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Background and purpose: We have no definitive treatment for dementia characterized by prolonged neuronal death due to cerebrovascular degeneration or the enormous accumulation of foreign matters, such as β-amyloid. Since these diseases develop slowly, we may be able to delay the onset and improve these symptoms by enhancing the energy metabolism of individual neurons to assist their viabilities. We examined the effect of TND1128, a derivative of 5-deazaflavin, proven to have the self-redox ability as a possible candidate for a direct activator for mitochondrial energy synthesis. Experimental Approach: We prepared brain slices obtained from mice 22 ± 2 hours pretreated with TND1128 or β−ΝΜΝ used as an active control. We measured Ca2+ concentrations in the cytoplasm ([Ca2+]cyt) and mitochondria ([Ca2+]mit) by using fluorescence Ca2+ indicators, Fura4F, and X-rhod-1, respectively, and examined the protective effects of TND1128 and β−ΝΜΝ on overloaded cytosolic and mitochondrial Ca2+ by repeating 80K exposure. Key Results: TND1128 (0.01, 0.1, and 1 mg/kg s.c.) mitigates the dynamics of both [Ca2+]cyt and [Ca2+]mit in a dose-dependent manner. β−ΝΜΝ (10, 30, and 100mg/kg s.c.) showed significant dose-dependent facilitatory effects on the recovery of [Ca2+]cyt during washing for 5 minutes. However, there was no significant effect on the [Ca2+]mit dynamics. Conclusion and implications: TND1128 works as a cofactor for activating cellular energy production machinery. TND1128 would rescue deteriorating neurons in various cerebrovascular disorder-related diseases, including Alzheimer’s disease and Parkinson’s disease. Furthermore, TND1128 will rescue patients with disorders of respiratory organs, such as pulmonary emphysema and COVID-19, which causes respiratory disability

Our aim in the present study was to clarify the activity-dependent and function-associated retention of stable isotopes (SIs) in rat brain regions. We measured regional distributions of the natural stable isotopes 15N and 13C in brain using a mass spectrometer with a dual inlet system and a double collector for ratiometry, and compared them with distributions obtained from internal organs and skeletal muscle. Although levels of 15N and 13C were very high in brain regions of prenatal rats, and robustly decreased after birth, developmental changes in brain regions became obvious when the ratio of 15N to 13C (abbreviated as 15N/13C) in each brain region was compared. A high correlation was observed between free motor activity and 15N/13C in the hippocampus, cerebrum, and striatum. A significantly higher 15N/13C was also observed in the hippocampus and striatum of rats with higher intelligence, which was evaluated by radial maze learning. Furthermore, 15N/13C in brain regions of trained rats were significantly higher than those of untrained age-matched rats. Our study suggests that the 15N/13C in a specific brain region may reflect the physiological feature of the region. This ratio may hence be applicable as a maker for pathological research on undiagnosed brain diseases.

Our aim in the present study was to clarify the activity-dependent and function-associated retention of stable isotopes (SIs) in rat brain regions. We measured regional distributions of the natural stable isotopes

Indium zinc oxide (IZO) is one of the most promising anode materials for organic light-emitting diodes (OLEDs). In the present paper, IZO–Ag–IZO multilayer films were prepared by sputtering and their electrical and optical properties were investigated. The multilayer that consisted of a 30-nm-thick top IZO layer sputtered under Ar, a 14-nm-thick Ag layer, and a 30-nm-thick IZO sputtered under O2 was observed to exhibit the highest figure of merit among the investigated multilayer films because of its low sheet resistance (3.6 Ω/sq) and high transmittance (86.9%). OLEDs fabricated using the developed IZO/Ag/IZO anode exhibited a higher luminance and a greater current density than those fabricated using an anode composed of a single layer of IZO or indium tin oxide. As a result, we observed that the IZO/Ag/IZO multilayer exhibits good electrical and optical properties as a transparent conductive film and is suitable for use as an anode in OLEDs.

The mechanism of brain edema is complex and still remains unclear. Our aim was to investigate the regional differences of cell volume and intracellular Ca2+ concentration ([Ca2+ ]i ) dynamics during hypotonic stress in male mouse hemi-brain slices. Brain slices were loaded with the fluorescence Ca2+ indicator fura-2, and cell volume and [Ca2+ ]i in the lateral cerebral cortex (LCC) and hippocampal CA1 (CA1) region were measured simultaneously during exposure to hypotonic stress using Ca2+ insensitive (F360) and Ca2+ sensitive fluorescence (F380), respectively. Brain cell swelling induced by hypotonic stress was followed by a regulatory volume change that coincided with an increase in [Ca2+ ]i . The degrees of change in cell volume and [Ca2+ ]i were significantly different between the LCC and CA1. The increase in cell volume and [Ca2+ ]i in the LCC, but not in the CA1, was decreased by the transient receptor potential channel blockers LaCl3 and GdCl3 . The increase in [Ca2+ ]i in both the LCC and CA1, was significantly decreased by the intracellular Ca2+ modulators thapsigargin and xestospongin C. The K+ channel activator isoflurane and Cl- channel blocker NPPB significantly decreased [Ca2+ ]i in the LCC. This study demonstrated that, between cells located in the LCC and in the CA1, the characteristics of brain edema induced by hypotonic stress are different. This can be ascribed to the different contribution of volume sensitive G-protein coupled receptor and stretch sensitive Ca2+ channels.

