GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca²⁺]_i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca²⁺]_i, within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca²⁺]_i. The increases in [Ca²⁺]_i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca²⁺]_i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca²⁺]_i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca²⁺]_i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [C²⁺]_i and are able to respond to neuronally released neurotransmitters. (c) 1995 Wiley-Liss, Inc.     

Ionotropic and Metabotropic Glutamate Receptor Agonist-Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca²⁺ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10^?6?10^?4 M) evoked a dose-dependent increase in [Ca²⁺]_i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca²⁺]_i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg²⁺. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca²⁺]_i increase in a Ca²⁺-free condition, suggesting Ca²⁺ release from intracellular Ca²⁺ stores. This was further evidenced by [Ca²⁺]_i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca²⁺. Furthermore, the quisqualate-induced Ca²⁺ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca²⁺ release from intracellular Ca²⁺ stores and the activation of the latter two types induces Ca²⁺ entry. These dual mechanisms of Ca²⁺ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.     

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca²⁺]_i) that was communicated to surrounding cells. Following propagation of the Ca²⁺ wave, many cells showed asynchronous oscillations in [Ca²⁺]_i. Dantrolene sodium (10 μM) inhibited the increase in [Ca²⁺]_i associated with this Ca²⁺ wave by 60-80%, and prevented subsequent Ca²⁺ oscillations. Despite the markedly decreased magnitude of the increase in [Ca²⁺]_i, the rate of propagation and the extent of communication of the Ca²⁺ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca²⁺]_i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca²⁺]_i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca²⁺ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca²⁺]_i in most cells that was followed by sustained oscillations in [Ca²⁺]_i in some cells. Dantrolene (10 μM) inhibited this initial [Ca²⁺]_i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca²⁺ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca²⁺ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP₃) mediates the propagation of Ca²⁺ waves whereas Ca²⁺ -induced Ca²⁺ release amplifies Ca²⁺ waves and generates subsequent Ca²⁺ oscillations.     

Activation of alpha-2 adrenergic receptors stimulates GABA release by astrocytes

Norepinephrine is one of the key neurotransmitters in the hippocampus, but its role in the functioning of the neuroglial networks remains unclear. Here we show that norepinephrine suppresses NH₄Cl-induced oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca²⁺]_i oscillations is mediated by activation of alpha-2 adrenergic receptors. Furthermore, UK 14,304, an agonist of alpha-2 adrenergic receptors, evokes a biphasic [Ca²⁺]_i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca²⁺]_i rise, mediated by G_iβγ subunit and subsequent PLC-induced mobilization of Ca²⁺ from internal stores, and a plateau phase, mediated by a Ca²⁺ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca²⁺ response in astrocytes and suppression of [Ca²⁺]_i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of G_βγ-signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca²⁺]_i oscillations in neurons but does not suppress them. The same effect is observed in the presence of bicuculline, a GABA(A) receptor antagonist. We demonstrate that UK 14,304 application increases the frequency and amplitude of slow outward chloride currents in neurons, indicating the release of GABA by astrocytes. Thus, our findings indicate that the activation of astrocytic alpha-2 adrenergic receptors stimulates GABA release from astrocytes via G_iβγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity.     

lntracellular ca2+ changes in cultured vascular smooth muscle cells by treatment with various spasmogens

Abstract

In order to evaluate various spasmogenswhich are candidates for cerebral vasospasm after subarachnoid haemorrhage, the intracellular calcium mobilizations were examined in cultured vascular smooth muscle cells preloaded with a fluorescent Ca²⁺ probe fura-2. Endothelin, oxyhaemoglobin, 5-hydroxytryptamine, norepinephrine, prostaglandin F₂α, leukotrienes C₄ and D₄ produced dose-dependent increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, bilirubin did not induce any significant [Ca²⁺]_i elevation. The maximal levels of [Ca²⁺]_i peak attained by endothelin or oxyhaemoglobin were higher than those of other compounds. Endothelin was the most potent in that it induced a high sustained [Ca²⁺]_i elevation at much lower concentrations compared with others. The combination of oxyhaemoglobin and endothelin induced a transient increase in [Ca²⁺]_i followed by a sustained lower plateau, then the [Ca²⁺]_i level was again increased slowly followed by a sustained higher plateau which lasted for more than 10 min after the exposure. These results suggest that endothelin and/or oxyhaemoglobin may play a crucial role in contraction of vascular smooth muscle after subarachnoid haemorrhage.     

