [Ca2+]in oscillation of MT cells induces the growth of filopodia and subsequently the formation of new presynaptic terminals. Furthermore, tetrodotoxin or the deprivation of extracellular calcium blocked bicuculline-induced synapse formation. The present results indicate that the long-lasting [Ca2+]in oscillation caused by bicuculline-induced disinhibition of cultured MT cells is significantly implicated in the mechanism underlying synapse formation on cultured AOB neurons. Our established culture system of AOB neurons will aid in clarifying the mechanism of synapse formation between AOB neurons and the molecular mechanism of pheromonal memory formation.

Aluminum is environmentally abundant but not an essential trace element. Although there is increasing evidence suggesting the implication of aluminum in the pathogenesis of Alzheimer’s disease, it is still controversial. We found and report here that aluminum maltolate, a stable and hydrophilic aluminum complex, causes death of primary cultured rat hippocampal neurons in a time- and dose-dependent manner. Degenerated neurons were TUNEL-positive. Immunohistochemical detection of synapsin I and microtubule associated protein 2 revealed the synapse loss between neurons intoxicated by aluminum maltolate. To explore the mechanism underlying its neurotoxicity, we administered various pharmacological compounds prior to the application of aluminum maltolate, and found that brain-derived neurotrophic factor (BDNF) markedly attenuated the neurotoxicity. Furthermore, aluminum maltolate inhibited the elevation of intracellular calcium levels caused by BDNF. Our results suggest the involvement of BDNF in the molecular mechanism underlying neurotoxicity induced by aluminum maltolate.

The mechanism of ATP-induced long-term potentiation (LTP) was studied pharmacologically using guinea-pig hippocampal slices. LTP, induced in CA1 neurons by 10 min application of 10 microM ATP, was blocked by co-application of the N-methyl-D-aspartate (NMDA) receptor antagonist, D,L-2-amino-5-phosphonovalerate (5 or 50 microM). In ATP-induced LTP, the delivery of test synaptic inputs (once every 20 s) to CA1 neurons could be replaced by co-application of NMDA (100 nM) during ATP perfusion. These results suggest that, in CA1 neurons, a co-operative effect between extracellular ATP and activation of NMDA receptors is required to trigger the process involved in ATP-induced LTP. In addition, ATP-induced LTP was blocked by co-application of an ecto-protein kinase inhibitor, K-252b (40 or 200 nM), whereas a P2X purinoceptor antagonist, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid 4-sodium (50 microM), or a P2Y purinoceptor antagonist, basilen blue (10 microM), had no effect.The results of the present study, therefore, indicate that the mechanisms of ATP-induced LTP involve the modulation of NMDA receptors/Ca(2+) channels and the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor/Ca(2+) channel.

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.

It has been proposed that synchronous oscillations of groups of neurons corresponding to sensory information and changes in temporal pattern of oscillations are important for processing of the information in the cortex. However, it has not been determined yet how the temporal or spatial pattern of such oscillations are regulated. We observed spontaneous synchronous oscillations of Ca2+ transients, which were caused by bursts of action potentials of neurons, even in cultured cortical neurons. The frequency of synchronous Ca2+ oscillations increased with development of synapses in cultured neurons and was highly correlated to the number of synapses formed in the same culture.

Recent epidemiological, neuropathological, and biochemical studies have suggested a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimer's disease. However, this relationship remains controversial. To investigate detailed characteristics of neurotoxicity of aluminum, we used primary cultured neurons of rat cerebral cortex as an in vitro model system for the observation of morphological changes induced by chronic exposure to aluminum. Although the exposure to aluminum chloride (10-100 microM) for 1 week did not cause marked neuronal death, degeneration of neuritic processes and accumulation of tau protein and beta-amyloid protein appeared after chronic exposure to 50 microM aluminum chloride for more than 3 weeks. We also investigated the polymerization of beta-amyloid protein in vitro using the immunoblotting technique. We thus found that aluminum induced conformational changes in beta-amyloid protein and enhanced its aggregation in vitro. The aggregated beta-amyloid protein was dissolved by the addition of desferrioxamine, a chelator of aluminum. The aggregated beta-amyloid protein pre-incubated with aluminum formed fibrillar deposits on the surface of cultured neurons.

The etiology of Alzheimer's disease has been suggested to be linked to the neurodegeneration induced by beta-amyloid protein (AbetaP), however, the mechanism underlying the latter remains unknown. We have previously shown the direct incorporation of AbetaP into neuronal membranes of immortalized hypothalamic neurons (GT1-7 cells) associated with the formation of calcium-permeable pores, and the elevation of the intracellular calcium concentrations in the GT1-7 cells. Taking together our results and those of numerous other studies, we hypothesize that the disruption of calcium homeostasis by AbetaP-channels may be the molecular basis of the neurotoxicity of AbetaP, and the development of Alzheimer's disease. It is also proposed that the constituents of membrane lipids may play important roles in the process of this channel formation. Our hypothesis may also explain the mechanism of development of other 'conformational diseases', such as prion disease or type 2 diabetes mellitus, which share some common features with Alzheimer's disease.

