Acute treatment with 17beta-estradiol attenuates astrocyte-astrocyte and astrocyte-neuron communication

Astrocytes are now recognized as dynamic signaling elements in the brain. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. The ovarian steroid hormone, 17beta-estradiol, in addition to its rapid actions on neuronal electrical activity can rapidly alter astrocyte intracellular calcium concentration ([Ca2+]i) through a membrane-associated estrogen receptor. Using calcium imaging and electrophysiological techniques, we investigated the functional consequences of acute treatment with estradiol on astrocyte-astrocyte and astrocyte-neuron communication in mixed hippocampal cultures. Mechanical stimulation of an astrocyte evoked a [Ca2+]i rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a [Ca2+]i wave. Following acute treatment with estradiol, the amplitude of the [Ca2+]i elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the [Ca2+]i rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. Mechanical stimulation of astrocytes induced [Ca2+]i elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated [Ca2+]i rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in the presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings may indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol-mediated hormonal control.     

Involvement of intracellular calcium stores during oxygen/glucose deprivation in striatal large aspiny interneurons.

Striatal large aspiny interneurons were recorded from a slice preparation using a combined electrophysiologic and microfluorometric approach. The role of intracellular Ca2+ stores was analyzed during combined oxygen/glucose deprivation (OGD). Before addressing the role of the stores during energy deprivation, the authors investigated their function under physiologic conditions. Trains of depolarizing current pulses caused bursts of action potentials coupled to transient increases in intracellular calcium concentration ([Ca2+]i). In the presence of cyclopiazonic acid (30 micromol/L), a selective inhibitor of the sarcoendoplasmic reticulum Ca2+ pumps, or when ryanodine receptors were directly blocked with ryanodine (20 [micromol/L), the [Ca2+]i transients were progressively smaller in amplitude, suggesting that [Ca2+]i released from intracellular stores helps to maintain a critical level of [Ca2+]i during physiologic firing activity. As the authors have recently reported, brief exposure to combined OGD induced a membrane hyperpolarization coupled to an increase in [Ca2+]i. In the presence of cyclopiazonic acid or ryanodine, the hyperpolarization and the rise in [Ca2+]i induced by OGD were consistently reduced. These data support the hypothesis that Ca2+ release from ryanodine-sensitive Ca2+ pools is involved not only in the potentiation of the Ca2+ signals resulting from cell depolarization, but also in the amplification of the [Ca2+]i rise and of the concurrent membrane hyperpolarization observed in course of OGD in striatal large aspiny interneurons.     

Role of Na+-H+ and Na+-Ca2+ exchange in hypoxia-related acute astrocyte death

Cultured astrocytes do not succumb to hypoxia/zero glucose for up to 24 h, yet astrocyte death following injury can occur within 1 h. It was previously demonstrated that astrocyte loss can occur quickly when the gaseous and interstitial ionic changes of transient brain ischemia are simulated: After a 20-40-min exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), most cells died within 30 min after return to normal saline (i.e., "reperfusion"). Astrocyte death required external Ca2+ and was blocked by KB-R7943, an inhibitor of reversed Na+-Ca2+ exchange, suggesting that injury was triggered by a rise in [Ca2+]i. In the present study, we confirmed the elevation of [Ca2+]i during reperfusion and studied the role of Na+-Ca2+ and Na+-H+ exchange in this process. Upon reperfusion, elevation of [Ca2+]i was detectable by Fura-2 and was blocked by KB-R7943. The low-affinity Ca2+ indicator Fura-FF indicated a mean [Ca2+]i rise to 4.8+/-0.4 microM. Loading astrocytes with Fura-2 provided significant protection from injury, presumably due to the high affinity of the dye for Ca2+. Injury was prevented by the Na+-H+ exchange inhibitors ethyl isopropyl amiloride or HOE-694, and the rise of [Ca2+]i at the onset of reperfusion was blocked by HOE-694. Acidic reperfusion media was also protective. These data are consistent with Na+ loading via Na+-H+ exchange, fostering reversal of Na+-Ca2+ exchange and cytotoxic elevation of [Ca2+]i. The results indicate that mechanisms involved in pH regulation may play a role in the fate of astrocytes following acute CNS injuries.     

Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ

Astrocytes closely contact neurons where they respond to neuronally released glutamate in immature brain slices. In previous studies, neither metabotropic nor ionotropic glutamate receptor-mediated responses were detected by imaging Ca2+ in astrocytes from mature (P21-P42) animals, suggesting astrocyte glutamate receptors only contribute to hippocampus physiology during development. In contrast to Ca2+ imaging, published electrophysiological experiments suggest P30-P35 astrocytes have alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. For this study, we imaged astrocytes in P31-P38 hippocampal slices to determine if metabotropic and ionotropic glutamate receptor activation elevates intracellular calcium in mature astrocytes. Drugs were perfused while [Ca2+]i was monitored (confocal imaging) in cells loaded with Calcium Green 1-AM. Imaged cells were subsequently identified as astrocytes by GFAP/S-100 immunostaining. Astrocytic Ca2+ increased after glutamate application in the presence of a glutamate uptake inhibitor. An agonist at group I/II metabotropic glutamate receptors, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), elicited Ca2+ increases as did group I agonist 3,5-dihydroxyphenylglycine (DHPG), suggesting that mature astrocytes respond to glutamate via metabotropic glutamate receptors. AMPA also elicited Ca2+ elevations that were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and occurred after treatment with omega-conotoxin MVIIC to block neurotransmitter release. These results demonstrate that astrocytes in mature hippocampus have functional ionotropic and metabotropic glutamate receptors that regulate astrocytic calcium levels. Glutamatergic regulation of astrocytic [Ca2+]i may be involved in synapse modeling, long-term potentiation, excitotoxicity and other events dependent on glutamatergic transmission in adult hippocampus.     

Activation of endothelin receptors by sarafotoxin regulates Ca2+ homeostasis in cerebellar astrocytes.

We carried out experiments designed to investigate the effects of sarafotoxin-6B (SFTx) on [Ca2+]i in cerebellar astrocytes using the Ca2+ indicator fura-2. Both endothelin-1 and sarafotoxin-6B increased [Ca2+]i in individual cerebellar astrocytes in cell culture. The shape of the response was variable but usually consisted of an initial peak of [Ca2+]i followed by an extended plateau increase in [Ca2+]i. In Ca(2+)-free medium only the initial peak was observed. If Ca2+ was subsequently readmitted to the external medium a plateau was now formed. When external Ca2+ was removed during a plateau, [Ca2+]i rapidly declined; replacing the external Ca2+ reversed this decline. The plateau was also reversibly reduced by addition of Ni2+ (5 mM) to the external medium. Addition of 50 mM K+ produced a small increase in [Ca2+]i in most cells. This response was blocked by nimodipine. However, nimodipine only slightly blocked the plateau increase in [Ca2+]i that was formed following activation of endothelin receptors. Furthermore, perfusion of cells with 50 mM K+ during the plateau portion of a response to SFTx reduced [Ca2+]i. In some cells addition of a phorbol ester produced a sustained increase in [Ca2+]i that was blocked by nimodipine. In conclusion, activation of endothelin receptors by SFTx in cerebellar astrocytes produces both Ca2+ mobilization and Ca2+ influx. The pathway for Ca2+ influx is predominantly a non-voltage-dependent one, although some entry through a dihydropyridine-sensitive pathway also appears to occur. Furthermore, activation of protein kinase C in cerebellar astrocytes activates voltage-sensitive Ca2+ channels.     

Hypoxic regulation of Ca2+ signaling in cultured rat astrocytes

Acute hypoxia modulates various cell processes, such as cell excitability, through the regulation of ion channel activity. Given the central role of Ca2+ signaling in the physiological functioning of astrocytes, we have investigated how acute hypoxia regulates such signaling, and compared results with those evoked by bradykinin (BK), an agonist whose ability to liberate Ca2+ from intracellular stores is well documented. In Ca2+-free perfusate, BK evoked rises of [Ca2+]i in all cells examined. Hypoxia produced smaller rises of [Ca2+]i in most cells, but always suppressed subsequent rises of [Ca2+]i induced by BK. Thapsigargin pre-treatment of cells prevented any rise of [Ca2+]i evoked by either BK or hypoxia. Restoration of Ca2+ to the perfusate following a period of acute hypoxia always evoked capacitative Ca2+ entry. During mitochondrial inhibition (due to exposure to carbonyl cyanide p-trifluromethoxyphenyl hydrazone (FCCP) and oligomycin), rises in [Ca2+]i (observed in Ca2+-free perfusate) evoked by hypoxia or by BK, were significantly enhanced, and hypoxia always evoked responses. Our data indicate that hypoxia triggers Ca2+ release from endoplasmic reticulum stores, efficiently buffered by mitochondria. Such liberation of Ca2+ is sufficient to trigger capacitative Ca2+ entry. These findings indicate that the local O2 level is a key determinant of astrocyte Ca2+ signaling, likely modulating Ca2+-dependent astrocyte functions in the central nervous system.     