The purpose of this study was to determine whether calmodulin (CaM) plays a role in neurotransmitter release by examining the effect that ophiobolin A (OBA), a CaM antagonist, on neurotransmitter release from clonal rat pheochromocytoma PC12 cells, primary cortical neurons, and primary cerebellar granule cells. OBA inhibited Ca²⁺/CaM-dependent phosphorylation of cAMP response element binding protein in all cell types tested. Moreover, Ca²⁺-dependent release of dopamine and acetylcholine from PC12 cells were remarkably reduced by OBA in a dose-dependent and temporal manner, but neurotransmitter release partially recovered with the addition of CaM in membrane permeabilized PC12 cells. OBA and several synthetic CaM antagonists suppressed Ca²⁺-dependent glutamate release from cerebral cortical neurons, but not from cerebellar granule cells. Myosin Va, a CaM binding protein, localized to synaptic vesicles of PC12 cells and cerebral cortical neurons, but not in cerebellar granule cells. OBA suppressed Ca²⁺-induced myosin Va dissociation from secretory vesicles, and inhibited secretory vesicle motility in PC12 cells. These results suggest that CaM, although not essential, regulates neurotransmitter release in a subset of neurons and secretory cells, and myosin Va is a possible target of OBA in this process.

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

We examined the protective effects of the immunosuppressants cyclosporin A (CsA) and FK506 on abnormal cytosolic Ca2+ ([Ca2+]c) and mitochondrial Ca2+ concentration ([Ca2+]m) dynamics induced by ischemia or high l-glutamate concentration in mouse brain slice preparations. We used fura-4F and rhod-2 as indicators for [Ca2+]c and [Ca2+]m, respectively, in their acetoxymethylester form. Slice preparations loaded with either of these two indicators were exposed to ischemic artificial cerebrospinal fluid (oxygen- and glucose-deprived medium) for 12 min or to aerobic medium with high l-glutamate concentration (isotonic 20 mM l-glutamate) for 5 min. CsA (1 – 10 μM) showed significant protective effects on the maximum increase in ischemia-induced [Ca2+]c and [Ca2+]m. FK506 (10 μM) showed significant protective effects on the [Ca2+]m increase, but not on the ischemia-induced [Ca2+]c increase. Both immunosuppressants showed almost equal protective effects on the [Ca2+]c and [Ca2+]m increases induced by high l-glutamate concentration. These results suggest that the protective effects of CsA and FK506 on Ca2+ overloading may be dependent upon the common pharmacological sites of actions relating to their effects as immunosuppressants. The small, but significant depressant effects of these drugs could give us important clues for rescuing critical brain damage induced by Ca2+ overloading. Keywords:: ischemia, glutamate, Ca2+ overloading, cyclosporin A, FK506

Previously, we reported upregulation of astrocyte [Ca(2+)](i) oscillation by growth factors (i.e., conversion of glutamate-induced sustained [Ca(2+)](i) increase in astrocytes cultured in a defined medium to [Ca(2+)](i) oscillation by EGF and bFGF treatment over 48 h) (Morita et al., (2003) J Neurosci 23:10944-10952). As our previous study also showed that these growth factors increase intracellular Ca(2+) stores, this study was performed to investigate the mechanism underlying loading of intracellular Ca(2+) stores in astrocytes, especially sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA), as a candidate mechanism by which growth factors upregulate [Ca(2+)](i) oscillation. The results indicated that the growth factors upregulated a SERCA inhibitor-sensitive component of [Ca(2+)](i) clearance, and increased expression of the SERCA subtype, SERCA2b. Furthermore, treating the growth factor-treated astrocytes with a low concentration of SERCA inhibitor to partially inhibit SERCA reduced the level of intracellular Ca(2+) storage and reversed glutamate-induced [Ca(2+)](i) oscillations to sustained [Ca(2+)](i) increases. Thus, the upregulation of [Ca(2+)](i) oscillations was attributed to the upregulation of SERCA activities. These results indicated that these growth factors regulate the pattern of glutamate-induced astrocyte [Ca(2+)](i) increases via SERCA2b expression.

Novel fluorescent compounds based on quinolinecarboxylic acid were developed. These compounds were easily prepared in two or three steps from commercially available compounds. Compound 2 showed a ratiometric response to zinc(II) salt in aprotic solvent.

The production and further metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] require several calcium-dependent enzymes, but little is known about subsequent calcium-dependent changes in cellular Ins(1,4,5)P3. To study the calcium dependence of muscarinic acetylcholine receptor-induced Ins(1,4,5)P3 increases in PC12h cells, we utilized an Ins(1,4,5)P3 imaging system based on fluorescence resonance energy transfer and using green fluorescent protein variants fused with the pleckstrin homology domain of phospholipase C-δ1. The intracellular calcium concentration, monitored by calcium imaging, was adjusted by thapsigargin pretreatment or alterations in extracellular calcium concentration, enabling rapid receptor-independent changes in calcium concentration via store-operated calcium influx. We found that Ins(1,4,5)P3 production was increased by a combination of receptor- and calcium-dependent components, rather than by calcium alone. The level of Ins(1,4,5)P3 induced by the receptor was found to be half that induced by the combined receptor and calcium components. Increases in calcium levels prior to receptor activation did not affect the subsequent receptor-induced Ins(1,4,5)P3 increase, indicating that calcium does not influence Ins(1,4,5)P3 production without receptor activation. Removal of both the receptor agonists and calcium rapidly restored calcium and Ins(1,4,5)P3 levels, whereas removal of calcium alone restored calcium to its basal concentration. Similar calcium-dependent increases in Ins(1,4,5)P3 were also observed in Chinese hamster ovary cells expressing m1 muscarinic acetylcholine receptor, indicating that the observed calcium dependence is common to Ins(1,4,5)P3 production. To our knowledge, our results are the first showing receptor- and calcium-dependent components within cellular Ins(1,4,5)P3.

