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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 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

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.

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.

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.

Our novel fluorescent indicator, DAF-FM, permits the bioimag-ing of nitric oxide (NO) in living cells with high resolution inspace and time, with stable intensity above pH 5.8. Amembrane-permeable derivative, DAF-FM DA, was appliedto imaging of NO generated in rat hippocampal slices byexposure to an aglycemic medium. NO production wasobserved mainly in the CA1 area, and was dependent on theconcentration of O 2 . During exposure to an anoxic-aglycemicmedium, NO was hardly produced, while marked elevation ofintracellular Ca 21 was observed. Production of NO increasedsharply as soon as the perfusate was changed to the normalmedium. These results suggest that NO synthase is activatedafter reperfusion rather than during ischemia.Keywords: diaminofluorescein, hippocampus, imaging,ischemia, nitric oxide, oxygen.J. Neurochem. (2001) 76, 1404–1410. Energy deprivation in neurons exposed to ischemia shouldlead to depolarization of the cell membrane because ofthe inhibition of Na 1 /K 1 -ATPase. Such depolarization isknown to result in elevation of intracellular free Ca

To develop an assay system that allows the N-methyl-D-aspartate (NMDA) receptor subtype-selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRepsilon1/zeta1-GluRepsilon4/zeta1) heat-indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)-1-phenyl-2[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide, was less selective for the GluRepsilon1/zeta1: the IC(50) values for the GluRepsilon1/zeta1-GluRepsilon4/zeta1 were 41.7, 13.3, 12.6 and 11.5 microM, respectively, while two well-known antagonists, DL-2-amino-5-phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.

We examined the development of cholinergic neuronal functions and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) responses in cultured embryonic rat septal cells. Choline acetyltransferase activity was increased from 4 to 6 days in culture and reached a plateau at day 8. Acetylcholine release was increased from 6 to 8 days in culture. AMPA-induced increase in intracellular Ca 2+ level was observed at 3 days in culture and most of the AMPA-responsive cells coincided with high-K + responsive cells. These results suggest that cholinergic neurons develop their neuronal functions about 8 days under cultured conditions, and functional expression of AMPA receptors precedes the cholinergic functional development.

A novel 17-mer peptide ligand for cyclic AMP was designed using the amino acid sequences of essential subsites in various cyclic AMP-dependent protein kinase (protein kinase A) families. The Au disk electrode, which was modified with the designed 17-mer oligopeptide, responded to cyclic AMP but virtually did not respond to any other cyclic nucleotides using the ion channel sensor mechanism. On the other hand, a scrambled peptide, which had the same amino acid composition as and had an amino acid sequence different from the 17-mer oligopeptide, did not respond to any nucleotides. This indicates that the designed 17-mer peptide actually acted as a selective ligand for cyclic AMP. This ligand-designing strategy using peptide sequences in target-binding proteins may possibly be extended to the design of peptide ligands for other second messengers.