Impact of progestins on estradiol potentiation of the glutamate calcium response

One mechanism by which estrogen may modulate cognitive function is through potentiation of glutamate-mediated rises in intracellular calcium ([Ca2+]i) with resultant effects on neuronal morphology and signaling. Since progesterone is a component of hormone replacement therapy (HRT), we sought to determine whether therapeutically relevant progestins attenuated or blocked estrogen potentiation of glutamate-induced [Ca2+]i rises. 17beta-estradiol and progesterone, alone or in combination, significantly potentiated the rise in [Ca2+]i. When co-administered, progesterone attenuated the estrogen response to the level seen with progesterone alone. In contrast, medroxyprogesterone acetate (MPA) had no effect when administered alone and completely blocked the 17beta-estradiol-induced potentiation when co-administered. These results may have important implications for effective use of HRT to maintain cognitive function during menopause and aging.     

Fluorescence measurement of changes in intracellular calcium induced by excitatory amino acids in cultured cortical astrocytes

Population response of [Ca2+]i in cultured cortical astrocytes to excitatory amino acids was measured at room temperature using the calcium-sensitive dye fura-2. Quisqualic acid (QA), glutamate (Glu), and kainic acid (KA) caused a peak increase in [Ca2+]i in the order QA greater than Glu greater than KA. No response to N-methyl-D-aspartic acid (NMDA) was observed whether or not Mg2+ was present externally. Both QA and Glu (100 microM) frequently elicited a decaying oscillatory [Ca2+]i response during sustained agonist application; the period of oscillations initially was 23.5 sec and increased as the response was damped. Comparatively, the [Ca2+]i response to KA was nonoscillatory. Both responses to Glu and KA were reduced slightly by antagonist gamma-D-glutamylaminomethyl-sulfonic acid (1 mM), but virtually were abolished by kynurenic acid (3 mM). Replacement of external Na+ by choline had no significant effect on the Glu response. Removal of external Ca2+ reduced the peak response to QA, Glu, and KA to 40, 34, and 18%, respectively; and markedly reduced the degree of QA- and Glu-induced [Ca2+]i oscillations. Pretreatment with phorbol esters, a potent activator of protein kinase C, blocked the [Ca2+]i response to Glu but not KA. It is concluded that cortical astrocytes express Glu receptors of the non-NMDA type in culture and that receptor activation leads to Ca2+ influx and release of internal Ca2+. Mobilization of Ca2+ apparently occurs via the known Glu-mediated hydrolysis of inositol lipids, which may come under negative-feed-back control by protein kinase C activation. Oscillatory [Ca2+]i signaling offers the possibility of a dynamic population response in an electrically coupled glial network.     

