川原 正博

Zinc is an essential trace element that is abundantly present in the brain. In spite of its importance for normal brain functions, it is widely recognized that excess zinc is neurotoxic. Numerous studies have indicated that zinc is crucial for neuronal injury after transient global ischemia and is linked with the pathogenesis of vascular type of dementia. We have investigated the molecular mechanisms of zinc-induced neurotoxicity in vitro and have explored substances that protect zinc-induced neurotoxicity. Pharmacological evidence based on results of our own and numerous other studies has indicated the significance of Ca²⁺ dyshomeostasis in the mechanism of zinc-induced neuronal injury. The introduction of zinc into neurons is reportedly mediated through several types of Ca²⁺-permeable channels. Ca²⁺ channel blockers attenuate zinc-induced neurotoxicity. Furthermore, calcium overload attenuates zinc neurotoxicity, and vice versa. In this paper, we review the routes of zinc entry and mechanisms of zinc-induced neuronal death in relation with calcium homeostasis. The possible role of carnosine (β-alanyl histidine), a dipeptide that is present in the brain, as an endogenous protective substance for neuronal injury is also discussed.

Aluminum(Al)is the 3^rd most abundant on the earth. However, it is not essential and harmful to life beings. Al has been suspected to have a relation with various neurodegenerative diseases. The increased concentration of Al in the soil is implicated in the death of plants or fishes caused by acid rain. The toxic effects of Al are largely influenced by its chemical speciation; the concentration and the type of ligands coexist. Polynuclear hydroxy-aluminum complexes such as aluminum tridecamer(Al₁₃; [AlO₄Al₁₂(OH)₂₄(H₂O)₁₂]⁷⁺)is formed in the water under partially neutralization, and is more toxic in the growth of plant roots compared to monomeric Al³⁺ ion. In this study, we investigated the formation and the stability of Al₁₃ using ²⁷Al-NMR and developed a pulse-exposure method to apply chemically-identified Al₁₃ to primary cultured neurons of rat cerebral cortex. After 14 days of the exposure at 1 h, Al₁₃-intoxicated neurons exhibited significant decrease in cell viability. Meanwhile, monomeric Al³⁺ did not cause significant cell death. Our results will aid for the understanding the molecular mechanism of Al neurotoxicity.

Zinc is an extremely most abundant trace element in the brain. Substantial amounts of zinc exist in the presynaptic vesicles, and are released with glutamate during the neuronal excitation. Synaptically-released zinc is believed to play crucial roles in normal brain functions. Therefore, zinc deficiency impairs brain development and capabilities of learning and mernory. Notwithstanding, recent Studies have indicated that excess zinc is linked with several neurodegenerative diseases and has a causative role in delayed neuronal death after transient global ischemia. We have developed the sensitive assay system for zinc neurotoxicity ill vitro using GT 1 -7 cells (immortalized hypothalamic neurons) to elucidate the functions of zinc in neurodegenerative diseases. Pharmacological experiments have exhibited the involvement of energy failure, metal-metal interaction, and disruption of calcium homeostasis in zinc-induced neurotoxicity. It is inferred that zinc might play its part in brain functions as Janus, an ancient Roman god with two faces, and that zinc homeostasis is essential for the neuronal survival. Our assay system provides a good method for screening the protective substances of zinc neurotoxicity as a therapeutic target of the global ischemia.

Aluminum is environmentally abundant, but not an essential element. Aluminum has been associated with several neurodegenerative diseases, such as dialysis encephalopathy, amyotrophic lateral sclerosis and Parkinsonism dementia in the Kii peninsula and Guam, and in particular, Alzheimer's disease. Although this association remains controversial, there is increasing evidence which suggest; the implication of metal homeostasis in the pathogenesis of Alzheimer's disease. Aluminum, zinc, copper, and iron cause the conformational changes of Alzheimer's amyloid-beta protein. Al causes the accumulation of tau protein and amyloid-beta protein in experimental animals. Aluminum induces neuronal apoptosis in vivo as well as in vitro. Furthermore, a relationship between aluminum and the iron-homeostasis or calcium-homeostasis has been suggested. Based on these findings, the characteristics of aluminum neurotoxicity are reviewed, and the potential link between aluminum and neurodegenerative diseases is reconsidered.