Adverse effects of an active fragment of parathyroid hormone (PTH1–34), a blood Ca2+ level-regulating hormone, were examined using rat hippocampal slices in organotypic culture. Exposure of cultured slice preparations to 0.1 μM PTH1–34 for 60 min resulted in a gradual increase in the intracellular Ca2+ concentration ([Ca2+]i); this effect was most obvious in the apical dendritic region of CA1 subfield. When PTH1–34 at a lower concentration (1 nM) was added to the culture medium and its toxic effects examined using a propidium iodide intercalation method, significant toxicity was seen 3 days after exposure and increased with time. Cells in the CA1 region seemed more vulnerable to the hormone than cells in other regions. At 1 week of exposure, the toxic effects were dose-dependent over the range of 0.1 pM to 0.1 μM, the minimum effective dose being 10 pM. The adverse effects were not induced either by the inactive fragment, PTH39–84, or by an active fragment of PTH-related peptide (PTHrP1–34), an intrinsic ligand of the brain PTH receptor. The PTH1–34-induced adverse effects were significantly inhibited by co-administration of 10 μM nifedipine, an L-type Ca2+ channel blocker, but not by co-administration of blockers of the other types of Ca2+ channel. The present study demonstrates that sustained high levels of PTH in the brain might cause degeneration of specific brain regions due to Ca2+ overloading via activation of dihydropyridine-sensitive Ca2+ channels, and suggests that PTH may be a risk factor for senile dementia. Keywords: Parathyroid hormone, PTH1–34, PTHrP, hippocampal organotypic culture, intracellular Ca2+ concentration, L-type Ca2+ channel, nifedipine, propidium iodide, senile dementia Introduction Evidence demonstrating dysfunction of Ca2+ homeostasis during aging is accumulating, and a relationship with the incidence of dementia has been suggested (Fujita, 1990). It is now well established that an increase in the intracellular Ca2+ concentration [Ca2+]i leads to neurotoxicity (Choi, 1988; Ogura et al., 1988), and aging appears to be a major risk factor for Alzheimer's disease and several other neurodegenerative conditions, which suggests that an increase in [Ca2+]i could contribute to the increased vulnerability of aged neurons (Kipen et al., 1995; Satrustegui et al., 1996; Thibault & Landfield 1996; Porter et al., 1997). The regulation of blood Ca2+ levels in normal subjects results from well-designed Ca2+ homeostatic mechanisms that depend on specific hormones, such as parathyroid hormone (PTH) and calcitonin, which regulate the absorption of dietary Ca2+, reuptake of Ca2+ from the renal tube and Ca2+ retention in osteocytes. However, in elderly subjects, especially those with osteoporosis, and in Alzheimer patients, it has been shown that blood Ca2+ levels are lower and thus PTH levels higher, than in normal subjects (Desterhorft et al., 1994). In a previous study, we demonstrated that PTH causes a gradual increase in the [Ca2+]i by activating dihydropyridine-sensitive Ca2+ channels via specific PTH receptors in the brain (Hirasawa et al., 1998). This result suggested to us that high blood PTH levels might cause an increase in the [Ca2+]i in brain cells and thus induce degeneration of cells in the brain. Since the blood-brain-barrier in elderly subjects is reported to be less tight than in younger subjects (Ueno et al., 1997), the adverse effects of PTH may yield important clues in elucidating the causal relationship between the deterioration in Ca2+ homeostasis and the incidence of senile dementia. In the present study, we examined the adverse effects of PTH1–34, an active fragment of PTH, on organotypic hippocampal slice cultures using the propidium iodide (PI) intercalation method, and found that PTH1–34 had a marked toxic effect on the tissue due to dihydropyridine-sensitive Ca2+ channel activation.

The biological functions of nitric oxide in the neuronal system remain controversial. Using a novel fluorescence indicator, DAF-2 DA, for direct detection of NO, we examined both acute rat brain slices and organotypic culture of brain slices to ascertain NO production sites. The fluorescence intensity in the CA1 region of the hippocampus was augmented, especially after stimulation with NMDA, in acute brain slices. This NO production in the CA1 region was also confirmed in cultured hippocampus. This is the first direct evidence of NO production in the CA1 region. There were also fluorescent cells in the cerebral cortex after stimulation with NMDA. Imaging techniques using DAF-2 DA should be very useful for the clarification of neuronal NO functions.

Because glial cells have been characterized by electrophysiological studies as being silent and inactive, neuroscientists have overlooked the roles of these cells in the dynamic function of the central nervous system. Recent measurements of intracellular Ca2+ concentration, however, revealed the dynamic and active features of the glial cells. Some populations of these cells gave rise to increase in intracellular Ca2+ concentration by stimulation with neurotransmitters such as glutamate, acetylcholine, serotonin, noradrenaline and histamine through the activation of specific neurotransmitter receptors distributed on the glial cells. Although the roles of the increased intracellular Ca2+ concentration in glial cells have not been elucidated, these properties suggested the functional participation of glial cells in synaptic modulation and plasticity.