大鼠小直径痛觉三叉神经节神经元同时存在三磷酸肌醇敏感性钙库和阿诺碱敏感性钙库？P2X和P2Y嘌呤受体介导细胞内钙信号转导通路的变化

背景： 面部创伤或面部手术等可能损伤三叉神经系统而导致三叉神经疼痛,由于其疼痛剧烈难忍、易复发,长期以来一直为口腔临床治疗的一大难题。现有大量研究发现嘌呤类受体与三叉神经痛相关,目前对其作用机制知之甚少。 目的：探讨在小直径三叉神经节神经元中嘌呤类受体介导钙信号途径。 方法：用Fura-2荧光染料通过显微镜荧光测定技术实时检测急性分离成年大鼠小直径三叉神经节神经元的细胞内钙离子浓度。 结果：用正常外液或去除细胞外Ca2+灌流细胞,分别给予thapsigargin（1 μmol/L）,内质网钙泵ATP酶抑制剂,和咖啡因（20 mmol/L）,ryanodine受体激动剂,均能够引起细胞内游离钙离子浓度（[Ca2+]i）不同程度地升高。ATP（100 μmol/L）也能够产生类似的效应。在去除细胞外Ca2+条件下,ATP引起的[Ca2+]i升高可被thapsigargin可逆性地抑制,而不能被咖啡因抑制;然而在正常外液环境中,ATP引起的细胞内[Ca2+]i 升高不能完全地被thapsigargin抑制。 结论：在痛觉三叉神经节神经元中,嘌呤类受体介导的[Ca2+]i升高有两条途径,一种途径是通过代谢型P2Y受体作用于三磷酸肌醇敏感性钙库;另一种途径是通过离子型受体P2X受体引起外钙内流。     

Neuroprotection and intracellular Ca2+ modulation with fructose-1,6-bisphosphate during in vitro hypoxia-ischemia involves phospholipase C-dependent signaling.

The neuroprotectant fructose-1,6-bisphosphate (FBP) preserves cellular [ATP] and prevents catastrophic increases in [Ca2+]i during hypoxia. Because FBP does not enter neurons or glia, the mechanism of protection is not clear. In this study, we show that FBP's capacity to protect neurons and stabilize [Ca2+]i during hypoxia derives from signaling by a phospholipase-C-intracellular Ca2+-protein kinases pathway, rather than Ca2+ chelation or glutamate receptor inhibition. FBP reduced [Ca2+]i changes in hypoxic hippocampal neurons, regardless of [Ca2+]e, and preserved cellular integrity as measured by trypan blue or propidium iodide exclusion and [ATP]. FBP also prevented hypoxia-induced increases in [Ca2+]i when glucose was absent and when [Ca2+]e was increased to negate Ca2+ chelation by FBP. These protective effects were observed equally in postnatal day 2 (P2) and P16 neurons. Inhibiting glycolysis with iodoacetate eliminated the protective effects of FBP in P16 neurons. FBP did not alter Ca2+ influx stimulated by brief applications of NMDA or glutamate during normoxia or hypoxia, but did reduce the increase in [Ca2+]i produced by 10 min of glutamate exposure during hypoxia. Because FBP increases basal [Ca2+]i and stimulates membrane lipid hydrolysis, we tested whether FBP's protective action was dependent on phospholipase C signaling. The phospholipase C inhibitor U73122 prevented FBP-induced increases in [Ca2+]i and eliminated FBP's ability to stabilize [Ca2+]i and increase survival during anoxia. Similarly, FBP's protection was eliminated in the presence of the mitogen/extracellular signal protein kinase (MEK) inhibitor U0126. We conclude that FBP may produce neuroprotection via activation of neuroprotective signaling pathways that modulate Ca2+ homeostasis.     

Ca2+ signaling regulated by an ATP-dependent autocrine mechanism in astrocytes

Although the mechanisms of Ca2+ wave propagation in astrocytes induced by mechanical stimulation have been well studied, it is still not known how the [Ca2+]i increases in the stimulated cells. Here, we have analyzed the mechanisms of [Ca2+]i increase in single, isolated astrocytes. Our results showed that there was an autocrine mechanism of Ca2+ regulation mediated by ATP in mechanically stimulated astrocytes. This autocrine mechanism induced the activation of phospholipase C via a G-protein, resulting in Ca2+ release from intracellular Ca2+ stores. A second pathway mediating a [Ca2+]i increase was via a Ca2+ influx from the extracellular space, which, interestingly, suppressed an intracellular Ca2+ oscillation. These two different Ca2+ cascades are involved in signal transduction and may function separately during intercellular communication.     

Astrocytic modulation of potassium under seizures

The contribution of an impaired astrocytic K^+ regulation system to epileptic neuronal hyperexcitability has been increasingly recognized in the last decade.A defective K^+ regulation leads to an elevated extracellular K^+ concentration([K^+]o).When[K^+]o reaches peaks of 10-12 mM,it is strongly associated with seizure initiation during hypersynchronous neuronal activities.On the other hand,reactive astrocytes during a seizure attack restrict influx of K^+ across the membrane both passively and actively.In addition to decreased K^+ buffering,aberrant Ca^2+ signaling and declined glutamate transport have also been observed in astrogliosis in epileptic specimens,precipitating an increased neuronal discharge and induction of seizures.This review aims to provide an overview of experimental findings that implicated astrocytic modulation of extracellular K^+ in the mechanism of epileptogenesis.     