It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest human tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase II alpha cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain.

胎盤組織に局在するカテプシン(システインプロテアーゼ)は、ここ数年で8種類も同定されたが、いずれも遺伝子レベルの発現が確認されているだけである。本研究では、妊娠後11.5日から15.5日までのマウス胎盤でのみ発現するカテプシン-P/J (Cat-P/J)および-6 (Cat-6) に注目し、組換えタンパク質の製作による機能解明を目指した。両組換えタンパク質は大腸菌pET発現系で製作した。蛍光基質での解析により、Cat-Pはカテプシンの典型的基質であるZ-Phe-Arg-MCAとαスロンビンの基質であるBoc-Val-Pro-Arg-MCAの両者を、Cat-6は後者のみを切断することが明らかになった。これらの活性は非常に弱いものであったが、両者の基質特異性の差が初めて明らかになった。次に高活性の組換え体を得る目的で、昆虫細胞発現系での製作を試みた。組換えCat-P/Jの発現は、感染開始から4日目の培地中で最大となることを確認したが、精製するには至らなかった。

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. (C) 2004 Elsevier B.V. All rights reserved.

著者らは,カルシウム・イメージング法を用いて,ラット大脳皮質初代培養神経細胞中のシナプス数を定量的かつ簡便にアッセイする系を確立し,この系を用いてシナプス形成に関わる分子群の探索を行ってきた.その結果,MAP1B(微小管関連タンパク質1B)がシナプス形成時に特異的にリン酸化されることが判明した.更に,このアッセイ系を,化学物質による甲状腺機能の撹乱作用の解析,フェロモン記憶形成に関わる副嗅球神経回路網の機能解析などに応用してきた.その方法と応用例について述べた

Conformational changes of Alzheimer's beta-amyloid protein (Ab P) enhance its neurotoxicity and play important roles in the pathogenesis of Alzheimer's disease. Recent studies have suggested that a common mechanism is based on diverse "conformational diseases". They share similarity in their formation of beta-sheet containing amyloid fibrils by disease-related proteins and the introduction of apoptotic degeneration. Numerous studies, including our own, have revealed that A beta P and several disease-related proteins are capable of directly incorporating into membranes, forming calcium-permeable ion channels, and causing abnormal elevation of intracellular calcium levels. This article reviews the current understanding of the pathology of conformational diseases based on the hypothesis that the disruption of calcium homeostasis through amyloid channels may form the molecular basis of neurotoxicity of A beta P and other disease-related proteins. The roles of membrane lipids and potential development of preventive agents are also discussed.

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.

Aggregation and subsequent conformational change of Alzheimer's beta-amyloid protein (AbetaP) enhance its neurotoxicity. Therefore, factors that inhibit or promote conformational changes of AbetaP play crucial roles in the pathogenesis of Alzheimer's disease (AD). Moreover, recent studies have suggested that a common mechanism is based on the diverse diseases termed "conformational diseases" including neurodegenerative diseases such as AD, prion diseases, Parkinson's disease, and Huntington's disease. These diseases share similarity in the formation of beta-sheet containing amyloid fibrils by disease-related proteins and the introduction of apoptotic degeneration. Aluminum, an environmental risk factor for AD, is a widely used cross-linker that causes conformational changes of AbetaP and other proteins. This report reviews and discusses characteristics of aluminum-induced conformational changes of AbetaP and their implication in pathogenesis of AD. Taking together our results and those of numerous other studies, we hypothesize that aluminum-induced conformational changes enhance the neurotoxicity of AbetaP and lead to development of AD.

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 micrombule 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. (C) 2003 Elsevier Inc. All rights reserved.

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 MTcells 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.