1. Real-time monitoring of dopamine (DA) release from rat striatal slices demonstrated that endothelin (ET)-3 (0.1-10 microM) produced a biphasic DA release consisting of transient and sustained components. When extracellular Ca2+ was removed, the sustained but not transient response remarkably decreased. 2. ET-3 (1-10 microM) stimulated an increase in the intracellular Ca2+ concentration ([Ca(2+)]i), which also consisted of two components. The external Ca2+ depletion inhibited primarily the sustained component of the Ca2+ response to ET-3. 3. ET-3 increased inositol 1,4,5-trisphosphate (IP3) concentrations in striatal slices. This response peaked at 10 to 20 sec and returned to the basal level 2 min after stimulation, an event which was in good accord with a prompt and transient phase of both cytosolic Ca2+ activity and DA release evoked by ET-3. 4. Thus, ET-3 produces a transient and a sustained release of DA from striatal slices by stimulating intracellular Ca2+ mobilization via IP3 formation and extracellular Ca2+ influx, respectively.

Platelet-activating factor (PAF), an alkylether phospholipid, is produced in the brain when it is subjected to various stimuli. Using a Xenopus oocyte expression system, we obtained evidence for functional PAF receptor mRNA expression in rat brain. The presence of the PAF receptor was confirmed and shown to be quite ubiquitous in the CNS by RNA blot and radioligand binding studies. To investigate the neuronal functions of PAF, intracellular Ca2+ increase elicited by nanomolar PAF application was analyzed in cultured rat hippocampal cells. Fractions of NMDA-responsive cells and non-NMDA-responsive cells were shown to respond to PAF, suggesting a potential role for PAF in the Ca2+ signaling pathway in the hippocampus.

The distribution of functional glutamate receptor subtypes was visualized as regional changes in intracellular Ca2+ concentration induced by administration of a specific agonist for each subtype. An image of the fura-2 fluorescence from a whole hippocampal slice was directly excited at 340 or 380 nm through an optical fibre and observed via a low-magnification objective lens (x4). A heterogeneous distribution of N-methyl-D-aspartate (NMDA) and non-NMDA subtypes was demonstrated by the ratio of fluorescence intensities before and after administration of each agonist. This 'macro' image analysis can reveal distributions of multiple functional receptors in a living brain slice.

The effect of dietary supplementation with polyunsaturated fatty acids (PUFAs) on long-term potentiation (LTP) and calcium mobilization in hippocampal slices from aged rats was assessed. LTP magnitude was significantly greater in PUFA-supplemented animals compared to age-matched controls (OCs). LTP did not differ among PUFA-supplemented groups. Calcium mobilization was estimated following membrane depolarization and selective activation of NMDA receptors. The resting level of [Ca2+](i) was slightly elevated in aged preparations compared to young controls (YCs). The transient increase in [Ca2+](i) in CA1 was significantly smaller in aged rats than in YC. The maximum increase in [Ca2+](i) in the CA1 and dentate gyrus (DG) did not differ among aged groups. The maximum increase in [Ca2+](i) and the calcium buffering ability were significantly greater in YC than in the aged rats. Selective activation of NMDA receptors induced regional differences in Ca2+ elevation. In the DG, Ca2+ elevation in OA was comparable to that in YC, and significantly higher than that in OC, suggesting that long-term arachidonic acid supplementation rescues the reduced neurogenesis in the DG. The decay in the depolarization and NMDA-induced increase in [Ca2+](i) was prolonged in aged CA1 and DG.

Here, we show that alpha(1)-adrenoceptor agonists suppress stress-induced death of mouse embryonic brain-derived neural progenitor cells (NPCs). NPCs highly expressed both alpha(1A)- and alpha(1B)-adrenoceptor genes, whereas the gene encoding alpha(1D)-adrenoceptor was expressed at low levels. Application of the alpha(1)-adrenoceptor agonists phenylephrine and cirazoline significantly promoted cell survival of embryonic NPCs that had been exposed to stress, as measured by a lactate dehydrogenase release assay, but had no remarkable effect on differentiation of the NPCs. Both phenylephrine and cirazoline protected NPCs from death induced by growth factor deprivation, N2 nutrient deprivation, tunicamycin treatment or staurosporine treatment. Phenylephrine and cirazoline treatments both maximally reduced stress-induced cell death by approximately 60% but did not change the percentage of undifferentiated cells as measured by nestin staining. Moreover, phenylephrine and cirazoline treatments did not affect the cellular activities of caspase-3 and caspase-7 but markedly reduced propidium iodide penetration into the cytoplasm, suggesting that alpha(1)-adrenoceptor agonists inhibit caspase-3/7-independent death of the embryonic NPCs.