A growing number of reports suggest that elevated levels of extracellular Alzheimer's beta-amyloid protein alter the homeostasis of free [Ca(2+)](i) in different cell types of the mammalian brain. In line with these results, we have previously shown that AbetaP[1-40] forms cation-selective channels (Ca(2+) included) across artificial planar bilayers formed from acidic phospholipids and across excised membrane patches from immortalized hypothalamic GnRH neurons (GT1-7 cells), suggesting that the nonregulated Ca(2+)-influx through these spontaneously formed "amyloid channels" may provide a mechanism to explain its toxicity (1). We have now found and report here that the application of AbetaP[1-40] to GT1-7 neurons consistently elevates [Ca(2+)](i) levels. We also found that human islet amylin and the prion protein fragment (PrP106-126), peptides that acquire beta-pleated sheet conformation in water solutions and have been reported to form ion channels across planar bilayer membranes, also increase cytosolic free calcium in GT1-7 neurons. Searching for protective agents, we found that soluble cholesterol, known to decrease the fluidity of the cell membrane, inhibits AbetaP[1-40]-evoked [Ca(2+)](i) rise. These results suggest that unregulated Ca(2+) entry across amyloid channels may be a common mechanism causing cell death, not only in diseases of the third age, including Alzheimer's disease and type 2 diabetes mellitus, but also in prion-induced diseases.

We previously demonstrated that cultured human fibroblasts internalize iron via transferrin-independent iron uptake (Tf-IU), redox, and receptor-mediated endocytosis uptake systems [Oshiro, S., Nakajima, H., Markello, T., Krasnewich, D., Bernardini, I., and Gahl, W.A. (1993) J. Biol. Chem. 268, 21586 21591]. Of these iron transport systems, the Tf-IU system is involved in the accumulation of transition metals in various mammalian cells. It is also known that in experimental animals fed aluminum (Al), Al at micromolar level selectively accumulates in the brain. In the present study, we examined the effects of Al accumulated in the brain cells on iron transport by the Tf-IU system and iron metabolism, using primary cultures from fetal rat cerebral cortex. Pretreatment of cells with 200 microM Al-nitrilotriacetate upregulated the Tf-IU system for iron. Moreover, of various metals tested, Al markedly upregulated the Tf-IU activity. To examine the influence of Al on iron metabolism, the interaction between Al accumulated in the cells and iron-responsive element binding protein (IRE-BP), a cellular iron regulator, was examined by Northern blot analysis, and activity assay: Al decreased the Tf receptor mRNA level and increased the aconitase activity of IRE-BP. The increase of aconitase activity by Al was also observed in vitro. These results suggest that Al accumulated in cortical cells affects iron metabolism.

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 stimulation of ganglioside biosynthesis through activating GM3, GD3 and GQ1b synthases. In a similar time course, the activity of p42 mitogen-activated protein kinase was also enhanced by L-PDMP. To evaluate the efficacy of this drug in long-term memory, rats were trained for 2 weeks using an 8-arm radial maze task, and then forebrain ischemia was induced by 4-vessel occlusion. Treatment with L-PDMP starting 24 hours after the transient ischemia ameliorated the deficit of a well-learned spatial memory, demonstrating the potential therapeutic intervention of the ceramide analog for neurodegenerative disorders.

The involvement of ecto-protein kinase activity in activity-dependent long-term potentiation (LTP) was studied in CA1 neurons of guinea-pig hippocampal slices. Application of 5 microM K-252b, an ecto-protein kinase inhibitor, blocked LTP induced by a theta-burst stimulation (3 bursts composed of 5 pulses at 100 Hz with inter-burst intervals of 200 ms). On the other hand, under 10 microM RK682, an ecto-phosphatase inhibitor, a robust LTP was induced by a weak theta-burst stimulation (3 bursts composed of 3 pulses) which was just at the threshold for the induction of LTP in the control perfusate. These findings suggest that ATP released from presynaptic terminals during the burst stimulation plays an important role in the induction of LTP through phosphorylation of extracellular domains of synaptic membrane proteins, as the substrate for ecto-protein kinase.

The mechanism of ATP-induced long-term potentiation was studied pharmacologically using guinea-pig hippocampal slices. Application of 1-10 microM ATP for 10 min transiently depressed and then slowly augmented the synaptic transmission in CA1 neurons leading to long-term potentiation (LTP). This ATP-induced LTP was blocked by the addition of K-252b, an ecto-protein kinase inhibitor, but was enhanced by the addition of RK682, an ecto-phosphatase inhibitor, both of which do not permeate the cell membrane. These results suggest that ATP applied to the perfusate provides enough substrate for ecto-protein kinase to induce LTP through phosphorylation of extracellular domains of membrane proteins in CA1 neurons.