Bovine serum albumin and lysophosphatidic acid stimulate calcium mobilization and reversal of cAMP-induced stellation in rat spinal cord astrocytes

We report that lysophosphatidic acid (LPA) stimulates dynamic calcium (Ca²⁺) fluctuations and morphological rearrangements in astrocytes derived from neonatal rat spinal cord. Addition of 10 μM LPA elicited single Ca²⁺ transients, or biphasic oscillations and sustained increases in intracellular Ca²⁺ ([Ca²⁺]_i). The biphasic Ca²⁺ response consisted of an initial release from intracellular stores, often followed by a sustained elevation or continued oscillations that required Ca²⁺ flux across the cell membrane. The type of Ca²⁺ response, but not the overall magnitude, was dependent on LPA concentration. Higher concentrations (>10 μM) often elicited sustained increases in [Ca²⁺]_i, while lower concentrations stimulated oscillations or single Ca²⁺ transients. It has previously been established that agents that elevate cyclic adenosine monophosphate (cAMP) induce flat astrocytes to adopt a more stellate morphology. LPA can completely reverse this morphological change at a half-maximal concentration of 215 nM. Inhibiting LPA-induced [Ca²⁺]_i fluctuations using BAPTA-AM to buffer [Ca²⁺]_i and EGTA in the bath to prevent transmembrane flux had little effect on the ability of LPA to reverse stellation. LPA is found bound to serum albumin, in which crude preparations have been shown to induce various physiological responses in a number of cell types. Many of the activities have been attributed to albumin-associated lipid factors including LPA. We show that lipid factors associated with BSA can mimic the effect of LPA in both Ca²⁺ mobilization and reversal of cAMP-induced stellation. GLIA 20:163–172, 1997. © 1997 Wiley-Liss Inc.     

Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells

Previous investigators have reported that intracellular pH responds to hypoxia with a heterogenous pattern in individual glomus cells of the carotid body. The aim of the present study was to examine whether hypoxia had similar effects on cytosolic calcium ([Ca²⁺]_i) in glomus cells, and if so, whether a heterogenous response pattern is also seen in other cell types. Experiments were performed on glomus cells from adult rat carotid bodies, rat pheochromocytoma (PC12) and vascular smooth muscle (A7r5) cells. Changes in [Ca²⁺]_i in individual cells were determined by fluorescence imaging using Fura-2. Glomus cells were identified by catecholamine fluorescence. [Ca²⁺]_i in glomus cells increased in response to hypoxia (pO₂ = 35 ± 8mmHg; 5 min), whereas hypoxia induced decreases in [Ca²⁺]_i were not seen. Increases in [Ca²⁺]_i were observed in 20% of the isolated cells and strings of cells, but clustered glomus cells never responded. The magnitude of the calcium change in responding cells was proportional to the hypoxic stimulus. Under a given hypoxic challenge, there were marked variations in the response pattern between glomus cells. The response pattern characteristic of any given cell was reproducible. At comparable levels of hypoxia, PC12 cells also responded with an increase in [Ca²⁺]_i with a heterogenous response pattern similar to that seen in glomus cells. In contrast, increases in [Ca²⁺]_i in A7r5 cells could be seen only with sustained hypoxia ( ∼ 20 min), and little heterogeneity in the response patterns was evident. These results demonstrate that: (a) hypoxia increases cytosolic calcium in glomus cells; (b) response patterns were heterogeneous in individual cells; and (c) the pattern of the hypoxia-induced changes in [Ca²⁺]_i is cell specific. These results suggest that hypoxia-induced increases in [Ca²⁺]_i are faster in secretory than in non-secretory cells.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