Evidence that glutamate and ATP release from astrocytes can occur via gap junction hemichannels (GJHCs) is accumulating. However, the GJHC is still only one possible release mechanism and has not been detected in some studies, although this may be because the levels were below those detectable by the system used. Because of these conflicting results, we hypothesized that release from astrocyte GJHCs might depend on different astrocyte states, and screened for factors affecting astrocyte GJHC activity by measuring fluorescent dye leakage via GJHCs using a conventional method for GJHC acivation, i.e. removal of extracellular divalent cations. Astrocytes cultured in Dulbecco's minimal essential medium containing 10% fetal calf serum, a medium widely used for astrocyte studies, did not show dye leakage, whereas those cultured in a defined medium showed substantial dye leakage, which was confirmed pharmacologically to be due to GJHCs and not to P2x7 receptors. EGF and bFGF inhibited the GJHC activity via the mitogen-activated protein kinase cascade, and the effect of the growth factors was reversed by interleukin-1β. These factors altered GJHC activity within 10 min, but did not affect connexin 43 expression. GJHC activity in hippocampal slice culture preparations was measured using the same methods and found to be regulated in a similar manner. These results indicate that astrocyte GJHC activity is regulated by brain environmental factors. © 2007 Wiley-Liss, Inc.

We previously showed that lipopolysaccharide (LPS) and pro-inflammatory cytokines utilized different mechanisms for the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) in cultured rat astrocytes. To further characterize these regulatory pathways, we tested the effects of inhibitory factors (anti-inflammatory cytokines, cellular cAMP, and glucocorticoid) on aspects of iNOS expression (from transcription to enzyme activity) during LPS- and cytokine-induced astrocyte NO production. Anti-inflammatory cytokines (transforming growth factor-beta and interleukin-4) suppressed both LPS- and cytokine-induced NO production by reducing iNOS protein expression without affecting mRNA levels. Increased cellular cAMP levels, induced by noradrenaline or forskolin, suppressed LPS-induced, but not cytokine-induced, NO production without affecting iNOS protein expression. The glucocorticoid analog, dexamethasone, suppressed LPS-induced, but not cytokine-induced, NO production by reducing iNOS promoter activity. These different mechanisms would allow the fine control of NO concentration in the brain, as well as accounting for the multiple roles of NO in brain physiology and pathology. Moreover, these mechanisms provide useful therapeutic targets for the treatment of neurodegenerative diseases.

The effect of bifemelane hydrochloride (bifemelane) was examined on human origin astrocyte clonal cells (Kings-1). Bifemelane (125 – 1000 µM) induced a dose-dependent increase in the intracellular calcium concentration ([Ca2+]i). In the highest concentration (1000 µM), the drug caused the second large increase in [Ca2+]i during the washing. The increase that occurred during the administration partially remained in the Ca2+-free medium and was blocked by 2-aminoethoxydiphenyl borate (2-APB), an IP3-receptor blocker, indicating that the source of Ca2+for the increase could be ascribed to the intracellular store. The increase in [Ca2+]i was not observed during washing with Ca2+-free medium, but was observed when the washing was performed with Ca2+-containing medium. Bifemelane caused a dose-dependent ATP release, but histamine and carbachol, which induced a large increase in [Ca2+]i, had no effects on the ATP release. The effects on the [Ca2+]i were blocked by pretreatment with pyridoxal phosphate-6-azophenyl-2’,4’ disulfonic acid, a P2-receptor antagonist. Although the mechanisms of ATP release induced by the drug have not been elucidated yet, the present results demonstrate that the increase in [Ca2+]i induced by bifemelane is not due to its direct effect on the cells, but is dependent upon the ATP released from the cells. Keywords:: human astrocyte, bifemelane, ATP release, store operated Ca2+ entry, intracellular Ca2+ store

