Yoichiro Kuroda

Effects of aluminum on the neurotoxicity of primary cultured neurons and on the aggregation of β-amyloid protein

Recent epidemiological, neuropathological, and biochemical studies have suggested a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimers 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.

Behavioral Alterations in Response to Fear-Provoking Stimuli and Tranylcypromine Induced by Perinatal Exposure to Bisphenol A and Nonylphenol in Male Rats

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.

Frequency of synchronous oscillations of neuronal activity increases during development and is correlated to the number of synapses in cultured cortical neuron networks.

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.

Formation and maturation of synapses in primary cultures of rat cerebral cortical cells: an electron microscopic study.

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)

Hetero-oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains

Adenosine and ATP modulate cellular and tissue functions via specific P1 and P2 receptors, respectively. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the direct interaction between P1 and P2 receptors. We recently demonstrated that the Gi/o‐coupled adenosine A1 receptor (A1R) and Gq/11‐coupled P2Y1 receptor (P2Y1R) form a heteromeric complex with a unique pharmacology in cotransfected HEK293T cells using the coimmunoprecipitation of differentially epitope‐tagged forms of the receptor [Yoshioka et al. (2001) Proc. Natl. Acad. Sci. USA 98, 7617–7622], although it remained to be determined whether this hetero‐oligomerization occurs in vivo. In the present study, we first demonstrated a high degree of colocalization of A1R and P2Y1R by double immunofluorescence experiments with confocal laser microscopy in rat cortex, hippocampus and cerebellum in addition to primary cultures of cortical neurons. Then, a direct association of A1R with P2Y1R was shown in coimmunoprecipitation studies using membrane extracts from these regions of rat brain. Together, these results suggest the widespread colocalization of A1R and P2Y1R in rat brain, and both receptors can exist in the same neuron, and therefore associate as hetero‐oligomeric complexes in the rat brain.

Nicotine-Like Effects of the Neonicotinoid Insecticides Acetamiprid and Imidacloprid on Cerebellar Neurons from Neonatal Rats

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.

Modification of parallel activity elicited by propagating bursts in developing networks of rat cortical neurones

Networks of cultured cortical neurones exhibit regular, synchronized, propagating bursts which are synaptically mediated, and which are hypothesized to play a part in activity‐dependent formation of connections during development in vivo. The relationship between the strength of synaptic connections and the characteristics of synchronized propagating bursting, however, is unclear. Modification of synchronized activity in cortical cultures in response to electrical stimulation was examined using multisite electrode array recording. By measuring the response of the network to weak, localized, test stimulation (TS), we observed a potentiation of activity following a relatively stronger inducing stimulation (IS). This potentiation was evident as an increased probability of eliciting bursts by TS, an increased frequency of spontaneous bursts and number of spikes per burst, and increased speed of burst propagation, and it lasted for at least 20 min. Changing the parameters of IS revealed that high frequency tetanic stimulation is not necessary to induce potentiation, while it is essential for IS to produce a regeneratively propagating burst. The results provide a direct demonstration of modification of both the spatial and temporal characteristics of synchronized network activity, and suggest an important physiological role for propagating synchronized bursting, as a mechanism for inducing plastic modifications in the developing cortex.

Intracellular calcium changes in neuronal cells induced by Alzheimer's beta-amyloid protein are blocked by estradiol and cholesterol.

Abstract1. The elevation of intracellular Ca2+ levels ([Ca2+]i) in immortalized hypothalamic neurons (GT1–7 cells) after exposure to Alzheimers ß-amyloid protein (AßP[25–35]) was investigated using a multisite fluorometry system.2. The marked rise in [Ca2+]i appeared afterexposure to 5–20-μM AßP[25–35]. Analysis of the spatiotemporal patterns of [Ca2+]i changes revealed that the magnitude and the latency of the response to AßP in each cell werehighly heterogeneous.3. The preadministration of 17ß-estradiol, 17α-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 AßP channels may be the molecular basis of the neurotoxicity of AßP and of the pathogenesis of Alzheimers disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.

Thyroid hormone-dependent development of mouse cerebellar Purkinje cells in vitro

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.

Attention-deficit and hyperactive neurobehavioural characteristics induced by perinatal hypothyroidism in rats.

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.