Spontaneous intracellular calcium oscillations in cortical astrocytes from a patient with intractable childhood epilepsy (Rasmussen's Encephalitis)

Many studies have demonstrated that astrocytes respond with fluctuations in intracellular calcium concentration ([Ca²⁺]_i) and membrane potential following the application of a number of ligands. Moreover, calcium (Ca²⁺) waves that spread through astrocytic syncitia have been described in numerous reports. We had the rare opportunity to study Ca²⁺ responses in astrocytes obtained from a patient diagnosed with Rasmussen's encephalitis, a rare form of intractable epilepsy. Using the ratiometric fluorescent indicator fura-2, we observed large spontaneous [Ca²⁺]_i oscillations. The mean time between initial rise in [Ca²⁺]_i and the return to baseline was 5.1 ± 0.19 minutes (SEM; n = 201) and [Ca²⁺]_i increased to a mean level of 271 ± 8 nM (SEM; n = 201) from a baseline of 136 ± 6 nM (SEM; n = 201). Removal of Ca²⁺ from the perfusion solution combined with the addition of the Ca²⁺ chelator EGTA (2 mM) completely but reversibly eliminated all oscillations suggesting the fluctuations were dependent on Ca²⁺ flux across the membrane. The percentage of cells undergoing spontaneous changes in [Ca²⁺]_i decreased over time in culture. At 10–11 days post-surgery, approximately 70% of the cells were exhibiting this behavior, and by day 23 transients were no longer observed. We did not observe comparable spontaneous [Ca²⁺]_i oscillations in rat cortical astrocytes. The potential that the spontaneous [Ca²⁺]_i oscillations observed may be a unique feature of epileptic tissues is discussed. GLIA 21:332–337, 1997. © 1997 Wiley-Liss, Inc.     

SHORT COMUNICATION Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons

We investigated the effects of oxygen (O₂)/glucose deprivation on intracellular sodium concentration ([Na⁺]_i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O₂/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na⁺]_i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (d -amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na⁺/Ca²⁺-exchanger, affected these responses to O₂/glucose. The present results demonstrate that, in cortical neurons, O₂/glucose deprivation induces an early rise in [Na⁺]_i which cannot be ascribed to the activity of voltage gated Na⁺-channels, glutamate receptors or of the Na⁺/Ca²⁺-exchanger.     

P2U nucleotide receptor activation in rat glial cell line induces [Ca2+]i oscillations which depend on cytosolic pH

In single rat glioma cells, the signal transduction process activated by the UTP sensitive purinergic nucleotide receptor was studied by determining [Ca²⁺]_i by Fura-2 fluorescence and measuring pH by BCECF fluorescence to elucidate the control of [Ca²⁺]_i oscillations by intracellular pH. Addition of UTP for long time periods (some min) causes a [Ca²⁺]_i response composed of i) an initial large peak and a following sustained increase (160 s duration), and ii) subsequent regular [Ca²⁺]_i oscillations (amplitude 107 nM, frequency 1.5 oscillations per min). The maintenance of the [Ca²⁺]_i oscillations depends on the continued presence of agonist. The oscillations are abolished by reducing extracellular Ca²⁺ concentration. The interaction of UTP receptors and bradykinin receptors during the [Ca²⁺]_i oscillations was investigated because previous studies have already shown that the peptide causes comparable [Ca²⁺]_i oscillations. During [Ca²⁺]_i oscillations induced by UTP or bradykinin, long-term admission of both hormones (400–500 s) causes a large initial response superimposed on regular [Ca²⁺]_i oscillations. Short pulses (12 s) of the second agonist given in any phase of the oscillations induce large [Ca²⁺]_i peaks. In both cases, the following oscillations are not disturbed. The influence of cytosolic pH was studied by alkalinizing pH_i by application of NH₄Cl. [Ca²⁺]_i oscillations stop after addition of NH₄Cl. Recovery of NH₄Cl-induced alkalinization is reduced by furosemide. To the same degree, the interruption of [Ca²⁺]_i oscillations is significantly prolonged in the presence of furosemide. Thus cytosolic alkalinization suppresses hormone-induced [Ca²⁺]_i oscillations in rat glioma cells. The understanding of the molecular mechanism of this interference of pH should provide an important contribution for unravelling the function of cytosolic pH in cellular signal transduction. © 1996 Wiley-Liss, Inc.     