When a synapse is stimulated in rapid succession, the second post-synaptic response can be larger than the first and termed paired-pulse facilitation. It has been reported that the paired-pulse ratio (PPR), which is the ratio of the amplitude of the second response to that of the first, depends on the probability of vesicular release at the synapse, and PPR has been used as an easy measure of the release probability. To re-examine the relation of PPR with transmitter release probability, we made whole-cell recordings from astrocytes and pyramidal neurons in the CA1 area of rat hippocampal slices, and studied responses evoked by paired-pulse stimulus of the Schaffer collaterals. In a control condition in which blockers for ionotropic glutamate receptors were added to the artificial cerebrospinal fluid, synaptically induced transporter currents (STCs) recorded from astrocytes showed PPF with similar dependency on stimulus interval as the AMPA-receptor-mediated excitatory post-synaptic currents (AMPA-EPSCs) recorded from pyramidal neurons. When the transmitter release was enhanced by raising Ca2+ concentration in the bathing medium or by applying 8-CPT, an adenosine A1 receptor antagonist, the PPR of the neuronal AMPA-EPSCs decreased significantly. In the same condition, although the amplitude of STCs was significantly increased, the PPR of STCs did not show significant change. The PPR of AMPA-EPSCs, however, recovered by lowering the stimulus intensity or by applying low concentration of NBQX, a competitive antagonist for AMPA-receptor. These results imply that the PPR of transmitter release at Schaffer collateral synapses stays constant as the release probability was altered.

Nitric oxide (NO) production by astrocytes is a significant factor affecting brain physiology and pathology, but the mechanism by which it is regulated is not known. Previous studies using different specimens and stimuli might have described different aspects of a complex system. We investigated the effect of culture and stimulus conditions on NO production by cultured astrocytes and identified two combinations of these allowing NO production. Lipopolysaccharide (LPS)-induced NO production required a high seeding cell density and was independent of the serum concentration, whereas that induced by proinflammatory cytokines required simultaneous treatment with interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma and low-serum conditions but was less affected by the seeding density. These two pathways showed differential sensitivity to protein kinase inhibitors. Both LPS and cytokines induced expression of inducible nitric oxide synthase (iNOS). Although LPS-induced iNOS expression required a high seeding cell density, cytokine-induced iNOS expression, in contrast to NO production, was not affected by the serum concentration. These results suggest that astrocytes interact with the environment and alter their responsiveness to NO production-inducing stimuli by regulating iNOS expression and activity. This is the first evidence for the selective use of two different regulatory pathways in any cell type.

To study calcium imaging data of cell populations that have various response patterns in peak amplitude and frequency of calcium oscillation in response to stimulation, comprehensive characterization based on statistical analysis of each response is important. In cultures of cells that are flat and in contact with each other, it is difficult to distinguish individual cells in calcium imaging data. We have developed a novel method to determine areas corresponding to individual cells in calcium imaging data. Rat neonatal cerebral astrocytes were filled with the calcium indicator Fura2, stained with acridine orange, and illuminated with UV light. The cell nuclei were clearly visualized. In addition, the images of these nuclei were useful for analyzing concentration-dependent alteration of calcium oscillation of cultured astrocytes in response to glutamate. This novel method may be useful for studying factors affecting calcium response patterns of cultured cell populations, including culture conditions, stimulus paradigms, and synthetic compounds. Keywords:: calcium oscillation, astrocyte, glutamate, Fura2, acridine orange

A series of analogs of omega-conotoxin GVIA, a peptide neurotoxin having 27 amino acid residues with three disulfide bridges, were synthesized by replacing each amino acid residue except for Cys and Hyp with Ala. CD spectra were virtually identical between native and all of the analogs, indicating the overall conformations were not changed by the substitutions. The inhibitory effects of these analogs on 125I-omega-conotoxin GVIA binding to chick brain synaptic plasma membranes showed that replacement of Tyr13 with Ala drastically lowered the affinity of the toxin to the N-type Ca2+ channel. Substitution of Tyr13 with Phe also showed reduction of the affinity, indicating that the hydroxyl group of Tyr13 is critical for binding. Since Lys2 is also important for binding (Sato, K. Park, N.-G., Kohno, T. Maeda, T., Kim, J.-I., Kato, R., and Takahashi, M. (1993) Biochem. Biophys. Res. Commun. 194, 1292-1296), we propose a two-point binding model in which Tyr13 and Lys2 interact with specific amino acid residues of the Ca2+ channel through hydrogen bonding and ionic interaction, respectively.