The effects of progesterone on intracellular Ca²⁺ levels have been examined in immortalized hypothalamic neurons (GT1-7) with fluorescence microscopy. The effects observed were classified into two types: 1) the rapid effects and 2) the slow effects. To observe the rapid effects of progesterone, we added these reagents to cells at 10 μM. Upon stimulation with progesterone, the increase in intracellular Ca²⁺ level was observed at around 40% of cells within two minutes. The progesterone-induced Ca²⁺ signals were also observed with membrane-impermeable progesterone conjugated to BSA. These results suggest that progesterone stimulates Ca²⁺ signals in a nongenomic way by acting on a target in the plasma membrane. Next, to observe the slow effects, cells were incubated with progesterone at 100 nM for 72 hours. The incubation for 72 hours significantly increased the intracellular Ca²⁺ level and stimulated the spontaneous fluctuations. Further, the 72h incubation significantly suppressed GABA-induced Ca²⁺ signals. We have also measured effects of tributyltin, a possible analogue of steroid hormones, on intracellular Ca²⁺ levels in GT1-7 cells. The effects were similar to those of progesterone, implying that tributyltin might mimic the progesterone action on intracellular Ca²⁺ in GT1-7 cells.

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 muM) caused significant death of immortalized hypothalamic neuronal cells (GT1-7 cells) in a dose- and time-dependent manner. LD(5)0 was estimated to be 34 muM. 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.

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 muM) caused significant death of immortalized hypothalamic neuronal cells (GT1-7 cells) in a dose- and time-dependent manner. LD(5)0 was estimated to be 34 muM. 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.

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 muM) 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 muM 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. (C) 2001 Elsevier Science Inc.

1. The elevation of intracellular Ca2+ levels ([Ca2+](i)) in immortalized hypothalamic neurons (GT1-7 cells) after exposure to Alzheimer's beta -amyloid protein (A betaP[25-35]) was investigated using a multisite fluorometry system. 2. The marked rise in [Ca2+](i) appeared after exposure to 5-20-muM A betaP[25-35]. Analysis of the spatiotemporal patterns of [Ca2+](i) changes revealed that the magnitude and the latency of the response to A betaP in each cell were highly heterogeneous. 3. The preadministration of 17 beta -estradiol, 17 alpha -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 A betaP channels may be the molecular basis of the neurotoxicity of A betaP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.

The etiology of Alzheimer's disease has been suggested to be linked to the neurodegeneration induced by β-amyloid protein (AβP), however, the mechanism underlying the latter remains unknown. We have previously shown the direct incorporation of AβP 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 AβP-channels may be the molecular basis of the neurotoxicity of AβP, 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. Copyright (C) 2000 Elsevier Science Inc.

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. (C) 2000 Elsevier Science B.V. All rights reserved.

A growing number of reports suggest that elevated levels of extracellular Alzheimer's beta-amyloid protein alter the homeostasis of free [Ca2+](i) in different cell types of the mammalian brain. In line with these results, we have previously shown that A beta P[1-40] forms cation-selective channels (Ca2+ 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 Ca2+-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 A beta P[1-40] to GT1-7 neurons consistently elevates [Ca2+](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 A beta P[1-40]-evoked [Ca2+](i) rise. These results suggest that unregulated Ca2+ 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, are examined the effects of Al accumulated in the brain cells on iron transport by the TI-IU system and iron metabolism, using primary cultures from fetal rat cerebral cortex. Pretreatment of cells with 200 mu M Al-nitrilotriacetate upregulated the Tf-IU system for iron. Moreover, of various metals tested, Al markedly upregulated the TB-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.

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.

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.

Synapse formation between cultured rat cortical neurons is inhibited by the continuous application of K-252b, an ecto-protein kinase inhibitor, which can not 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, [gamma-P-33]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. (C) 1994 Academic Press, Inc.

The aggregation of amploid 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.

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. (C) 1994 Academic Press, Inc.

'Szheimer's disease has aroused much interest among neuroscientists within the recent decades. The deposition of amyloid β‐protein in senile plaques is one of the major h'slmarks of the disease, and is thought to be a primary factor for its occurrence. Recent studies have shown that amyloid β‐protein causes degeneration of cultured hippocamp's neurons, and that its neurotoxicity is enhanced by its aggregation and by conformation's changes. Therefore, factors that stimulate or inhibit amyloid β‐protein aggregation may play key roles in the etiology of 'Szheimer's disease. Moreover, environment's factors, such as 'suminum and zinc, as well as genetic factors may 'sso be involved in the pathogenesis of the disease. Copyright © 1994, Wiley Blackwell. All rights reserved

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.