Phosphoinositide-3 kinase (PI3K) and phospholipase C (PLC) utilize the same phosphoinositides as substrates to produce different signaling molecules. These enzymes are activated by a similar set of cell signaling mechanisms, i.e., tyrosine kinases and G proteins, and affect common cell functions, including proliferation, motility, and intracellular trafficking. Despite these similarities, the interplay between these enzymes is not well understood. To address this issue, the effects of the PI3K inhibitor LY294002 on carbachol-induced calcium increase in PC12h cells were examined. As carbachol stimulates both Gq- and Gi-coupled muscarinic acetylcholine receptors (mAChRs), PI3K and PLC are activated simultaneously in this protocol. LY294002 was found to reduce the carbachol-induced calcium increase, and the reduction was attributed to suppression of calcium entry. As LY294002 did not affect either carbachol-induced calcium release or calcium entry induced by calcium store depletion, this agent was found to suppress calcium entry directly activated by mAChRs. Although PI3K was supposed to compete for substrates with PLC, the PI3K inhibitor did not enhance PLC-dependent cellular responses. As LY294002 was still effective by treating cells after carbachol stimulation, it is likely that this agent blocks the calcium entry channels directly. Keywords:: calcium entry, phosphoinositide-3 kinase, muscarinic acetylcholine receptor, PC12, phospholipase C

この総説では多様なグリア細胞のうち，アストロサイトに焦点を合わせた研究の成果を中心として医薬品開発への手がかりとしての重要性を解説した．小泉はアストロサイトのP2Y1受容体を制御し正常に維持する薬物が脳虚血等の酸化ストレスによる神経細胞およびアストロサイトの機能障害保護作用を持つ可能性を示唆した．工藤はアストロサイトが保有するグリオトランスミッターやサイトカインを遊離させる薬物の探索によって新しい中枢神経作用薬を発見することが可能であることを示唆した．和田はグリア細胞に存在するGPCRの研究が中枢神経系薬理学的研究に極めて重要な意味を持つことをNtsr2，PAC1およびVPAC2を実例として示した．橋本は統合失調症発症のグルタミン酸仮説の要になるNMDA受容体機能低下の原因がグリア細胞で合成されるD-セリンの減少やキヌレン酸の増加にある可能性を示唆し，統合失調症の新しい治療薬の開発にはグリア細胞に焦点を当てることが重要であることも示唆した．

Evidence increasingly shows that astrocytes play a pivotal role in brain physiology and pathology via calcium dependent processes, thus the characterization of the calcium dynamics in astrocytes is of growing importance. We have previously reported that the epidermal growth factor and basic fibroblast growth factor up-regulate the oscillation of the calcium releases that are induced by stimuli, including glutamate in cultured astrocytes. This calcium oscillation is assumed to involve protein kinase C (PKC), which is activated together with the calcium releases as a consequence of inositol phospholipid hydrolysis. In the present study, this issue has been investigated pharmacologically by using astrocytes cultured with and without the growth factors. The pharmacological activation of PKC largely reduced the glutamate-induced oscillatory and non-oscillatory calcium increases. Meanwhile, PKC inhibitors increased the total amounts of both calcium increases without affecting the peak amplitudes and converted the calcium oscillations to non-oscillatory sustained calcium increases by abolishing the falling phases of the repetitive calcium increases. Furthermore, the pharmacological effects were consistent between both glutamate- and histamine-induced calcium oscillations. These results suggest that PKC up-regulates the removal of cytosolic calcium in astrocytes, and this up-regulation is essential for calcium oscillation in astrocytes cultured with growth factors.

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 104/cm2 showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 104/cm2 largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a component of the ubiquitin system, which has a fundamental role in regulating various biological activities. However, the functional role of the ubiquitin system in neurogenesis is not known. Here we show that UCH-L1 regulates the morphology of neural progenitor cells (NPCs) and mediates neurogenesis. UCH-L1 was expressed in cultured NPCs as well as in embryonic brain. Its expression pattern in the ventricular zone (VZ) changed between embryonic day (E) 14 and E16, which corresponds to the transition from neurogenesis to gliogenesis. At E14, UCH-L1 was highly expressed in the ventricular zone, where neurogenesis actively occurs; whereas its expression was prominent in the cortical plate at E16. UCH-L1 was very weakly detected in the VZ at E16, which corresponds to the start of gliogenesis. In cultured proliferating NPCs, UCH-L1 was co-expressed with nestin, a marker of undifferentiated cells. In differentiating cells, UCH-L1 was highly co-expressed with the early neuronal marker TuJ1. Furthermore, when UCH-L1 was induced in nestin-positive progenitor cells, the number and length of cellular processes of the progenitors decreased, suggesting that the progenitor cells were differentiating. In addition, NPCs derived from gad (UCH-L1-deficient) mice had longer processes compared with controls. The ability of UCH-L1 to regulate the morphology of nestin-positive progenitors was dependent on its binding affinity for ubiquitin but not on hydrolase activity; this result was also confirmed using gad-mouse-derived NPCs. These results suggest that UCH-L1 spatially mediates and enhances neurogenesis in the embryonic brain by regulating progenitor cell morphology.