Synapse formation between cultured rat cortical neurons is inhibited by the continuous application of K-252b, an ecto-protein kinase inhibitor, which cannot permeate the cell membrane. In order to identify the phosphorylated membrane proteins which are necessary for synapse formation, endogenous substrates for ecto-protein kinase activity were investigated. To detect phosphorylation of proteins containing extracellular domains, [γ-33P]ATP was applied to the medium for brief periods. Proteins were then separated by SDS polyacrylamide gel electrophoresis and detected by autoradiography. Some bands showed immediate phosphorylation and this phosphorylation was suppressed by the addition of K-252b to the medium. We examined partial amino acid sequences of these substrates. The band with the highest molecular weight, whose phosphorylation was strongly inhibited by K-252b, was identified as microtubule-associated protein (MAP) 1B. These results suggest the possibility that the phosphorylation of extracellular domains of MAP1B is involved in synaptogenesis between cortical neurons.

Gangliosides are major components of nerve cell membranes and are especially rich in synaptic areas. In order to evaluate the role of endogenous gangliosides in synapse formation, endoglycoceramidase (EGCase) was used to remove oligosaccharides of gangliosides from the cell surface. We have reported previously that synapse formation between cultured rat cerebral cortical neurons can be estimated by the synchronous oscillation of synaptic activity monitored by fura-2 calcium imaging. Continuous application of endoglycoceramidase (EGCase) together with its activator protein dose-dependently decreased the frequency of synchronous oscillations without any morphological changes in neurons and their neurites. The result suggests that oligosaccharides liberated from glycosphingolipids on cultured cortical cell surface with EGCase are important for synapse formation between cortical neurons.

Cerebral cortical cells from fetal rats (18 days) were cultured for 3, 7, 14, 21, 28, 35 days in vitro (DIV) and the development of synapses was examined morphologically by electron microscopy. At 3 DIV, no synapses could be recognized. An immature type of synapse was found at 7 DIV which thereafter developed morphologically. The length of the synaptic contact zone (SCZ) increased with DIV from 271 +/- 11.4 nm (mean +/- SEM) at 7 DIV to 410 +/- 11.4 nm at 35 DIV. The number of synaptic vesicles per terminal also increased with DIV: 10.0 +/- 1.2 at 7 DIV, 35.7 +/- 3.4 at 21 DIV, and 53.3 +/- 4.5 at 35 DIV. The time course of numerical density of synapses was examined quantitatively by electron microscopy. Synaptic density was very low at 7 DIV. It was significantly increased at 14 DIV and thereafter showed variable changes. Four culture series showed decreases after 14 DIV, but one series showed a further increase at 21 DIV followed by a decrease at 28 DIV. The mean density of synapses at each DIV was as follows: 1780 +/- 86/10(6) microns3 at 7 DIV, 4244 +/- 595/10(6) microns3 at 14 DIV, 2285 +/- 674/10(6) microns3 at 21 DIV, 2552 +/- 646/10(6) microns3 at 28 DIV, and 2080 +/- 532/10(6) microns3 at 35 DIV. Neuronal cell density was counted in each culture to calculate the relative number of synapses per neuron. The cell density decreased with age from 301 +/- 51/10(6) microns3 at 7 DIV to 39 +/- 9/10(6) microns3 at 35 DIV.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of adenosine A2 receptor antagonist (CP-66713) on long-term potentiation were studied using guinea pig hippocampal slices in a perfusion system. Tetanic stimulation of Schaffer collateral input which was applied during perfusion of CP-66713 (10 μM), did not induce long-term potentiation but rather long-term depression of evoked synaptic potentials (field EPSP), but induced long-term potentiation of the population spike in CA1 neurons. Thus, adenosine derivatives which accumulate in the synaptic cleft during the tetanic stimulation may be involved in induction of the long-term potentiation via A2 receptors at the synapse. The clear discrimination between long-term depression of the field EPSP and long-term potentiation of the population spike suggests EPSP-spike potentiation at the postsynaptic sites.

Cerebral cortical cells from fetal rats (14 days) were cultured in vitro (7–104 days). Localization of synaptophysin was examined immunocytochemicaly using a monoclonal antibody (SY-38) against synaptophysin. Synaptophysin-immunoreactive (SP-IR) spots were distributed on the surface of the cells. Electron-microscopic observation showed that the SP-IR staining was localized at presynaptic terminals, indicating that the SP-IR spots observed will, fluorescence microscopy represent presynaptic terminals. The time-course of the distribution of SP-IR spots was examined. SP-IR spots could not be observed in cells at 7 days in vitro (DIV). At 14 DIV, SP-IR spots could be first observed on the surface of cell bodies and neurites. Thereafter, the density of SP-IR spots on the cell bodies and neurites increased with DIV. At 42 and 104 DIV, SP-IR spots were almost continued, surrounding the neurites and cell bodies of the cultured cells. These immunocytochemical studies suggest that the dissociated 14-day fetal cerebral cortical neurons make synapses by at least 14 DIV and that the density of synapses increases with time.