The perfusion of adenosine triphosphate (ATP) induces long-term potentiation (LTP) in CA1 synapses of hippocampal slices, whereas the perfusion of ATP plus d , l -2-amino-5-phosphonovaleric acid (AP5) can result in the formation of long-term depression (LTD). To clarify the difference in change of intracellular calcium concentration ([Ca 2+ ] i ) corresponding to induction of LTP or LTD, we measured [Ca 2+ ] i during the perfusion of ATP or ATP+AP5, while simultaneously recording evoked field potentials. In both cases, ATP (or ATP+AP5) perfusion transiently increased [Ca 2+ ] i but the extent of increase of [Ca 2+ ] i by ATP was larger than that caused by ATP+AP5. Thus, the larger rise in [Ca 2+ ] i induces LTP but the smaller rise induces LTD. These results are consistent with the Ca 2+ hypothesis as proposed by Lisman (Trends Neurosci. 17 (1994) 406).

Flux of K+ and changes in intracellular Ca2+ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K+ occurred at the initiation of sperm motility which is induced by decrease in external K+ and the K+ efflux and sperm motility were inhibited by K+ channel blockers. Intracellular Ca2+ increased within a short period in K(+)-free condition, and the accumulation of 45Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K+ and the increase in intracellular Ca2+ were suppressed when external K+ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K+ through K+ channel and subsequent increase in intracellular Ca2+ are prerequisite for the initiation of sperm motility.

Protein kinase C (PKC) activities, especially, substrates and PKC isozymes, associated with postsynaptic density (PSD) fractions isolated from rat cerebral cortex, hippocampus, or cerebellum were investigated. The 17k M(r) major substrate for PKC was associated with PSD fractions prepared from cerebral cortex and hippocampus, and several substrates including 18k M(r) protein were associated with PSD fraction isolated from cerebellum. The content of 17k M(r) substrate was extremely low in the PSD fraction prepared from cerebellum. PKCs-beta and gamma were associated with PSD fractions and PKC-alpha was virtually absent in the fraction prepared from the three different regions of the brain. All of PKCs-alpha, beta, and gamma were associated with synaptosome fractions. The 36k M(r) bands immunoreactive with anti-PKC-beta antibody, probably degradation products of native PKC-beta, were detected in both the PSD and synaptosome fractions from the three regions, and the ratio of the degradation fragments to native PKC molecule was higher in PSD fractions than in synaptosome fractions. The results suggest postsynaptic roles of PKCs-beta and gamma and involvement of proteolytic activation of PKC-beta in the postsynaptic signal processing.

The expression of a receptor subtype for one type of excitatory amino acid agonist, t-ACPD, was examined in developing Purkinje cells of cerebellar slices. The t-ACPD-induced responses were compared with those induced by QA in current response, single cell Ca2+ imaging and changes in the miniature currents in the same preparation. It was found that t-ACPD induced a single component of inward current, and an increase in the frequency of miniature currents associated with the presence of external Ca2+, but was ineffective at mobilizing intracellular Ca2+ even in the presence of external Ca2+. The present study suggests the expression of at least two types of metabotropic receptors in the Purkinje cell region, one of which, expressed in the Purkinje cell dendrites, is highly sensitive to QA, but relatively insensitive to t-ACPD, and the other of which is a t-ACPD-sensitive receptor expressed on the presynaptic terminals of the neurons making synapses onto Purkinje cells.

Both an enhancement of Ca 2+ -independent kinase activity in the supernatant fraction and enhanced breakdown of type β kinase C (PKC-β) were observed in the hippocampus after induction of tetanus-induced long-term potentiation (LTP) in the hippocampal CA1 region of rat. The enhanced activity was inhibited by the PKC-specific inhibitor, PKC 19–36 . Both phenomena were also observed simultaneously in the in vitro model system in which hippocampal homogenate was treated with CaCl 2 , and both enhancements were inhibited by the addition of calpain inhibitors, leupeptin and benzyloxycarbonyl-Leu-Met-H. The results suggest that Ca 2+ -independent kinase activity enhanced in the supernatant fraction during LTP derives from the catalytic fragment of PKC-β released by calpain.