Diverse excitatory and inhibitory neuronal responses are mediated via Gq-coupled receptors, but the lack of a systematic comparison of different receptors or neurons has hindered a better understanding of these responses. Such a comparison may be provided by an exogenous receptor that is activated by compounds that have no effect on endogenous receptors. We therefore expressed an invertebrate biogenic amine receptor, the Drosophila octopamine receptor, in rat cortical neurons and compared octopamine receptor-mediated responses with those mediated by the group I metabotropic glutamate receptor, the endogenous Gq-coupled receptor in rat cortical neurons. Stimulation of either receptor did not result in a calcium response in octopamine receptor-expressing neurons, although octopamine preferentially elicited a calcium increase in octopamine receptor-expressing PC12h cells, while enhancing the neuronal depolarization-induced calcium increase and the electrical excitability. The increased excitability was caused by inward currents resulting from a reduction in the leak current, which was voltage-independent and blocked by genistein, a non-selective tyrosine kinase inhibitor. These results show that, in cortical neurons, exogenous octopamine receptor in mushroom bodies activated the same cell signaling pathway as endogenous metabotropic glutamate receptor, suggesting that the diverse neuronal responses mediated by Gq-coupled receptors are due to the properties of different neurons, rather than to the properties of the receptors.

We investigated the effects of bifemelane, a nootropic drug, on the intracellular calcium concentration ([Ca2+]i) in rat cerebral astrocytes using a Ca2+ imaging device. At concentrations of 10 – 30 µM, bifemelane induced a slow onset and small increase in the [Ca2+]i, while at higher concentrations (100 – 300 µM), it induced a rapid transient increase in the [Ca2+]i during administration and a second large increase was seen during drug washout. The first peak was observed in Ca2+-free medium, but its onset was significantly delayed, and no second peak was seen. Neither of these effects was seen in cells treated with thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, in Ca2+-free medium. When thapsigargin-treated astrocytes were returned to normal medium containing Ca2+ (1.8 mM), the [Ca2+]i increased significantly, and this effect was reversely inhibited by bifemelane. We conclude that bifemelane causes the first peak by stimulating release from intracellular Ca2+ stores and the second by capacitive entry through store–operated Ca2+ channels. Although the detail mechanisms of action of the drug are still unknown, bifemelane will be provided as a pharmacological tool for basic studies on astrocytes. Keywords:: bifemelane, astrocyte, Ca2+, capacitive entry, nootropic drug

Synaptic transmission is conducted by neurotransmitters released from presynaptic nerve terminals by means of Ca2+-dependent exocytosis of synaptic vesicles. Formation of a complex of soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins, including vesicle-associated membrane protein-2 (VAMP-2) in the synaptic vesicle membrane, and syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane, is essential for exocytosis. Ionomycin treatment of cultured rat cerebellar granule cells led to cleavage of SNAP-25, but not syntaxin 1 and VAMP-2, that was dependent on extracellular Ca2+. Cleavage was also induced by N-methyl-D-aspartate (NMDA) treatment, but not by depolarization. The use of various site-specific antibodies to SNAP-25, suggested that the cleavage site was in the N-terminal domain of SNAP-25. Calpain inhibitors abolished the Ca2+-dependent cleavage of SNAP-25 and markedly facilitated Ca2+-dependent glutamate (Glu) release from cerebellar granule cells. These results suggest that calpain may play an important role in the long-lasting regulation of synaptic transmission by suppressing neurotransmitter release, possibly through the proteolytic cleavage of SNAP-25.

We have investigated whether the intracellular calcium concentration ([Ca2+]i) oscillations induced in astrocytes using the metabotropic glutamate-receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) are Ca2+-dependent, using three different Ca2+ indicators with different affinities for Ca2+. When rat hippocampal cells in culture were loaded with fura-2 (Kd: 145 nM), two-thirds of the cells showed obvious oscillatory increase in [Ca2+]i during t-ACPD-administration. Those cells were identified as astrocytes by immunohistochemistry in our previous paper. In cells loaded with fura-2FF (Kd: 25,000 nM), a similar percentage of t-ACPD-responsive cells showed oscillatory [Ca2+]i changes. However, in cells loaded with quin-2 (Kd: 60 nM), t-ACPD induced no oscillatory responses, but some cells showed a small transient increase in the [Ca2+]i. The same small transient [Ca2+]i increase was seen in cells loaded with both fura-2FF and BAPTA, a Ca2+ chelator (Kd: 135 nM). These findings indicate the involvement of [Ca2+]i-dependent regulatory mechanisms in the induction of the t-ACPD-induced oscillatory change in the [Ca2+]i in astrocytes. Keywords:: astrocyte, calcium oscillation, metabotropic glutamate receptor, calcium indicator, cell culture