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of G_s. Measurements of intracellular free calcium concentration ([Ca²⁺]_i) reveal that mastoparan induces a dramatic elevation of [Ca²⁺]_i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca²⁺]_i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca²⁺]_i is maintained in the absence of extracellular Ca²⁺, suggesting that the rise of [Ca²⁺]_i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca²⁺]_i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca²⁺]_i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca²⁺]_i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca²⁺]_i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca²⁺ release from intracellular stores, probably via activation of PLC and IP₃. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.     

Characterization of the hypo-osmolarity-induced Ca2+ response in cultured rat astrocytes

The influence of astrocyte swelling on the cytosolic free calcium concentration [Ca²⁺]_i was studied at the single cell level. Sudden exposure of normo-osmotically (305 mosmol/l) cultured astrocytes to hypo-osmotic medium induced a biphasic increase in cytosolic calcium with an initial peak followed by a sustained plateau. The response was osmolarity dependent and was maximal at 205 mosmol/l with respect to [Ca²⁺]_i and the percentage of responding cells. Other modes of astrocyte swelling [gradual adjustment of hypo-osmolarity, normo-osmotic exposure of hyper-osmotic (405 mosmol/l) maintained cells] produced a much weaker [Ca²⁺]_i response. Change from 405 to 205 mosmol/l, however, resulted in the entire peak and an increased plateau. Experiments with Ca²⁺-free medium and after pretreatment with BAPTA-AM, thapsigargin, phorbol myristate acetate, or nimodipine revealed that the peak mainly resulted from depletion of intracellular Ca²⁺ stores, whereas the plateau was probably due to capacitative Ca²⁺ entry and Ca²⁺ influx independent of store depletion including a nimodipin-sensitive component. Prior depletion of ryanodine-, bradykinin- or ATP-sensitive stores revealed that the initial hypo-osmolarity-induced Ca²⁺-release was from a Ca²⁺ pool also affected by ATP and bradykinin, but not by ryanodine. The recent finding, that the hypo-osmolarity-induced [Ca²⁺]_i response was completely maintained if phospholipase C-mediated phosphatidylinositol hydrolysis was blocked, suggests that hypo-osmolarity may exert an inositol (1,4,5) triphosphate-independent access to these stores. GLIA 20:51-58, 1997. © 1997 Wiley-Liss, Inc.     

Receptor-activated Ca2+ increases in vibrodissociated cortical astrocytes: A nonenzymatic method for acute isolation of astrocytes