We have previously suggested that kainic acid (KA) increases the fluorescence of a fluoroprobe for Ca2+ via activation of the non-NMDA (N-methyl-D-aspartic acid) receptor in cultured hippocampal cells. The present study provides further evidence to support this suggestion. KA and NMDA increased the fluorescence occasionally in different subcellular loci of a single cell for each agonist, excluding the possibility that KA activated the NMDA receptor. KA elevated fluorescence in the cell whose membrane was voltage-clamped, thus eliminating possible Ca2+ entry through voltage-dependent channels. A monophasic response to KA was found to occur in the cells identified immunochemically as neurons. Even when the cells were cultured at different embryonic stages, KA responsiveness reached a peak at a rather fixed timepoint through the total age of neurons, while NMDA responsiveness continued to increase. This suggests that the Ca(2+)-permeable non-NMDA receptor is expressed according to a genetic schedule, but not to an artifactual effect of cell culture.

Using primary rat cerebellar cell cultures we observed that trans-1-amino-cyclopentyl-1, 3-dicarboxylic acid (t-ACPD) was able to induce an increase in intracellular [Ca2+] in different cell types. This response was not abolished by external Ca2+ withdrawal, indicating that t-ACPD triggered the release of intracellularly stored Ca2+. In neurons the t-ACPD response was monophasic and inhibited by L-2-amino-4-phosphonobutyrate (APB). In astrocytes, characterized by their immunoreactivity to antisera to glial fibrillary acidic protein and S-100 protein, the response was oscillatory and resistant to APB application. These results suggest the presence of glutamate metabotropic receptor subtypes in the mammalian brain.

The postnatal development of calcium-mobilizing systems was studied by both microfluorometric imaging analysis of Ca2+ on living rat cerebellar slices and immunohistochemical labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate binding protein (IP3BP) in fixed rat cerebellum. Stimulation with quisqualate (QA) or N- methyl- d -aspartate (NMDA) enhanced the Ca2+ level only diffusely on postnatal day (PND) 3, but more discretely on PNDs 7 and 15. On PND 21, QA-induced responses were localized in the molecular layer especially, but not in the granular layer. By contrast, NMDA mobilized Ca2+ prominently in the granular layer, but only weakly in the molecular layer. Localized expression of PIP2 in the molecular layer paralleled QA-induced Ca2+ mobilization, but IP3BP was expressed more diffusely. The present study offers the first direct evidence that PIP2, but not IP3BP, is essential for QA-induced Ca2+ mobilization in the cerebellar cortex.

Microscopic fluorometry of cultured rat hippocampal neurones revealed that the intracellular concentration of Ca 2+ ([Ca 2+ ] i ) rises in response to the activation of excitatory amino acid receptor (EAA-R), which included not only N- methyl- d -aspartate subspecies but also kainate and quisqualate subspecies of EAA-R. Each EAA-R mediated [Ca 2+ ] i rise consisted of the components dependent on and independent of the activity of the voltage-dependent Ca 2+ channel. The receptor-mediated voltage-independent [Ca 2+ ] i rise may be related to the modulation of synaptic transmission efficacy.

Microfluorometry with fura-2 was applied to study the action of the anticonvulsant (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) on N-methyl-D-aspartate (NMDA)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured mouse hippocampal neurons. MK-801 caused a potent and long-lasting blockade of the NMDA-activated [Ca2+]i elevation in a selective manner, not affecting the [Ca2+]i rise induced by quisqualate or kainate. Blockade and recovery from the blockade by MK-801 showed use dependency; the degree of blockade was dependent on the presence of NMDA. The use-dependent onset of antagonism was, however, highly sensitive to the bath temperature. MK-801 applied in the absence of NMDA had no effect on the response to subsequent application of NMDA at 22 degrees C, whereas it reduced the subsequent response to NMDA significantly at 37 degrees C. MK-801 interacted with the receptor-ion channel complex even when Mg2+, which is considered to block the open channel, had already blocked the NMDA-induced [Ca2+]i. The recovery from blockade by MK-801 was not accelerated by the application of 10 mM Mg2+ for 5 min. These results suggest that MK-801 can gain access to its binding site in the absence of NMDA at physiological temperature, and that this binding site is distinct from that for Mg2+.