The voltage-gated Ca(2+) channels responsible for synaptic transmission at CA3-CA1 synapses are mainly P/Q- and N-types. It has been shown that tonic inhibition of transmission due to activation of adenosine A(1) receptors occurs at this synapse. We have recently developed a technique to monitor synaptically released glutamate which is based on synaptically induced glial depolarisation. Using this technique, we have examined the effects of different voltage-gated Ca(2+) channel blockers on glutamate release. Under conditions in which the adenosine A(1) receptor was not blocked, omega-AgaIVA (a P/Q-type voltage-gated Ca(2+) channel blocker) suppressed synaptically induced glial depolarisation to a greater extent than omega-CgTxGVIA (an N-type voltage-gated Ca(2+) channel blocker) did. In contrast, in the presence of an adenosine A(1) receptor antagonist, omega-AgaIVA was less effective at suppressing synaptically induced glial depolarisation than omega-CgTxGVIA. These results indicate that, in the absence of adenosine A(1) receptor-mediated tonic inhibition, the contribution of N-type is much greater than that of P-type, and that N-types are the primary target of tonic inhibition in normal conditions in which adenosine A(1) receptor-mediated tonic inhibition is present.

Several reports emphasize the importance of differentiating between psychosis in schizophrenia and the psychotic form of narcolepsy. The failure to identify narcolepsy leads to the labeling of patients as refractory to standard treatments for schizophrenia and retards consideration of intervention for narcolepsy in which psychosis can improve with psychostimulant treatment. Psychosis in patients with narcolepsy can occur in three ways: (i) as the psychotic form of narcolepsy with hypnagogic and hypnopompic hallucinations; (ii) as a result of psychostimulant use in a patient with narcolepsy; and (iii) as the concurrent psychosis of schizophrenia in a patient with narcolepsy. The present case report describes a difficult-to-treat patient who likely had concurrent schizophrenia and narcolepsy. It then summarizes the literature related to the treatment of the three types of patients with psychosis associated with narcolepsy.

6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) is widely used as an antagonist on non-NMDA glutamate receptors. However, several studies have shown that CNQX increases the spontaneous inhibitory postsynaptic current frequency at hippocampal pyramidal neurons and cerebellar granule cells. Dentate granule cells are known to be another distinctive type of principal neurons in hippocampus, and receive dense synaptic input from hilar interneurons. Thus, we examined the effects of CNQX on the dentate granule cells and hilar interneurons with whole-cell recording. CNQX increased the frequency of GABAergic spontaneous postsynaptic currents (sPSCs) on the granule cells, and increased the resting potential and the action potential frequency of the hilar interneurons. These increases were not observed with other glutamate receptor antagonists. The increases in sPSC frequency may be caused by the depolarization and the action potentials of the interneurons.

We have developed a novel device for the quantification of edematous morphology changes in acute brain slices. We can also carry out real-time monitoring of detailed hippocampal cells. The device we developed is based on infrared differential interference contrast microscopy (IR-DIC) and a custom-made real-time computerized image-analysis system for quantification of the morphological dynamics of cells in slice preparations. We applied the coefficient of variation (CV) of light intensity in IR-DIC images to evaluate the change in morphological dynamics. We examined three kinds of edema in the CA1 region of rat hippocampal acute slices under conditions of hypotonic, strong excitation, and experimental ischemia, together with field excitatory postsynaptic potential (fEPSP) recording from radiatum in CA1 the region. There were notable close relationships among the edema formations, the light transmittance, the extent of changes in CV, and features of fEPSP during the three different insults. The present results indicate that CV is a reliable quantification index for edema formation in brain tissue and confirm that applying CV for the analysis in addition to the light transmittance analysis presents additional important information on brain tissue swelling.

Although mannitol has been used as an osmotherapeutic drug on brain injury, the clinical efficiency of the drug are still controversial. In the present study, we examined the effects of mannitol on the edema in a hippocampal slice due to brief ischemia. To evaluate the effects, we employed an image analysis system that consists of an infrared-differential interference contrast (IR-DIC) microscope, an infrared CCD camera, and a computer with custom-made software. By this system, severity of the edema can be quantified as the coefficient of variation (CV) of digitalized slice images. The dose-dependent improvement on the deteriorated hippocampal slices could be obtained by administration of mannitol (10, 50, and 100 mM) after 10-min ischemia. However, field excitatory postsynaptic potentials (fEPSP) in CA1 stratum radiatum, which disappeared during 10-min ischemia, were never recovered by mannitol after more than 20-min treatment. fEPSP were blocked by the effective dose of mannitol for morphological recovery, but the effects found to be reversible. Although we failed to find positive rescuing effects of mannitol on the synaptic activities after ischemia, the protective effects of the drug on ischemic edema may rescue the secondary damages around the infarct area. Keywords:: mannitol, ischemia, brain edema, hippocampus, image analysis