1. In Mg2+-free external solution, rat cortical neurons in cultured networks entered a stable firing mode, consisting of regular bursts of action potentials superimposed on long-lasting depolarizations. The average separation between bursts varied from culture to culture, but was usually between 5 and 20 s. The distribution of burst intervals followed a Gaussian or normal distribution, with a standard deviation of typically 10% of the average burst period. 2. A gradually depolarizing pacemaker potential was never observed between bursts, but the threshold for action potentials during the quiescent phase was greater-than-or-equal-to 10 mV above the resting potential. No progressive change in conductance or excitability was observed during the quiescent period. Intracellular stimulation of action potentials did not reproduce the long-lasting depolarization. 3. Switching from current clamp to voltage clamp at the resting potential revealed large postsynaptic currents, mainly excitatory but with a small inhibitory component, at the same phase and frequency as the spike bursts, showing that periodic synaptic input is responsible for the burst-depolarizations. The current could be eliminated by local application of 2-amino-5-phosphonovaleric acid (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the postsynaptic cell. In the presence of tetrodotoxin, irregular miniature excitatory postsynaptic currents were observed. 4. A fluorescent calcium indicator (fluo-3, 100 muM) was included in the whole-cell pipette solution, to allow simultaneous electrical and calcium measurements in the same cell. In current clamp, transient intracellular calcium increases were found, which were synchronized to the spike bursts. The Ca2+ rise lasted as long as the action potential burst, and was followed by an exponential decay considerably slower than that of the membrane potential. Calcium transients disappeared during voltage clamp at the resting potential, suggesting that calcium influx through voltage-dependent calcium channels greatly exceeds that through synaptic channels. 5. Multisite Ca2+ recording, after loading with fluo-3 acetoxymethyl (AM) ester, revealed that the onsets of burst-related calcium transients were synchronized in all active cells of each view-field, to within approximately 20 ms. Occasionally, secondary rhythms were observed in which only a subset of cells participated. The times to peak and the decay times of calcium transients varied among synchronized cells. 6. The pharmacology of the burst-related calcium transients was investigated by bath application of a variety of compounds. Tetrodotoxin (1 muM) produced reversible inhibition of transients, as did APV (100 muM) and Mg2+ (1 mM), implying that voltage-dependent sodium channels and N-methyl-D-aspartate (NMDA) receptor-mediated channels are essential. Bicuculline (20 muM) and strychnine (20 muM) both produced a reversible increase in the burst period, sometimes with a slight increase in the amplitude of transients, whereas muscimol (10 muM) reversibly arrested the transients. 7. To investigate the timing of action potential firing at many sites, we cultured cells on substrates with embedded arrays of transparent electrodes. Simultaneous recordings at up to eight sites showed extracellular action potential bursts coincident with intracellular action potentials. Differences in burst initiation times of approximately 20 ms were observed at physical separations of approximately 1 mm. The order of burst initiation at different channels and the detailed firing pattern changed from burst to burst, implying that the wave of excitation was initiated randomly. 8. The phase of periodic bursting could be locked to stimulating current pulses passed through individual sites in the electrode array, and periodic bursting could be initiated in silent cultures. 9. These results indicate that the periodic spike bursts and intracellular calcium transients are generated by periodic excitatory synaptic conductance transients, with a slow NMDA receptor-mediated component. The depolarization opens voltage-dependent calcium channels and the resulting calcium elevation persists during the interburst quiescent period. The random direction and timing of excitation and the lack of any observed pacemaker potential in cells could be explained if the prevalent spontaneous miniature excitatory synaptic events and tonically active NMDA channels act as random sources of excitation.

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) mum3 at 7 DIV, 4244 +/- 595/10(6) mum3 at 14 DIV, 2285 +/- 674/10(6) mum3 at 21 DIV, 2552 +/- 646/10(6) mum3 at 28 DIV, and 2080 +/- 532/10(6) mum3 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) mum3 at 7 DIV to 39 +/- 9/10(6) mum3 at 35 DIV. The number of synapse per neuron increased with DIV to the end of the 4th week (from 64.1 +/- 9.8 at 7 DIV to 1130.1 +/- 254.3 at 28 DIV). The time course of synapse formation in culture resembled that in the cerebral cortex in vivo.