Acute ischemic injury of astrocytes is mediated by Na-K-Cl cotransport and not Ca2+ influx at a key point in white matter development

Cerebral palsy is a common birth disorder that frequently involves ischemic-type injury to developing white matter (WM). Dead glial cells are a common feature of this injury and here we describe a novel form of acute ischemic cell death in developing WM astrocytes. Ischemia, modeled by the withdrawal of oxygen and glucose, evoked [Ca2+]i increases and cell death in astrocytes in post-natal day 10 (P10) rat optic nerve (RON). Removing extracellular Ca2+ prevented increases in [Ca2+]i but increased the amount of cell death. Astrocytes showed rapid [Na+]i increases during ischemia and cell death was reduced to control levels by substitution of extracellular Na+ or Cl- or by perfusion with bumetanide, a selective Na-K-Cl cotransport (NKCC) blocker. Astrocytes showed marked swelling during ischemia in the absence of extracellular Ca2+, which was blocked by bumetanide. Raising the extracellular osmolarity to limit water uptake reduced ischemic astrocyte death to control levels. Ultrastructural examination showed that post-ischemic astrocytes had lost their processes and frequently were necrotic, effects partially prevented by bumetanide. At this point in development, therefore, NKCC activation in astrocytes during ischemia produces an osmo-regulatory challenge. Astrocytes can subsequently regulate their cell volume in a Ca2+-dependent fashion but this will require ATP hydrolysis and does not protect the cells against acute cell death.     

Differential intracellular calcium responses to glutamate in type 1 and type 2 cultured brain astrocytes.

Fluorescence image analysis using the calcium indicator fluo-3 was used to examine changes in [Ca2+]i induced by glutamate in mixed glia populations cultured from neonatal rat brains. [Ca2+]i responses were correlated with glia type by performing immunohistochemistry using markers specific for type 1 and type 2 astrocytes on the same cells used in the imaging experiments. Glutamate (30-500 microM) induced two markedly different [Ca2+]i responses in the two astrocyte types: the response in type 1 astrocytes consisted of an initial fast transient followed by varying degrees of oscillations, whereas the predominant response in type 2 astrocytes was a slow rise in [Ca2+]i to a more or less sustained and nonoscillatory level. In some type 2 astrocytes, an initial spikelike transient similar to that in type 1 astrocytes was observed; the overall size of the spike, however, was smaller than in type 1 astrocytes. Two agonists for the ionotropic glutamate receptor, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate, elicited a 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX)-sensitive, external Ca(2+)-dependent, sustained [Ca2+]i rise in type 2 but not type 1 astrocytes. The initial spike in type 2 astrocytes was less dependent on external Ca2+ and not blocked by CNQX. [Na+]i as measured by the Na(+)-fluorescence dye SBFI, was elevated by kainate in both astrocyte types, though the increase was larger in type 2 astrocytes. This increase was reduced by CNQX, suggesting this [Ca2+]i increase was mediated, at least in part, by ionotropic glutamate receptors. The results are discussed in terms of the relative distribution of two classes of glutamate receptors on these two astrocyte types: one, the ionotropic class, is linked directly to an ion channel, and the other, the metabotropic class, induces internal mobilization of Ca2+ via inositol phospholipid hydrolysis.     

Estrogen receptor-alpha mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons

A mechanism underlying gender-related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP-induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERalpha and ERbeta expression in DRG neurons from wild-type (WT) female mice by RT-PCR. To study whether ERalpha or ERbeta mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERalpha knockout (ERalphaKO), and ERbetaKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17beta-Estradiol (E2) inhibited ATP-induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5-min exposure) and stereo specific; 17alpha-estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 microM) in WT mouse. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane-associated ER mediated the response. In DRG neurons from ERbetaKO mice, E2 attenuated the ATP-induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERalphaKO mice, E2 failed to inhibit the ATP-induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP-induced [Ca2+]i signaling is mediated by membrane-associated ERalpha.     