In response to neurotransmitters, astrocytes show various types of calcium increase (transient, oscillatory, and complex), the physiological significance of which is still controversial. To explore this variability, we examined factors affecting the calcium increase pattern in cultured astrocytes and investigated the consequences of the astrocytic calcium response in slice preparations. We found that growth factors (GFs) (EGF plus basic FGF) promoted calcium oscillation in response to glutamate, ATP, or thimerosal (which directly activates the inositol-1,4,5 triphosphate receptor) and that this effect was suppressed by pro-inflammatory cytokines (interleukin-1β or tumor necrosis factor-α), lipopolysaccharide, or a MEK (mitogen-activated protein kinase kinase) inhibitor, suggesting dual regulation of calcium oscillation in astrocytes by factors affecting brain function and pathology via the mitogen-activated protein kinase (MAPK) cascade. The calcium oscillation was accompanied by enlargement of the calcium store, cell proliferation, and the development of a hypertrophic morphology. The cytokines suppressed GF-induced MAPK-dependent immediate early gene promoter activation, but not phosphorylation of extracellular signal-regulated kinase (ERK), showing that they affected gene regulation by acting on the MAPK cascade downstream of ERK. In slice preparations, a metabotropic glutamate receptor agonist converted the spontaneous neuronal calcium increase, attributable to synaptic transmission, to an oscillatory response similar to that seen in astrocytes in culture, indicating that the calcium response in astrocytes acted as a feedback mechanism on the activity of neighboring neurons. This is the first evidence for a dual regulation of calcium oscillation by physiological factors and for the control of calcium dynamics actually being used in physiological processes.

To elucidate the spatial and temporal relationships between phosphatidyl inositol metabolism and changes in intracellular calcium levels, we developed a simultaneous imaging system using green fluorescent protein fused with the pleckstrin homology domain, and the fluorescent calcium indicator, FuraRed. The redistribution of the fusion protein, which represents the phosphatidyl inositol metabolism process, was quantified by calculating the coefficient of variance of the fluorescence over the entire cytosolic region, excluding the nucleus. This calculation increased the reproducibility, compared to the normalized fluorescence changes in arbitrarily selected cytosolic regions used in conventional analysis. The application of this method to analyzing the response of PC12h cells to a number of pharmacological stimuli showed that the extent of the phosphatidyl inositol metabolism was related to the calcium level, but not induced by calcium alone.

Cell surface glycoconjugates are thought to mediate cell-cell recognition and play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of gangliosides Neu5Acalpha 8Neu5Acalpha 3Galbeta 4GlcCer, Galbeta 3GalNAcbeta 4(Neu5Acalpha 8Neu5Acalpha 3)Galbeta 4GlcCer (GD1b), Neu5Acalpha 3Galbeta 3GalNAcbeta 4(Neu5Acalpha 8Neu5Acalpha 3)Galbeta 4GlcCer (GT1b) or its oligosaccharide portion induced a rapid and transient activation of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) in the subplasmalemma. Galbeta 3GalNAcbeta 4(Neu5Acalpha 3)Galbeta 4GlcCer (GM1), GalNAcbeta 4(Neu5Acalpha 3)Galbeta 4GlcCer, Neu5Acalpha 3Galbeta 4GlcCer, Neu5Acalpha 3Galbeta 3GalNAcbeta 4(Neu5Acalpha 3)Galbeta 4GlcCer (GD1a), and Neu5Acalpha 8Neu5Acalpha 3Galbeta 3GalNAcbeta 4(Neu5Acalpha 8Neu5Acalpha 3)-Galbeta 4GlcCer were ineffective. GT1b and GD1b stimulated transient elevation of bulk cytosolic Ca2+ levels while GM1 slightly elevated the levels and GD1a did not. Thus, the cytosolic Ca2+ elevation by the gangliosides may trigger the CaM-KII activation. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108-15 cells and primary hippocampal neurons, but not in glial cells. CaM-KII inhibitors blocked both CaM-KII activation and the subsequent filopodia formation. A small G-protein cdc42 was a potential downstream target of CaM-KII activated by the gangliosides. These results suggest that oligosaccharides of the gangliosides serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of CaM-KII followed by cdc42 up-regulation via a cell surface receptor-like component. The filopodia formation induced by the gangliosides may have a physiological relevance because long-term exposure of hippocampal neurons to GT1b oligosaccharide induced advanced dendritogenesis. Furthermore, exposure of cerebellar neurons to GT1b oligosaccharide facilitated CaM-KII-dependent dendritic outgrowth and branch formation of cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. Thus, the ganglioside/CaM-KII signal plays a role in modulating dendritic morphogenesis by inducing cdc42-mediated actin reorganization.

Previous studies have shown that spikes can be generated in the dendrites of CA1 pyramidal neurons. Some have suggested that, in response to synaptic inputs, spikes are initiated near the soma and propagate back into the dendrites, but some recent studies have shown that intense synaptic inputs initiate spikes in the dendrite. Here, we report the optical detection of spike propagation along the apical dendrites of hippocampal pyramidal neurons. Rat hippocampal slices were stained with the fluorescent voltage-sensitive dye, JPW1114, and optical signals monitored using a 16×16 photodiode array system at a frame rate of 4 kHz. A stimulating electrode was placed at the boundary between the stratum (str.) lacnosum-moleculare and the str. radiatum to stimulate the Schaffer collateral, and fast and slow signal components were detected in the dendritic and somatic regions. By comparing the optical signals with whole-cell recordings, we confirmed that the fast component was due to a population of dendritic spikes in pyramidal neurons. The fast component appeared in dendritic locations near the input sites in response to synaptic activation, and signal onset at the soma was delayed by a few milliseconds compared with that at the input sites. Local perfusion of a Na+ channel blocker near the soma eliminated the fast component at the soma, but had no effect on the fast component at the input sites. Our results indicate that dendritic spikes can be initiated in dendrites near the input site and propagate orthodromically toward the proximal dendrites and the soma.