Acute effects of lithium on synaptic transmissions in the CA1 neurones of rat hippocampus were examined. Perfusion of 2–10 mM lithium chloride (LiCl) produced a dose-dependent increase in the amplitude of field EPSPs, whereas change in the population spikes was variable. The increasing ratio of second field EPSP, which was examined by paired-pulse stimulation, was reduced about 10% by 5 mM LiCl. Intracellularly recorded EPSPs and IPSPs were facilitated by 10 mM LiCl, and the soma membrane was depolarized about 3.2 mV. Intracellular calcium concentration was measured in single hippocampal neurones using fura-2. Although calcium concentration at rest was approximately 30 mM and was increased to an average of 220 nM by 10 −5 M glutamate, 10 mM LiCl had no influence on it. The effects of Li on calcium-dependent processes were not manifested in this study. Variable changes of population spikes may be dependent on the balance between the excitatory and inhibitory postsynaptic potentials during lithium application.

An oligopeptide (17mer) which recognizes cyclic AMP was designed and applied to electrochemical detection of cAMP. Redox peak currents on cyclic voltammograms of ferrocyanide/ferricyanide redox couple with Au electrode which was modified with the peptide showed response dependently on the cAMP concentration, while the electrode showed only slight response to ATP that is a structural analog of cAMP.

The effect of lyoniol-A, a diterpenoid from an Ericaceous plant, on the de-efferented muscle spindle was studied to elucidate the mechanism responsible for abnormal behaviours in mammals resulting from administration of the agent. Increase in the frequency of afferent discharges in response to lyoniol-A (200–300 Hz) was much greater than that obtained on stretching the muscle or after suxamethonium. The result showed that lyoniol-A did not affect the extrafusal fibres nor intrafusal fibres and that adaptation, one of the characteristics of a muscle spindle, did not appear after an administration of the agent. In conclusion, lyoniol-A is a potent muscle spindle excitant that is likely to be a pharmacologically useful tool, and this action may largely responsible for the abnormal behaviours resulting from an administration of lyoniol-A. The experiment also dealt with lyoniol-B (deacetyl-lyoniol-A), grayanotoxin-I and veratridine.

1. In the isolated frog spinal cord perfused with kainic acid (KA, 5 × 10 −4 M) containing Ringer's solution, within 2 hr there were increases in the amplitude of the dorsal root depolarization, as induced by the GABA-agonists. 2. KA perfusion produced increases in the specific binding of [ 3 H]muscimol to crude synaptic membranes and incubation with KA for 3 hr did not increase [ 3 H]muscimol binding. 3. [ 3 H]GABA was released from KA-treated spinal cord slices in the presence of high K + . 4. KA-induced supersensitivity of the dorsal root to GABA may relate to direct actions on primary afferent terminals and not to denervation of GABAergic neurons.

1. The effects of palytoxin (PTX) isolated from Palythoa tubercurosa were tested on the isolated intra-arterially perfused spinal cord of the frog. The resting and evoked potentials were recorded by means of a sucrose-gap technique. 2. PTX caused a marked depolarization of both ventral and dorsal roots. The minimum effective concentration was extremely low, approximately 10(-11) M. During the depolarization the evoked ventral and dorsal root potentials were markedly reduced in amplitude. The ventral root reflex was first augmented and then decreased. 3. The depolarization caused by PTX was markedly reduced when the preparation was perfused with NaCl-deficient medium. However, tetrodotoxin (10(-7) M) only slightly inhibited the depolarization. 4. In a high Ca2+ medium (3.6 mM), the time required to reach the maximum depolarization evoked by PTX was significantly prolonged. In contrast, in a low Ca2+ medium (0.9 mM), PTX caused a marked depolarization soon after application. In a Ca2+-free, Mg2+ (9.0 mM) medium, PTX caused rhythmic oscillatory potentials in both ventral and dorsal roots. 5. The potency of N-acetyl PTX was one hundredth that of the parent compound. 6. It is suggested that PTX may interfere with the stabilizing action of Ca2+ on the neuronal membrane, consequently facilitating Na+ permeability. The primary amine in the PTX molecule may be important for its pharmacological action.