Low-[Mg2+]-induced Ca2+ fluctuations in organotypic hippocampal slice cultures

We show here by whole field monitoring of free intracellular Ca2+ ([Ca2+]i), locally recorded field potential (fp) and external [Ca2+], that low-[Mg2+] induces seizure like events (SLEs) accompanied by simultaneous fluctuations of [Ca2+]i and [Ca2+]e in cultured hippocampal slices. Within a SLE, complex [Ca2+]e fluctuations are seen throughout phases of Ca2+ depletion (tonic) and Ca2+ recovery (clonic) of the extracellular space. Information theory entropy-based analyses revealed strong asymmetric associations of [Ca2+]i and [Ca2+]e kinetics. By contrast, signal-associations between SLEs were found to be weak and of symmetric nature distinguishing seizure-like and interictal events by extensive coupling and decoupling of [Ca2+]i and [Ca2+]e fluctuations, respectively.     

Astrocytes in 17beta-estradiol treated mixed hippocampal cultures show attenuated calcium response to neuronal activity

Glial cells in the brain are capable of responding to hormonal signals. The ovarian steroid hormone 17beta-estradiol, in addition to its actions on neurons, can directly affect glial cells. Estrogen receptors have been described on both neurons and astrocytes, suggesting a complex interplay between these two in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration ([Ca(2+)](i)). Using simultaneous electrophysiology and calcium imaging techniques, we monitored neuronal activity evoked astrocyte ([Ca(2+)](i)) changes in mixed hippocampal cultures loaded with fluo-3 AM. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca(2+)](i)) elevations in astrocytes, which could be blocked by 200 microM MCPG and also 1 microM TTX. We compared astrocytic ([Ca(2+)](i)) transients in control and 24-hour estradiol treated cultures. The amplitude of the ([Ca(2+)](i)) transient, the number of responsive astrocytes, and the ([Ca(2+)](i)) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca(2+)](i)) rise in astrocytes in response to local application of the metabotropic glutamate receptor (mGluR) agonist t-ACPD was attenuated in estradiol treated cultures, suggesting functional changes in the astrocyte mGluR following 24-h treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca(2+)](i)) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling.     

Low concentrations of ethanol deplete type-2 astrocytes of intracellular free magnesium

The acute effects of low concentrations of ethanol on intracellular free magnesium ions ([Mg2+]i) in cultured type-2 astrocytes were studied by digital imaging microscopy using the Mg2+ fluorescent probe, mag-fura-2. In 0-mM ethanol, the basal level of [Mg+]i was 124.7+/-2.56 microM with a heterogeneous distribution within the cells. Treatment of the cells with 10 and 25 mM ethanol (10 min) resulted in rapid concentration-dependent reduction in [Mg2+]i; the greater the concentration of alcohol, the greater the depletion of [Mg2+]i. Exposure of cells to 10 and 25 mM resulted in approximately 27 and 50% reductions in [Mg2+]i, respectively. Reincubation in normal Mg2+-physiological buffer solution restored [Mg2+]i levels. These observations may suggest that acute "binge drinking" of ethanol, which often results in cerebral ischemia and stroke, may do so as a result of depletion of astrocytic [Mg2+]i, possibly producing disruption of the blood-brain barrier.     

Cytosolic Ca2+ under high glucose with suppressed Na+/K+ pump activity in rat sensory neurons

Cytosolic Ca2+ concentration ([Ca2+]i) was measured in isolated rat dorsal root ganglion (DRG) neurons using the fluorescent Ca2+ indicator fura-2. Exposure to high (50 mM) extracellular K+ evoked a robust increase in [Ca2+]i, which was almost totally abolished by concomitant presence of nisoldipine (10 microM) and omega-conotoxin GVIA (10 microM). Whereas either high (30 mM) D-glucose alone or ouabain (100 microM) alone did not appreciably affect the high K+-induced [Ca2+]i elevation, neurons pretreated with high D-glucose together with ouabain exhibited a significantly larger [Ca2+]i response to high K+ stimulation, which was almost completely inhibited by nisoldipine and omega-conotoxin GVIA. These results suggest that a combination of high glucose and suppressed Na+/K+ pump activity potentiates the [Ca2+]i elevation stimulated by activation of the voltage-gated Ca2+ channels in rat DRG neurons.