Previously, we reported that morning bright light therapy improved sleep time and cognitive function in Alzheimer type of dementia. We conducted a double blind study to examine the effects of melatonin on the sleep-wake rhythm, cognitive and non-cognitive functions in Alzheimer type of dementia. The subjects were 9 persons given a placebo (PLA), and 11 given melatonin ( 3 mg)(MLT). The mean age was 79.2+/-6.4 (17 females and 3 males). The drugs were given at 20: 30 each day for 4 weeks. We checked sleep time and activity by Actigraph through one week before and the 4th week after drug administration. Cognitive and non-cognitive functions were evaluated with the clinical dementia rating scale (CDR), and Mini Mental State Examination (MMSE), and the Alzheimer's Disease Assessment Scale (ADAS). We successfully recorded Actigraph data from 18 patients (PLA8, MLT10). The mean sleep time change ratio and SD of the administration of PLA in the night was-0.2+/-13.7%, and MLT was 33.2+/-37.6%. The mean activity counts and SD of the administration of PLA in the night was 29.8+/-77.0%; in MLT it was-44.9+/-21.9%. Melatonin significantly prolonged the sleep time (p=0.017) and decreased activity (p=0.014) in the night (21: 00-6: 00) in the MLT group, although no significant difference in sleep time or activity in the daytime (6: 00-21: 00) was recognized between the two groups. In comparison with ADAS cognition score changes, the mean change and SD in the PLA was 0.3+/-3.7; in MLT it was-4.3+/-3.6 points. In comparison with ADAS non-cognition score, the mean change and SD in the PLA group was-0.8+/-1.0, in the MLT group it was-4.1+/-2.2 points. There were also significant differences between the PLA and the MLT groups in the comparison with the score improvement of ADAS cognition (p=0.017) and non-cognition (p=0.002), otherwise there was no significant difference in improvement of MMSE between both groups. Melatonin administration had effect to improve sleep time and night activity, but no significant effect to improve daytime naps and activity. Although melatonin administration might has less strong effect on circadian rhythm than morning bright light therapy we previously reported, cognitive and non-cognitive functions were improved. Melatonin seemed to be useful for care of the Alzheimer type of dementia patients.

Long-term depression (LTD) of synaptic transmission is induced by low-frequency stimulation (LFS) of afferents lasting for a long time, typically for 10-15 min, in neocortical and hippocampal slices. It is suggested that calcineurin, Ca2+/calmodulin-dependent protein phosphatase, plays a role in the induction of LTD, based on the results that pharmacological or genetic manipulation of calcineurin activity interfered in its induction. However, questions as to why it takes so long to induce LTD and in which compartment of neurons calcineurin is activated remain unanswered. With a fluorescent indicator for calcineurin activity, we visualized the spatiotemporal pattern of its activation in living neurons in layer II/III of visual cortical slices of rats during the LFS of layer IV that induced LTD of synaptic responses. During LFS, the fluorescence intensity gradually increased with a latency of a few minutes in dendrites and soma of neurons, and remained increased during the whole observation period (10-25 min) after LFS. The onset latency of the increase in the soma was slower than that in the distal dendritic region. The LFS-induced rise in fluorescence was not observed in neurons which were loaded with inhibitors of calcineurin, indicating that the intensity of fluorescence reflects calcineurin activity. Control stimulation at 0.05 Hz and theta-burst stimulation did not significantly change the intensity of fluorescence. Only LFS-type inputs effectively activate calcineurin in postsynaptic neurons in an augmenting manner, and such a time-consuming activation of calcineurin may be a reason why long-lasting LFS is necessary for the induction of LTD.

The distribution of group I metabotropic glutamate receptors in rat hippocampal cells in culture was examined by calcium imaging and immunocytochemistry. To distinguish different cell types in the culture, the effects of t-ACPD ((1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid) and of NMDA (N-methyl-D-aspartate) were examined. About 40% of the cultured cells showed either a transient increase or a sustained or oscillatory increase in the intracellular calcium concentration ([Ca(2+)](i)) during t-ACPD administration, while about 60% of the cells showed a sustained [Ca(2+)](i) increase in response to NMDA. Cells that showed an oscillatory [Ca(2+)](i) change during t-ACPD administration did not respond to NMDA administration, while cells that showed a sustained [Ca(2+)](i) increase during NMDA administration did not show any oscillatory response to t-ACPD. After pharmacological examination using those two agonists, the cultured cells were subjected to immunocytochemistry using anti-GFAP and ant-MAP-2 antibodies to distinguish, respectively, astrocytes and neurons. All cells responding to NMDA with a sustained [Ca(2+)](i) increase were MAP-2-positive, whereas all cells showing either oscillatory or sustained [Ca(2+)](i) increase in response to t-ACPD were GFAP-positive. The present results show that, in these cultures, group I metabotropic glutamate receptors are mainly expressed on glial cells and contribute to dynamic [Ca(2+)](i) changes in astrocytes.