A new nonenzymatic method for the acute isolation of astrocytes from rat cerebral cortex is described. A vibratory device was used to dissociate the cells from thin brain slices, and the method yielded fresh and relatively well-preserved astrocytes without previous enzyme incubation. These cells were examined in a microspectrofluorometric system for measurement of changes in intracellular free calcium concentrations ([Ca²⁺]_i), and their expression of various neurotransmitter receptors was determined. Acutely isolated glial fibrillary acidic protein (GFAP)-positive astrocytes (p7–p18) were seen to respond to the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10^-4 M) with increases in [Ca²⁺]_i, and this response was blocked by (RS)-1-aminoindan-1,5 dicarboxylic acid (AIDA, 10^-3 M), an antagonist to group 1 metabotropic glutamate receptors. The δ-opioid receptor agonist D-Pen², D-Pen⁵-enkehalin (DPDPE, 10^-6 M) evoked [Ca²⁺]_i increases that were blocked by the δ-opioid antagonist ICI 174.388 (10^-5 M). The astrocytes failed to respond to 5-hydroxytryptamine (5-HT, 10^-5 M), although the same cells subsequently were found to respond to other agonists. Furthermore, [Ca²⁺]_i responses evoked by phenylephrine (10^-5 M) were blocked by prazosin (0.2⋅10^-6 M), suggesting the expression of α₁-adrenergic receptors on the acutely isolated astrocytes. The cells were also shown to react with [Ca²⁺]_i increases in response to depolarization with high extracellular potassium concentrations (50⋅10^-3 M). The signals induced by depolarization were not seen in Ca²⁺-free buffer, indicating the presence of voltage-activated calcium channels in these cells. Thus, the present study confirms some of the results earlier obtained in cell cultures, suggesting that cortical astrocytes in vivo express glutamate, opiate, and adrenergic receptors, coupled to increases in [Ca²⁺]_i, whereas no receptors for 5-HT could be detected. J. Neurosci. Res. 54:390–401, 1998. © 1998 Wiley-Liss, Inc.     

Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence

The intracellular free ([Ca²⁺]_i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca²⁺]_i was 109 nM on average under the resting condition and increased by raising the extracellular K⁺, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca²⁺]_i could be the result of opening of voltage-dependent Ca²⁺ channels. However, Ca²⁺ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca²⁺ channels (for both entries) or a limited number of the cells showing the latter response.     

Halothane-induced intracellular calcium release in cholinergic cells

Low concentrations of halothane and isoflurane can release acetylcholine in an extracellular Ca²⁺-independent manner. In the present study, a cholinergic cell line (SN56) was used to examine whether release of calcium from intracellular stores occurs in the presence of halothane. Changes in intracellular calcium concentration ([Ca²⁺]_i) were measured using fluo-3, a fluorescent calcium-sensitive dye and laser scanning confocal microscopy. Halothane, at sub-anesthetic concentrations (14, 28, 40 and 56 μM), increased [Ca²⁺]_i in SN56 cells. This effect remained even when the cells were perfused with medium lacking extracellular calcium, suggesting the involvement of intracellular Ca²⁺ sources. SN56 cells responded to ryanodine by increasing [Ca²⁺]_i and this effect was blocked by dantrolene, an inhibitor of Ca²⁺-release from ryanodine-sensitive stores. The effect of halothane was attenuated after the increase in [Ca²⁺]_i induced by ryanodine and it was suppressed by dantrolene, suggesting the participation of ryanodine-sensitive stores. Using cyclopiazonic acid, a Ca²⁺-ATPase inhibitor, we investigated whether the depletion of intracellular Ca²⁺ stores interfered with the effect of halothane. Cyclopiazonic acid significantly decreased the increase in [Ca²⁺]_i induced by the volatile anesthetic. It is suggested that sub-anesthetic concentrations of halothane may increase [Ca²⁺]_i by releasing Ca²⁺ from intracellular stores in cholinergic cells.     

An Interaction of Oxytocin Receptors with Metabotropic Glutamate Receptors in Hypothalamic Astrocytes