Cell surface glycoconjugates are thought to mediate cell-cell recognition and to play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of glyco-chains with an N-acetylgalactosamine (GalNAc) residue at the non-reducing termini (GalNAc-S) such as GalNAcbeta4(Neu5Acalpha3)Galbeta4GlcCer (GM2) ganglioside, its oligosaccharide portion, GalNAcbeta4Galbeta4GlcCer (Gg(3)) Cer, GalNAcalpha3GalNAcbeta3Galalpha4Galbeta4GlcCer (Gb(5)) Cer (Forssman hapten) and alpha1-4 linked oligomers of GalNAc, induced a rapid and transient activation of cAMP-dependent protein kinase (PKA) in subplasmalemma. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108-15 cells and primary cultured hippocampal neurons, but not in glial cells. A cAMP-dependent protein kinase (PKA) selective inhibitor and an adenylate cyclase inhibitor blocked both PKA activation and the subsequent filopodia formation. A small GTPase cdc42 was a potential downstream target of GalNAc-S-activated PKA. These results suggest that extracellular GalNAc-S serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of PKA followed by cdc42 up-regulation via a cell surface receptor-like component. Filopodia formation induced by GalNAc-S may have a physiological relevance because long-term exposure to GalNAc-S enhanced F-actin-rich dendrite generation of primary cultured hippocampal neurons, and PKA-dependent dendritic outgrowth and branch formation of primary cultured cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. These findings provide evidence for a novel GalNAc/PKA-signaling cascade in regulating some neuronal maturation.

A cell type-specific green fluorescent protein (GFP) expression system in rat cortical primary cultures has been developed for the fluorescence labeling of brain cells. Lipid-mediated transfection (lipofection) was employed, allowing the establishment of a convenient efficient system for the analysis of individual cells. To achieve cell type-specific labeling, GFP expression vectors containing the rat neuron-specific enolase (NSE) gene promoter, human glial fibril acidic protein (GFAP) gene promoter, human elongation factor (EF-1alpha) gene promoter, or human cytomegalovirus (CMV) immediate early promoter were constructed, and their specificities examined. Vectors containing the CMV or GFAP promoter resulted primarily in GFP expression in astrocytes, while those containing the EF1-alpha or NSE promoter resulted primarily in GFP expression in neurons. This labeling system was applied to the morphological analysis of living neurons and to cell type-selective calcium imaging. Confocal microscopy revealed that individual GFP-expressing neurons had processes, which were longer than 500 microm and bore spine-like protrusions. A calcium-indicating GFP variant, yellow cameleon (YC2.1), was expressed in the same system, and cell type-selective calcium imaging performed. On pharmacological stimulation, YC2.1-expressing neurons responded to depolarizing stimuli, but not to the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), while astrocytes responded only to tACPD.

To clarify the function of caspase-1-like proteases in neuronal cell death, it is important to be able to detect the activity in living organs by microscopic visualization. In the present study, we synthesized a novel fluorescent substrate sensitive to the caspase-1-like activity, which is easily introduced into cells constituting living organs by extracellular application. As a result, the substrate was shown to be useful in imaging the caspase-1-like activity in rat hippocampal slice cultures. After induction of cell death with glutamate, a significant increase in the activity was observed, especially in the pyramidal cells, suggesting the association of the activity with promotion of cell death.

In order to clarify the neural correlates and brain functions during non-rapid eye movement (NREM) sleep, regional cerebral blood flow (rCBF) were measured using positron emission tomography (PET) in healthy male subjects. During light NREM sleep, the rCBF in the midbrain, in contrast to that in the pons and thalamic nuclei, did not decrease when compared to that during wakefulness, while rCBF decreased in the left medial frontal gyrus, left inferior frontal gyrus, and left inferior parietal gyrus of the neocortex. During deep NREM sleep, the rCBF in the midbrain tegmentum decreased and there was a marked and bilateral decrease in the rCBF in all neocortical regions except for the peri-rolandic areas and the occipital lobe. Thus, the activity of the midbrain reticular formation may be maintained during light NREM sleep. Furthermore, selective deactivation of heteromodal association cortices including those related to language, may occur during NREM sleep, which supports the recent theory that sleep is a local, use-dependent process of the brain.

We studied the sleep effect, the carry over effect (sleepiness) on polysomnography (PSG), the sleep propensity test (SPT), the subjective sleep rating scale for the ultra short acting hypnotics triazolam and short acting hypnotics brotizolam. Ten healthy male volunteers were used for double blind crossover design by randomized allocation with a single oral administration. Informed consent was obtained from all subjects. Placebo (PL), triazolam (TL, 0.125 mg), triazolam (TH, 0.25 mg) and brotizolam (BR, 0.25 mg) were administered at 11 p.m. PSG was recorded from 11 p.m. to 7 a.m. in the next morning. Then, Stanford Sleepiness Scale (SSS) and Kwansei Gakuin Sleepiness Scale (KSS) were checked before 8 a.m., and SPT was recorded at 8:00–8:20 a.m. In PSG of total sleep, TH decreased the frequency of stage shifts significantly ( p p p p