Hypothalamic astrocytes play a critical role in the regulation and support of many different neuroendocrine events, and are affected by oestradiol. Both nuclear and membrane oestrogen receptors (ERs) are expressed in astrocytes. Upon oestradiol activation, membrane‐associated ER signals through the type 1a metabotropic glutamate receptor (mGluR1a) to induce an increase of free cytoplasmic calcium concentration ([Ca²⁺]_i). Because the expression of oxytocin receptors (OTRs) is modulated by oestradiol, we tested whether oestradiol also influences oxytocin signalling. Oxytocin at 1, 10, and 100 nm induced a [Ca²⁺]_i flux measured as a change in relative fluorescence [ΔF Ca²⁺ = 330 ± 17 relative fluorescent units (RFU), ΔF Ca²⁺ = 331 ± 22 RFU, and ΔF Ca²⁺ = 347 ± 13 RFU, respectively] in primary cultures of female post‐pubertal hypothalamic astrocytes. Interestingly, OTRs interacted with mGluRs. The mGluR1a antagonist, LY 367385 (20 nm ), blocked the oxytocin (1 nm )‐induced [Ca²⁺]_i flux (ΔF Ca²⁺ = 344 ± 19 versus 127 ± 11 RFU, P < 0.001). Conversely, the mGluR1a receptor agonist, (RS)‐3,5‐dihydroxyphenyl‐glycine (100 nm ), increased the oxytocin (1 nm )‐induced [Ca²⁺]_i response (ΔF Ca²⁺ = 670 ± 31 RFU) compared to either compound alone (P < 0.001). Because both oxytocin and oestradiol rapidly signal through the mGluR1a, we treated hypothalamic astrocytes sequentially with oxytocin and oestradiol to determine whether stimulation with one hormone affected the subsequent [Ca²⁺]_i response to the second hormone. Oestradiol treatment did not change the subsequent [Ca²⁺]_i flux to oxytocin (P > 0.05) and previous oxytocin exposure did not affect the [Ca²⁺]_i response to oestradiol (P > 0.05). Furthermore, simultaneous oestradiol and oxytocin stimulation failed to yield a synergistic [Ca²⁺]_i response. These results suggest that the OTR signals through the mGluR1a to release Ca²⁺ from intracellular stores and rapid, nongenomic oestradiol stimulation does not influence OTR signalling in astrocytes.     

Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells

The bacterial alkaloid staurosporine is widely employed as an inducer of apoptosis in many cell types including neurons. The intracellular cascades that mediate staurosporine-induced apoptosis are largely unknown. Exposure of cultured PC12 cells to staurosporine resulted in a rapid (min) and prolonged (1–6 hr) elevation of intracellular free calcium levels [Ca²⁺]_i, accumulation of mitochondrial reactive oxygen species (ROS), and decreased mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction (1–4 hr). These early events were followed by membrane lipid peroxidation, loss of mitochondrial transmembrane potential, and nuclear apoptotic changes. Treatment of cells with serum or nerve growth factor within 1–2 hr of staurosporine exposure resulted in recovery of [Ca²⁺]_i and ROS levels, and rescued the cells from apoptosis. The increased [Ca²⁺]_i and ROS production were required for staurosporine-induced apoptosis because the intracellular calcium chelator BAPTA and uric acid (an agent that scavenges peroxynitrite) each protected cells against apoptosis. The caspase inhibitor zVAD-fmk and the anti-apoptotic gene product Bcl-2 prevented the sustained [Ca²⁺]_i increase and ROS accumulation induced by staurosporine indicating that caspases act very early in the apoptotic process. Our data indicate that a [Ca²⁺]_i increase is an early and critical event in staurosporine-induced apoptosis that engages a cell death pathway involving ROS production, oxidative stress, and mitochondrial dysfunction. J. Neurosci. Res. 51:293–308, 1998. © 1998 Wiley-Liss, Inc.     

Developmental Loss of GABA- and Glycine-induced Depolarization and Ca2+ Transients in Embryonic Rat Dorsal Horn Neurons in Culture

More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) when maintained in culture for <1 week. This [Ca²⁺]_i response has previously been shown to be due to depolarization and subsequent Ca²⁺ entry through voltage-gated Ca²⁺ channels following activation of bicuculline-sensitive GABA_A receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca²⁺]_i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca²⁺]_i. The loss of the [Ca²⁺]_i response was not due to a change in the abundance or the properties of voltage-gated Ca²⁺ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca²⁺]_i transient in response to 30 mM K⁺. Nor was the loss of the [Ca²⁺]_i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca²⁺]_i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation.